ISOLATED NUCLEIC ACID MOLECULES USEFUL AS LEUKEMIA MARKERS AND IN BREAST CANCER PROGNOSIS

Abstract: The present invention relates to four novel human genes amplified and overexpressed in breast carcinoma and located on the q11-q21.3 region of chromosome 17. The four novel genes are useful in breast cancer prognosis. The present invention also relates to a fifth novel human gene expressed in breast carcinoma and located on chromosone 6q22-q23. A sixth novel gene is also described that is the murine homolog of the human D52 gene. The genes and gene fragments of the present invention are themselves useful as DNA and RNA probes for gene mapping by in situ hybridization with chromosomes and for detecting gene expression in human tissues (including breast and lymph node tissues) by Northern blot analysis.

Description: Isolated Nucleic Acid Molecules Useful as Leukemia Markers and in Breast Cancer Prognosis Field of the Invention The invention relates to four novel human genes amplified and overexpressed in breast carcinoma. The four genes are located at chromosome 17ql l-q21.3. The invention also relates to a fifth novel human gene expressed in breast carcinoma and located at chromosome 6q22-q23. A sixth novel gene is also described that is the murine homolog of the human D52 gene. Background of the Invention Despite earlier detection and a lower size of the primary tumors at the time of diagnosis (Nystrόm, L. etal, Lancet 341:973-97% (1993); Fletcher, S.W. et al, J. Natl. Cancer Inst. 55:1644-1656 (1993)), associated metastases remain the major cause of breast cancer mortality (Frost, P. & Levin, R., Lancet 339: 1458-- 1461 (1992)). The initial steps of transformation characterized by the malignant cell escape from normal cell cycle controls are driven by the expression of dominant oncogenes and/or the loss of tumor suppressor genes (Hunter, T. & Pines, J., Cell 79:573-582 (1994)). Tumor progression can be considered as the ability of the malignant cells to leave the primary tumoral site and, after migration through lymphatic or blood vessels, to grow at a distance in host tissue and form a secondary tumor (Fidler, IJ, Cancer Res. 50:6130-6138 (1990); Liotta, L. etal, Cell 64:327-336 (1991)). Progression to metastasis is dependent not only upon transformation but also upon the outcome of a cascade of interactions between the malignant cells and the host cells/tissues. These interactions may reflect molecular modification of synthesis and/or of activity of different gene products both in malignant and host cells. Several genes involved in the control of tumoral progression have been identified and shown to be implicated in cell adhesion, extracellular matrix degradation, immune surveillance, growth factor synthesis and/or angiogenesis (reviewed in, Hart, I.R. & Saini, A., Lancet 339: 1453-1461 (1992); Ponta, H. et al, B.B.A. 1198: 1-10 (1994); Bernstein, L.R. & Liotta, L.A, Curr. Opin. Oncol. 6:106-113 (1994); Brattain, M.G. etal, Curr. Opin. Oncol. 6: 7-81 (1994); and Fidler, LI & Ellis, L.M., Cell 79:185-188 (1994)). However, defining the mechanisms involved in the formation and growth of metastases is still a major challenge in breast cancer research (Rusciano, D. & Burger, MM, BioEssqys 74: 185-194 (1992); Hoskins, K. & Weber, B.L., Current Opinion in Oncology 6:554-559 (1994)). The processes leading to the formation of metastases are complex (Fidler, I.I, Cancer Res. 50:6130-6138 (1990); Liotta, L. et al., Cell 64:321 '-336 (1991)), and identifying the related molecular events is thus critical for the selection of optimal treatments. Summary of the Invention By differential screening of a cDNA library from breast cancer derived metastatic axillary lymph nodes, four clones (MLN 50, 51, 62 and 64) were isolated by the present inventors and determined to be co-localized at the ql l- q21.3 region of the chromosome 17 long arm. Several genes implicated in breast cancer progression have been assigned to the same portion of chromosome 17, most notably the oncogene c-erbB-2 in ql2 and the recently cloned tumor suppressor gene BRCA1 in q21. Additionally, the D53 gene was cloned by the present inventors from a cDNA library of primary infiltrating ductal breast carcinoma using a expressed sequence tag that was identified to be homologous to the previously identified D52 gene, and the D53 gene was localized to chromosome 6q22-q23. The four MLN genes of the present invention are useful as prognostic markers for breast cancer. Although no group of the art-known prognosticators completely fulfills the objective to fully distinguish high- and low-risk patients, combinations of the prognostic factors can improve the prediction of a patient's prognosis. Thus, by the invention, further prognostic markers are provided which can be added to the population of art-known prognosticators to more particularly distinguish between high- and low-risk breast cancer patients. By the invention, when compared to MLN 50, 51, 62, or 64 gene expression level or gene copy number in non-tumorigenic breast tissue, enhanced MLN 50, 51, 62, or 64 gene expression level or gene copy number in breast cancer tissue is indicative of a high-risk breast cancer patient. The invention further provides a method for distinguishing between different types of acute myeloid leukemia, which involves assaying leukemia cells for D52 or D53 gene expression; whereby, the presence of D52 transcripts (mRNA) or protein or the lack of D53 mRNA or protein indicates that the leukemia cells have myelocytic characteristics (such as HL-60 cells) and the presence of D53 mRNA or protein or the lack of D52 mRNA or protein indicates that the leukemia cells have erythroid characteristics (such as K-562 cells). Also provided are isolated nucleic acid molecules encoding MLN 50, 51, 62, 64, D53, or murine (m) D52 polypeptides whose amino acid sequences are shown in Figures 14, 21(A-D), 6, 16, 24(B) and 25(B), respectively. In another aspect, the invention provides isolated nucleic acid molecules encoding MLN 50, 51, 62, 64, or D53 polypeptides having an amino acid sequence as encoded by the cDNAs deposited as ATCC Deposit Nos. 97608, 97611, 97610, 97609 and 97607, respectively. Further embodiments of the invention include isolated nucleic acid molecules that are at least 90% and preferably at least 95%, 97%, 98% or 99% identical the above- described isolated nucleic acid molecules of the present invention. The present invention also relates to vectors which contain the above- described isolated nucleic acid molecules, host cells transformed with the vectors and the production of MLN 50, 51, 62, 64, mD52 or D53 polypeptides or fragments thereof by recombinant techniques. The present invention further provides an isolated MLN 50, 51, 62, 64, D53 or mD52 polypeptide having the amino acid sequence as shown in Figure 14, 21(A-D), 6, 16, 24(B) or 25(B), respectively. In a further aspect, an isolated MLN 50, 51, 62, 64 or D53 polypeptide is provided having an amino acid sequence as encoded by the cDNAs deposited as ATCC Deposit Nos. 97608, 97611, 97610, 97609 and 97607, respectively. Brief Description of the Figures Figure 1. Expression Analysis of the 10 MLN Genes. Northern blots contained 10 μg of total RNA isolated from MLN (lanes 1), NLN (lanes 2) and FA (lanes 3). Five filters have been prepared and each of them was successively hybridized using two MLN cDNA probes (MLN 62 and 50; MLN 74 and 51; MLN 19 and 64; MLN 10 and 137; MLN 4 and 70) and the internal loading control 36B4. rRNA size markers (S values) are indicated (left). Figure 2. Chromosomal Assignment of MLN SO, 51, 62 and 64 Genes by in Situ Hybridization. (A) Idiogram of the human G-banded chromosome 17 illustrating the distribution of labeled sites for MLN 50, 51, 62 and 64 cDNA probes. (B) Putative relative assignment of the MLN genes within the ql l-q21.3 region of the long arm of the chromosome 17. Figure 3. Expression Analysis of MLN SO, SI, 62 and 64 Genes Among Breast Cancer Cell Lines. Ten μg of total RNA from breast cancer cell lines were loaded in each lane. Hybridizations were carried out successively with probes corresponding to MLN 50, 51, 62 and 64. Control hybridizations were performed with MLN 19 (c-erbB-2), p53 and 36B4. Approximate sizes of the mRNAs are indicated in kb (right). Figure 4. Northern Blot Analysis ofCARTl mRNA in Human Breast Fibroadenomas, Carcinomas and Lymph Node Metastases. Each lane contained 10 μg of total RNA. From left to right, RNA samples from breast fibroadenomas (FA, lanes 1-6), carcinomas (BC, lanes 7-16) and metastatic lymph nodes (MLN, lanes 17 and 18) were loaded. Hybridization was carried out using 32 P cDNA probe for CART1. A 2000-base long CARTl transcript was expressed, at various levels, in some carcinomas (lanes 7, 11 and 13), and in one metastatic sample (lane 17). The 36B4 probe (Masiakowski, P. et al, Nucl Acids Res. 70:7895-7903 (1982)) was used as positive internal control. Autoradiography was for 2 days for hybridization of CARTl, whereas 36B4 hybridization was exposed for 16 hrs. Figure 5. In Situ Hybridization of CARTl mRNA in Human Breast Carcinoma and Axillary Lymph Node Metastasis. Sections of normal breast (A), in situ carcinoma (C), invasive carcinoma (B) and metastatic lymph node (D) were hybridized with antisense 35 S RNA probe specific for CART 1. CART 1 was strongly expressed in the tumoral epithelial cells, whereas the stromal part of the tumor was totally negative (B). CARTl transcripts were homogeneously distributed throughout the positive areas (B-D). Normal ducts were devoid of CARTl signal (A). No significant labeling above background was found when using sense human CARTl RNA probe (data not shown). Bright field (A-D). Figure 6. Nucleotide and Amino Acid Sequences of Human CARTl. Nucleotide sequence (SEQ ID NO:l) is numbered in the 5' to 3* direction and amino acid sequence (SEQ ID NO:2) in the open reading frame is designated by the one letter code. The underlined nucleotide sequences correspond to the Kozak and poly(A) addition signal sequences. Putative NLS sequences are bold- typed and broken underlined. The two C-rich regions are boxed and H and C residues are bold-typed. Restricted TRAF domain is grey-boxed. Arrow-heads indicate the splicing sites and asterisk the stop codon. Figure 7. Primary Structure of the CARTl C3HC3D Motif and Comparison with RING Finger Proteins from Various Species. These sequences are aligned to each other using the PileUp program (Feng, D.F. & Doolittle, RF., J. Mol. Evol. 25:351-360 (1987)). Bracket numbers indicate the respective position of the motif in each protein. Residues identical in all sequences are bold-typed, and the conservative residues (R/K; I/N/L; Y/F; D/E; Ν/Q; S/T) are grey-boxed. Gaps are used to optimize alignment: H, Homo (CARTl (SEQ ID ΝO:2), RING1 (SEQ ID NO: 13), BRCA1 (SEQ ID NO: 14), CD40bp (SEQ ID NO: 15), SS-A/Ro (SEQ ID NO:16), MEL18 (SEQ ID NO: 17)); M, Mus (1RAF2 (SEQ ID NO: 18), RPT- 1 (SEQ ED NO: 19)); X, Xenopus (XNF7 (SEQ ID NO:20)); D, Drosophila (SU(z)2 (SEQ ID NO:21)); S, Saccharomyces (RAD18 (SEQ ED NO:22)); D, Dictyosteliu (DG17 (SEQ ID NO:23). Figure 8. Pattern of Avail Digestion of the Full-Length CARTl cDNA. (A) Positions and sequence of Avail sites (bold-typed) in the full-length CARTl cDNA (SEQ IDNO:l). Corresponding protein sequence from residues 54 to 60 of SEQ ID NO:2 is indicated using one letter code. D is bold-typed. (B) Ethidium bromide staining of gel electrophoresis of the CARTl Avail digest. Molecular weight (m.w.) and CARTl fragments sizes are given on the left and right sides, respectively. Figure 9. Primary Structure of the Three Original HC3HC3 C-rich Motifs Present in CARTl and Comparison with Those ofCD40-bp, TRAF2 andDG17. Alignment and conventional symbols are as described in the Figure 7 legend above: CARTl (101-154) (SEQ ID NO:2); CARTl (155-208) (SEQ ID NO:2); CARTl (209-267) (SEQ ID NO:2); CD40bp (134-189) (SEQ ID NO:24); CD40bp (190-248) (SEQ ID NO:25); TRAF2 (124-176) (SEQ ID NO:26); TRAF2 (177-238) (SEQ ED NO:27); DG17 (193-250) (SEQ ID NO:28). Figure 10. Primary Structure of the Restricted TRAF Motif and Comparison with Those of CD40-bp, TRAF1 and TRAF2. Alignment and conventional symbols are as described in the Figure 7 legend above. Consensus sequence (SEQ ED NO:32) is indicated for CARTl (308-387) (SEQ ID NO:2), CD40bp (415-494) (SEQ ID NO:29), TRAF1 (260-339) (SEQ ID NO:30), and TRAF2 (352-431) (SEQ ID NO:31). Consensus sequence (SEQ ID NO:36) is indicated for CARTl (388-470) (SEQ ID NO:2), CD40bp (495-567) (SEQ ID NO:33), TRAF1 (340-409) (SEQ ID NO:34), and TRAF2 (432-501) (SEQ ID NO:35). Figure 11. Organization of the Human CARTl Gene and Protein. Schematic representation of the CARTl gene exonintron organization. Exons are numbered from 1 to 7. The correspondence between DNA coding sequences and protein domains are indicated (B, BamΗ ; ORF, open reading frame; UTR, untranslated region). Figure 12. Comparison of CARTl, CD40-bp, TRAF2 and DG17 Protein Structural Organization. The size and position of RING finger, CART motif, α helix and restricted TRAF domain are represented for each of these proteins, highlighting the similarity of their protein organization. Figure 13. Northern Blot Analysis of Lasp-1 mRNA Expression in Human Tissues. (A) Total RNA (10 g) extracted from breast-derived metastatic lymph node (lanes 1 and 2), breast carcinomas (lanes 3-12), fibroadenomas (lanes 13-17) and breast hyperplasia (lane 18) were loaded, transferred, and hybridized with 32 P-labeled probes specific for c-er_B-2, Lasp-1 and to the RNA loading control 36B4. Approximate transcript sizes are indicated (right). (B) Total RNA extracted from normal lymph node (lane 1), normal skin (lane 2), normal lung (lane 3), normal stomach (lane 4), normal colon (lane 5), normal liver (lane 6), SK-Br-3 (lane 7), BT-474 (lane 8) and MCF-7 (lane 9) were loaded, transferred, and hybridized with 32 P-labeled probes specific for c-erbB-2, Lasp-1 and to the RNA loading control 36B4. Approximate transcript sizes are indicated (right). Figure 14. Nucleotide and Amino Acid Sequences of Human Lasp-1. (A) Nucleotide sequence (SEQ ED NO: 3) and amino acid sequence (SEQ ID NO:4) of human Lasp-1. Nucleotides and amino acid residues are numbered on the left and right, respectively. The consensus residues involved in the LIM domain are underlined and bolded and in the SH3 domain re-bolded. Putative tyrosine residues in tyrosine kinase phosphorylation are underlined. An asterisk denotes the termination codon. The signal for polyadenylation is underlined. (B) Structure of Lasp-1 cDNA. The shaded box indicates the protein-coding region. The position of the different expressed sequences tags with homology to Lasp-1 are indicated with their corresponding length and accession numbers. Figure 15. Comparison of the Lasp-1 LIM and SH3 Domains with Other Proteins. (A) Comparison of Lasp-1 LIM domain (residues 1-51 of SEQ ID NO:4) with other LIM proteins: YLZ4 (1-51) (SEQ ED NO:37); hCR P (1- 55) (SEQ ED NO:38); rCRP2 (1-56) (SEQ ED NO:39); rCRP2 (119-180) (SEQ K> NO:40); TSF3 (5-64) (SEQ DD NO:41); TSF3 (104-162) (SEQ ID NO:42)). The consensus LIM domain residues are bolded, identical residues are dashed, (.) indicates gaps in the alignment. (B) Comparison of Lasp-1 SH3 domain (residues 196-261 of SEQ ED NO:4) with other proteins: YLZ3 (134-200) (SEQ ED NO:43); EMS1 (486-550) (SEQ ID NO:44); ABP1 (526-592) (SEQ ED NO:45); h/fyn (76-141) (SEQ ID NO:46); h/src (78-144) (SEQ ID NO:47); h/frg (71-135) (SEQ ID NO:48); h/yes (85-152) (SEQ ID NO:49). The identical residues are dashed, conserved or semiconserved residues in more then half or the aligned sequences are bolded, (.) indicates gaps in the alignment. Figure 16 Nucleotide and Amino Acid Sequences of Human MLN 64. Nucleotide sequence (SEQ ID NO:5) is numbered in the 5' to 3' direction and amino acid sequence (SEQ ID NO: 6) in the open reading frame is designated by the one letter code. The underlined nucleotide sequences correspond to the Kozak and poly(A) addition signal sequences. The dashed underlined nucleotide sequences correspond to the sequences which could be deleted; 0 new splicing site after deletion; ♦ sites of insertions. Synthetic peptide sequence is bold-typed. Arrowheads indicate the splicing sites and asterisk the stop codon. Figure 17. Organization of the Human MLN 64 Gene and Protein. Schematic representation of the MLN 64 gene exon/intron organization. Exons are numbered from 1 to 15 (hatched and open boxes for coding and noncoding exons, respectively). Arrows indicate the nucleotide substitution, exon deletion and intron insertion sites (a: exon 2, C/T substitution, b: exon 2, 137 bp 5* end deletion, c: exon 4, A/G substitution, d: exon 4, 13 bp 3' end deletion, e: intron 6, 199 bp 5' end insertion, f: complete exon 7 deletion, g and h: intron 9, 51 bp and 657 bp 5' end insertion). Figure 18. Northern Blot Analysis of MLN 64 mRNA in Human Breast Fibroadenomas, Carcinomas and Lymph Node Metastases. Each lane contained 10 μg of total RNA. From left to right, RNA samples from breast fibroadenomas (lanes 1-6), carcinomas (lanes 7-14), normal lymph nodes (lanes 15 and 16) and metastatic lymph nodes (lanes 17 and 18) are loaded. Hybridization was carried out using 32 P cDNA probe for MLN 64. A 2000-base long MLN 64 transcript is expressed, at various levels, in some carcinomas (lanes 6, 10 and 11), and in the metastatic samples (lanes 16 and 17). The same pattern of expression was observed using an erbB-2 probe. The 36B4 probe (Masiakowski, P. et al, Nucl Acids Res. 70:7895-7903 (1982)) was used as positive internal control. Autoradiography was for 2 days for hybridization of MLN 64 and erbB-2, whereas 36B4 hybridization was exposed for 16 hrs. Fϊgure 19. In Situ Hybridization of MLN 64 mRNA in Human Breast Carcinoma and Axillary Lymph Node Metastasis Sections of normal breast (A), in situ carcinoma (C), invasive carcinoma (B) and metastatic lymph node (D) were hybridized with antisense 35 S RNA probe specific for MLN 64. MLN 64 is strongly expressed in the tumoral epithelial cells, whereas the stromal part of the tumor is totally negative (B). MLN 64 transcripts are homogeneously distributed throughout the positive areas (B-D). Normal ducts are devoid of MLN 64 signal (A). No significant labeling above background was found when using sense human MLN 64 RNA probe (data not shown). Bright field (A-D). Figure 20. Immunohistochemistry of Human Breast Carcinoma and Axillary Lymph Node Metastasis. Sections of normal breast (A), in situ carcinoma (C), invasive carcinoma (B) and metastatic lymph node (D) were studied for the presence of MLN 64 protein, using a monoclonal antibody {see Materials and Methods). MLN 64 is strongly expressed in the tumoral epithelial cells, whereas the stromal part of the tumor is totally negative (B). MLN 64 protein was located in cytoplasmic bundles like structures (B-D). Normal ducts are devoid of MLN 64 staining (A). Figure 21 (A-D). Nucleotide and Amino Acid Sequences of Human MLN 51. Nucleotide sequence (SEQ ID NO:7) is numbered in the 5' to 3' direction. The length of the sequence is 4253 bases and includes an additional untranslated 233 nucleotides on the 5' end. Amino acid sequence (SEQ ID NO:8) is numbered in the 5' to 3' direction (underneath). The length of the sequence is 534 amino acids. Figure 22. Alignment of Expressed Sequence Tags (ESTs) with Homology to the CARTl cDNA Sequence. Nine ESTs with homology to part of the CARTl nucleotide sequence were identified in GenBank. The accession number and alignment relative to the CARTl gene are indicated. The CARTl ORF is boxed. Figure 23. Alignment of Expressed Sequence Tags (ESTs) with Homology to the MLN 51 cDNA Sequence. Three ESTs with homology to part of the MLN 51 nucleotide sequence were identified in GenBank. The accession number and alignment relative to the MLN 51 gene are indicated. Figure 24 (A)-(B). Diagrammatic Representation of 3 hD53 cDNAs. (A) Diagrammatic representation of 3 hD53 cDNAs, with clones 83289 and 116783 representing cDNAs isolated by the Washington University-Merck EST project, and clone Ul representing a cDNA isolated from the human breast carcinoma cDNA library during this study. Shaded regions indicate 5'-UTR sequence, solid regions indicate coding sequence and open regions indicate 3'-UTR sequence. The polyadenylation signals associated with polyA sequences are indicated, as is a clone 83289 deletion, and an Alu sequence in the 3 -UTR of clone 83289. (B) Nucleotide sequence (SEQ ED NO:9) and amino acid sequence (SEQ ID NO: 10) determined for the hD53 Ul cDNA. The predicted coding sequence is translated using the one letter code (in bold), with numbering in italics referring to the translated product, and all other numbering referring to the nucleotide sequence. Within the 3'-UTR, the polyadenylation signal (ATTAAA, nucleotides 1308-1313 of SEQ ID NO:9) is shown underlined and in bold, as is the corresponding site of polyA addition (nucleotide 1325). Figure 25 (A)-(B). Diagrammatic Representation of Two mD52 cDNAs. (A) Diagrammatic representation of two mD52 cDNAs isolated from the apoptotic mouse mammary gland cDNA library. Shaded regions indicate 5'-UTR sequence, solid regions indicate coding sequence and open regions indicate 3'-UTR sequence. The polyadenylation signals associated with polyA sequences are indicated. (B) Nucleotide sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12) determined for the mD52 Cl cDNA. The predicted coding sequence is translated using the one letter code (in bold), with numbering in italics referring to the translated product, and all other numbering referring to the nucleotide sequence. Within the 3 -UTR, two polyadenylation signals (ATTAAA, nucleotides 976-981, and AATAAA, nucleotides 2014-2019, both of SEQ ID NO: 11) are shown underlined and in bold, as are the corresponding sites of polyA addition (nucleotides 1012 and 2033 of SEQ ID NO:l 1). Figure 26 (A)-(B). Alignment of mD52, hD52 and hD53. (A) Alignment of mD52 (SEQ ID NO: 12), hD52 (SEQ ID NO:50) and hD53 (SEQ ID NO: 10) amino acid sequences, shown using the one-letter code, as produced by the program PileUp. Numbers above and below the sequences refer to amino acid positions in mD52 and hD53, respectively, with numbering being identical for the 3 sequences up to residue 127, and for hD52 and mD52 up to residue 171. Vertical lines and colons indicate residues identical or conserved, respectively, in mD52 and hD52, and/or in hD52 and hD53 sequences. The following substitutions were allowed: MILVA, GA, DE, TS, QN, YFW, RKH. The combined limits of the N-terminal PEST domains (Lys^-Arg 40 in mD52, Arg 10 - Arg 40 in hD52, and Met'-Lys 37 in hD53), coiled-coil domains (Glu∞-Leu 71 mD52, Ala∞-Leu 71 in hD52 and VaP-Leu 71 in hD53), and C-terminal PEST domains (Lys , 2 -Pro 185 in mD52, Lys 152 -Lys 179 in hD52 and Lys ^His '" n hD53) are indicated above the sequences. In addition, potential sites of N-glycosylation (Asn 163 and Asn 167 in mD52, Asn 167 in hD52, and Asn 82 in hD53) are shown underlined and in bold. Potential sites of phosphorylation by casein II kinase (Ser 26 , Thr 32 , Thr 44 , Ser 75 , Ser 06 in mD52; Ser 26 , Thr 30 , Ser 32 , Ser 75 SeH 36 , Thr 171 in hD52; Thr 17 , Ser 32 , Ser 58 , Ser 16 , Ser" 9 , Ser 174 , Thr 197 in hD53), protein kinase C (Thr , Thr 133 in mD52 and hD52; Thr 52 , Ser 58 , Ser 122 , Ser 131 , Thr 146 , Ser 160 ; Ser* 4 in hD53), cAMP- and cGMP-dependent kinase (Ser 100 in mD52 and hD52), and tyrosine kinase (Tyr 130 in hD53) are all shown in bold. (B) The aligned coiled-coil domains identified in mD52 (SEQ ID NO: 12), hD52 (SEQ ID NO:51) and hD53 (SEQ ED NO: 10) sequences, shown using the one-letter code. Numbers below the sequences refer to amino acid positions in the 3 sequences. The abcdefg heptad repeat pattern is indicated above the sequences, with positions a and d (frequently occupied by hydrophobic amino acids in coiled-coil domains) shown in bold, and positions e and g (frequently occupied by negatively and positively charged amino acids, respectively) are underlined. Where mD52, hD52 and hD53 sequences are in accordance with this consensus, the relevant residues are correspondingly shown in bold or underlined. Figure 27 (A)-(B). (A) Ideogram of the human G-banded chromosome 6 illustrating the distribution of labeled sites with the 116783 hD53 probe. (B) Localization of the mD52 gene to mouse chromosomes 3 and 8 by in situ hybridization. Diagrams of WMP mouse Rb (3; 12) and Rb (8; 9) chromosomes, indicating the distributions of labeled sites on chromosomes 3 and 8. Figure 28. The Effects of Estradiol Treatment on hD52 and hD53 Transcript Levels in Human Breast Carcinoma Cell Lines. Northern blot analyses were performed using 10 μg total RNA for each sample. The identity and size (in parenthesis) of each transcript is indicated to the right of each panel, whereas the corresponding duration of autoradiographic exposure is shown on the left. For each cell line, lane 1 indicates total RNA from cells grown for 6 days in normal media {see Materials and Methods), lane 2 indicates total RNA from cells grown for 1 day in normal media and for 5 days in phenol red-free DMEM with 10% steroid-depleted FCS and 0.6 μg/ml insulin, lane 3 is as for lane 2 except that for the last 3 days of culture, media were supplemented with 10 "9 M estradiol, and lane 4 is as for lane 2 except that for the last 3 days of culture, media were supplemented with 10 *8 M estradiol. ER+/ER- indicates the presence/absence of the estrogen receptor in the cell line(s) shown below. The hD52 and hD53 transcripts were co-expressed in the 3 cell lines, and transcript levels for both genes were similarly affected by estradiol stimulation/deprivation in MCF7 cells, and were not affected by the same treatments in BT-20 cells. Differing effects on hD52 and hD53 transcript levels were noted in the experiment using BT-474 cells. The estrogen-inducible__S2 gene was used as a control for the effectiveness of estradiol supplementations/deprivations. As expected, the presence of estradiol induced pS2 expression in ER+ cell lines, but not in the ER+ cell line BT-20. For all 3 cell lines, similar results were obtained in at least one other experiment performed on a separate occasion. Figure 29. The Effects ofTPA Treatment on hD52 or hD53 Transcript Levels in Human Leukemia Cell Lines. Northern blot analyses were performed using 10 μg total RNA for each sample. The identity and size (in parenthesis) of each transcript is indicated to the right of each panel, whereas the corresponding duration of autoradiographic exposure is shown on the left. Lanes marked (C) indicate total RNA from cells grown in normal media {see Materials and Methods), lanes marked (16) indicate total RNA from cells grown in media supplemented with 16 nM TPA, and lanes marked (160) indicate total RNA from cells grown in media supplemented with 160 nM TPA. Times shown above the lanes indicate when cells were harvested after the start of each experiment. (A) TPA treatment of HL-60 cells was found to decrease hD52 and transferrin receptor (TR) transcript levels after 18 hrs TPA treatment. hD53 transcripts were not detected in HL-60 cells. Similar results were obtained in at least one other experiment performed on a separate occasion. (B) TPA treatment of K-562 cells was found to decrease hD53 and transferrin receptor (TR) transcript levels after 24 hrs TPA treatment. hD52 transcripts were not detected in K-562 cells. Figure 30. Southern Blot Analysis of Three Representative Breast Cancer Tumor DNAs with Amplifications of Chromosomal Region 17qll-q21. (L) and (T) indicate matched Taql-digested DNA samples isolated from peripheral leukocytes and tumor tissue, respectively. Hybridizations were carried out successively with probes MLN 50, 51, 62, 64 and ERBB2. Case 309 shows amplifications for MLN 62, ERBB2 and MLN64. Case 1191 shows amplification for only MLN 62. Case 1512 shows amplifications for ERBB2 and MLN 64. Figure 31. 17qll-q21 Amplicon Maps in Human Breast Cancer. Lines correspond to each tumor sample, columns to each marker. The densitometrically determined gene dosages (amplification levels) were subdivided into four categories. White boxes represent a normal copy number, shaded boxes 2-5 times amplification, dark shaded boxes 6-10 times amplification, and black boxes > ten times amplification. The loci from 17ql l-q21 are ordered according to their chromosomal location, from the most centromeric locus (MLN 62) to the most telomeric locus (MLN 51). Figure 32. Nothern Blot Analysis of MLN 50, 51, 62, 64 and ERBB2 in Normal and Tumoral Breast Tissues. Nl and N2, normal breast tissues; T309, Tl 191 and T1512, breast tumor tissues. Hybridizations were carried out successively with probes MLN 50, 51, 62, 64 and ERBB2. Control hybridizations with the 36B4 probe showed that similar amounts of mRNA were loaded in each case. Right, approximate sizes of the mRNAs are indicated in kb. Case 309 shows overexpressions for MLN62, ERBB2 and MLN64, compared with normal breast tissues. Case 1191 shows overexpression for only MLN62. Case 1512 shows overexpressions for ERBB2 and MLN64. Detailed Description of the Invention Isolation and Localization of Six Novel Genes, MLN 50, 51, 62, 64, D53 and mD52 The present inventors have identified four genes, co-localized on the long arm of chromosome 17, which are amplified and overexpressed in malignant breast tissues. In order to identify and clone these genes involved in tumor progression, differential screening of a cDNA library from breast cancer derived metastatic axillary lymph nodes was performed. The method involved screening the MLN cDNA library using two probes representative of malignant (MLN) and nonmalignant (fibroadenomas; FA) breast tissues. FAs were selected as control tissues since, although nonmalignant, they are proliferating tissues, thereby miniπiizing the probability to identify mRNAs characteristic of cellular growth, but unrelated to the malignant process. The differential screening method is explained in detail in Example 1, below, and in Basset, P. et al, Nature 348:699-704 (1990), where it is described as allowing identification of the stromelysin-3 gene {see also, U.S. Pat. No. 5,236,844). Four differential clones (MLN 50, 51, 62 and 64) were isolated which correspond to cDNAs whose sequences do not belong to any previously characterized gene or protein family as determined by comparison to the combined GeneBankEMBL databanks. By in situ hybridization of metaphase cells, the four new genes of the present invention were determined to be co-located to the ql 1- q21.3 region of the chromosome 17 long arm. Several genes implicated in breast cancer progression have been assigned to the same portion of chromosome 17, most notably the oncogene c-erbB-2 in ql2 (Fukushige, S.I. et al, Mol. Cell. Biol. 6:955-958 (1986)) and the recently cloned tumor suppressor gene BRCA1 in q21 (Hall, J.M. et al, Science 250:1684-1689 (1990); and Miki, Y. et al, Science 266:66-71 (1994)). According to their chromosomal assignments, the present inventors mapped the four novel genes proximal (MLN 62 and 50) and distal (MLN 64 and 51) to the c-erbB-2 gene, and proximal to the BRCA1 gene. It has been shown previously that multiple chromosome segments on the chromosome 17 long arm are targets for amplification in breast tumorigenesis (Muleris, M. et al, Genes Chrom. Cancer 70:160-170 (1994); Kallioniemi, A. et al, Proc. Natl. Acad. Sci. USA 97:2156-2160 (1994)), and 17ql2 was found to be the most commonly amplified chromosomal band-region (Guan, X.Y. et al, Nat. Genet. 5:155-161 (1994)). Consistently, in breast cancers, c-erbB-2 overexpression is most often correlated to gene amplification (Slamon, D.I et al, Science 255:177-182 (1987); van de Nijver, M. et al, Mol. Cell. Biol 7:2019- 2023 (1987)). It is assumed in the art that DΝA amplification plays a crucial role in tumor progression by allowing cancer cells to upregulate numerous genes (Kallioniemi, A etal, Proc. Natl. Acad. Sci. USA 97:2156-2160 (1994); Lόnn, U. et al, Intl. J. Cancer 55:40-45 (1994)). Amplification is known to target oncogenes and genes involved in drug resistance. Frequency of gene amplification as well as gene copy number increase during breast cancer progression, notably in patients who do not respond to treatment, suggesting that overexpression of the amplified target genes confers a selective advantage to malignant cells (Lonn, U. et al, Intl. J. Cancer 55:40-45 (1994); Guan, X.Y. et al, Nat. Genet. 5:155-161 (1994)). In vivo, the four MLΝ genes showed amplification in 10-20% of breast carcinomas tested. The D52 gene has been isolated by differential screening of a cDΝA library from primary infiltrating ductal breast carcinoma (Byrne, I A. et al, Cancer Res. 55:2896-2903 (1995)) and found to be overexpressed and localized exclusively to cancer cells, and not to other cell types such as fibroblastic cells. By in situ hybridization of metaphase cells, D52 was localized to chromosome 8q21. This region of the human genome has been noted to be amplified in breast cancer cell lines, and it was suggested that the frequent gain of the entire chromosome 8q arm in breast carcinomas may indicate the existence of several important loci within this region (Kallioniemi, A. et al, Proc. Natl. Acad Sci. USA 97:2156-2160 (1994)). The present inventors have isolated a homolog of D52 by screening a cDΝA library from primary infiltrating ductal breast carcinoma with an expressed sequence tag (EST) that was identified to be homologous to the hD52 gene, followed by a secondary screening of the resulting positive clones. The method for cloning the D52 homolog is explained in detail in Example 5 below. One clone (D53) was isolated by the present inventors that encodes a protein sharing 52% identity to the D52 protein. By in situ hybridization of metaphase cells, the new gene of the present invention was determined to be localized to the q22-q23 region of chromosome 6. The present inventors have also isolated a murine homolog of the hD52 gene from an apoptotic mouse mammary gland cDNA library by screening with a fragment (containing 91 bp of 5 XJTR and 491 bp of coding sequence) of the hD52 gene. The method for cloning the murine (m) D52 is explained in detail in Example 5 below. The mD52 clone encodes a 185 amino acids protein sharing 82% homology with hD52. By in situ hybridization of murine metaphase cells, the mD52 gene of the present invention was determined to be localized to chromosome 3A1-3A2, as well as chromosome 8C. MLN 50, 51, 62 and 64 as Breast Cancer Prognosticators The four MLN genes of the present invention encode polypeptides which are useful as prognostic markers for breast cancer. It is known in the art that prognostic markers provide important information in the management of breast cancer patients (Elias et al, J. Histotechnol. 15(4):315-320 (1992)). For example, for application of systemic adjuvant therapy in primary breast cancer, identification of high- and low-risk patients is a major issue (McGuire, W.L., N. Engl. J. Med 320:525-527 (1989)). Several classical (tumor size, lymph node status, histopathology, steroid receptor status) and second-generation prognostic factors (proliferation rate, DΝA ploidy, oncogenes, growth factor receptors and some glycoproteins) are currently available for making therapeutic decisions (McGuire, W.L., Prognostic Factors for Recurrence and Survival, in EDUCATIONAL BOOKLET AMERICAN SOCIETY OF CLINICAL ONCOLOGY, 25th Annual Meeting, 89-92 (1989); Contesso et al, Eur. J. Clin. Oncol. 25:403-409 (1989)). Although no group of the art-known prognosticators completely fulfills the objective to f Hy distinguish high- and low-risk patients, combinations of the prognostic factors can improve the prediction of a patient's prognosis (McGuire, W.L., N. Engl. J. Med 320:525-527 (1989)). Thus, by the invention, further prognostic markers are provided which can be added to the population of art- known prognosticators to more particularly distinguish between high- and low- risk breast cancer patients. The present inventors have discovered that, in many instances, cells obtained from breast tumors contain significantly greater copy number of at least one of the four MLΝ genes and express significantly enhanced levels of MLΝ 50, 51, 62 or 64 mRΝA and/or protein when compared to cells obtained from "normal" breast tissue, i.e., non-tumorigenic breast tissue. Thus, the invention provides a method useful during breast cancer prognosis, which involves assaying a first MLΝ 50, 51, 62 or 64 gene expression level or gene copy number in breast tissue and comparing the gene expression level or gene copy number with a second MLΝ 50, 51, 62 or 64 gene expression level or gene copy number, whereby the relative levels of said first gene expression level or gene copy number over said second is a prognostic marker for breast cancer. The present inventors have not observed any unamplified tumor overexpression of the MLΝ 50, 51, 62 or 64 genes. Thus, while the inventors do not intend to be bound by theory, it appears that the four MLΝ genes could not be activated by mechanisms other than gene amplification in breast carcinoma such as, for example, by alteration of regulatory sequences of the genes. Accordingly, by the invention, gene amplification and enhanced gene expression over the standard is clinically relevant for breast cancer prognosis as independent studies have shown an association between the presence of amplification and an increased risk of relapse (Slamon et al, Science 235: 77 (1987); Ravdin & Chamness, Gene 759:19 (1995)). The methods of the invention can be used alone or together with other markers known in the art for breast cancer prognosis, including those discussed above. By "assaying MLΝ 50, 51, 62 or 64 gene expression level" is intended qualitatively or quantitatively measuring or estimating the MLΝ 50, 51, 62 or 64 protein level or MLΝ 50, 51, 62 or 64 mRΝA level in a first biological sample either directly or relatively by comparing to the MLN 50, 51, 62 or 64 protein level or mRNA level in a second biological sample. By "assaying MLN 50, 51, 62 or 64 gene copy number" is intended qualitatively or quantitatively measuring or estimating MLN 50, 51, 62 or 64 gene copy number in a first biological sample either directly or relatively by comparing to the MLN 50, 51, 62 or 64 gene copy number in a second biological sample. Preferably, the MLN 50, 51, 62 or 64 protein level, mRNA level, or gene copy number in the first biological sample is measured or estimated and compared to a second standard MLN 50, 51, 62 or 64 protein level, mRNA level, or gene copy number, the standard being taken from a second biological sample obtained from an individual not having breast cancer. As will be appreciated in the art, once a standard MLN 50, 51, 62 or 64 protein level, mRNA level, or gene copy number is known, it can be used repeatedly as a standard for comparison. It will also be appreciated in the art, however, that the first and second biological samples can both be obtained from individuals having breast cancer. In such a scenario, the relative MLN 50, 51, 62 or 64 protein levels, mRNA levels or gene copy numbers will provide a relative prognosis between the individuals. By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains MLN 50, 51, 62 or 64 protein; MLN 50, 51, 62 or 64 mRNA; or the MLN 50, 51, 62 or 64 gene. Preferably, the biological sample includes tumorigenic or non-tumorigenic breast tissue. Methods for obtaining tissue biopsies are well known in the art. The present invention is useful as a prognostic indicator for breast cancer in mammals. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans. Assaying MLN 50, 51, 62 or 64 gene copy number can occur according to any known technique such as, for example, by visualizing extrachromosomal double minutes (dmin) or integrated homogeneously staining regions (hsrs) (Gebhart et al, Breast Cancer Res. Treat. 5:125 (1986); Dutrillaux et al, Cancer Genet. Cytogenet. 49:203 (1990)). Other techniques such as comparative genomic hybridization (CGH) and a strategy based on chromosome microdissection and fluorescence in situ hybridization can also be used to search for regions of increased DNA copy number in tumor cells (Guan et al, Nature Genet. 5:155 (1994); Muleris et /., Genes Chrom. Cancer 70:160 (1994)). DNA probes that hybridize to the four MLN genes can be prepared as described below. Total cellular RNA can be isolated from normal and tumorigenic breast tissue using any suitable technique such as the single-step guanidinium- thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). The LiCL/urea method described in Auffray and Rougeon, Eur. J. Biochem. 707:303 (1980) can also be used. MLN 50, 51, 62 or 64 mRNA levels are then assayed using any appropriate method. These include Northern blot analysis, SI nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR). Northern blot analysis can be performed as described in Harada et al, Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biological sample as described above. For the Northern blot, the RNA is denatured in an appropriate buffer (such as glyoxal/dimethyl, sulfoxide/sodium phosphate buffer), subjected to agarose gel electrophoresis, and transferred onto a nitrocellulose or nylon filter. MLN 50, 51, 62 or 64 DNA labeled according to any appropriate method (such as the 32 P-multiprimed DNA labeling system (Amersham)) is used as probe. After hybridization, the filter is washed and exposed to x-ray film. MLN 50, 51, 62 or 64 DNA for use as probes according to the present invention are described below. Where a fragment is used, the DNA probe will be at least about 15-30 nucleotides in length, and preferably, at least about 50 nucleotides in length. SI mapping can be performed as described in Fujita et al, Cell 49:357- 367 (1987). To prepare probe DNA for use in SI mapping, the sense strand of MLN 50, 51, 62 or 64 cDNA is used as a template to synthesize labeled antisense DNA. The antisense DNA can then be digested using an appropriate restriction endonuclease to generate further DNA probes of a desired length. Such antisense probes are useful for visualizing protected bands corresponding to MLN 50, 51, 62 or 64 mRNA. Northern blot analysis can be performed as described above. Alternatively, MLN 50, 51, 62 or 64 mRNA levels are assayed using the RT-PCR method described in Makino et al, Technique 2:295-301 (1990). By this method, the radioactivities of the amplification products in the polyacrylamide gel bands are linearly related to the initial concentration of the target mRNA. Briefly, this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are then subject to PCR using labeled primers. Alternatively, rather than labeling the primers, a labeled dNTP can be included in the PCR reaction mixture. PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of the appropriate bands (corresponding to the MLN 50, 51, 62 or 64 mRNA) is quantified using an imaging analyzer. RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan. Any set of oligonucleotide primers which will amplify reverse transcribed MLN 50, 51, 62 or 64 mRNA can be used and can be designed by reference to the MLN 50, 51, 62 or 64 DNA sequence provided below. Assaying MLN 50, 51, 62 or 64 protein levels in a biological sample can occur using any art-known method. Preferred are antibody-based techniques. For example, MLN 50, 51, 62 or 64 protein expression in tissues can be studied with classical immunohistological methods. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological examination is obtained. Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of MLN 50, 51, 62 or 64 protein for Western-blot or dot/slot assay (Jalkanen, M., et al, J. Cell. Biol 707:976-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 705:3087-3096 (1987)). In this technique, which is based on the use of cationic solid phases, quantitation of MLN 50, 51, 62 or 64 protein can be accomplished using isolated MLN 50, 51, 62 or 64 as a standard. This technique can also be applied to body fluids. With these samples, a molar concentration of MLN 50, 51, 62 or 64 protein will aid to set standard values of MLN 50, 51, 62 or 64 protein content for different body fluids, like serum, plasma, urine, spinal fluid, etc. The normal appearance of MLN 50, 51, 62 or 64 amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject. Other antibody-based methods useful for detecting MLN 50, 51, 62 or 64 gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, a monoclonal antibody can be used both as an immunoabsorbent and as an enzyme-labeled probe to detect and quantify the MLN 50, 51, 62 or 64 protein. The amount of MLN 50, 51, 62 or 64 protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm. Such an ELISA for detecting a tumor antigen is described in Iacobelli et al, Breast Cancer Research and Treatment 77:19-30 (1988). In another ELISA assay, two distinct monoclonal antibodies can be used to detect MLN 50, 51, 62 or 64 protein in a body fluid. In this assay, one of the antibodies is used as the immunoabsorbent and the other as the enzyme-labeled probe. The above techniques may be conducted essentially as a "one-step" or "two-step" assay. The "one-step" assay involves contacting MLN 50, 51, 62 or 64 protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody. The "two-step" assay involves washing before contacting the mixture with the labeled antibody. Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample. Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), salphee ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ("Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to assaying MLN 50, 51, 62 or 64 protein levels in a biological sample obtained from an individual, MLN 50, 51, 62 or 64 protein can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of MLN 50, 51, 62 or 64 protein include those detectable by X-radiography, NMR or ESR For X-radiography, suitable labels include radioisotopes such as barium or caesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. An antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, m l, U2 In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for breast cancer. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ""Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the protein. In vivo tumor imaging is described in S.W. Burchiel et al, Immunopharmacokinetics of Radiolabelled Antibodies and Their Fragments, in TUMOR IMAGING: THE RADIOCHEMICAL DETECTION OF CANCER (S.W. Burchiel and B.A. Rhodes, eds., Masson Publishing Inc. (1982)). Antibodies for use in the present invention can be raised against the intact MLN 50, 51, 62 or 64 protein or an antigenic polypeptide fragment thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier. As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to the MLN 50, 51, 62 or 64 protein. Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl Med 24:316-325 (1983)). Thus, these fragments are preferred. The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the MLN 50, 51, 62 or 64 protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of MLN 50, 51, 62 or 64 is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or MLN 50, 51, 62 or 64-binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256:495 (1975); Kohler et al, Eur. J. Immunol. 6:511 (1976); Kohler etal, Eur. J. Immunol. 6:292 (1976); Hammerling et al, MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS, 563-681 (Elsevier, N.Y., 1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with a MLN 50, 51, 62 or 64 antigen or, more preferably, with a cell expressing the antigen. Suitable cells can be recognized by their capacity to bind anti-MLN 50, 51, 62 or 64 antibody. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 °C), and supplemented with about 10 μg/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al, Gastroenterology 50:225- 232 (1981). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the MLN 50, 51 , 62 or 64 antigen. It will be appreciated that Fab and F(ab') 2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments). Alternatively, antigen binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry. Where in vivo imaging is used to detect levels of MLN 50, 51, 62 or 64 protein in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, Morrison, Science 229:1202 (1985); Oi et al, BioTec niques 4:214 (1986); Cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al, EP 171496; Morrison et al, EP 173494; Neuberger et al, WO 8601533; Robinson et al, WO 8702671; Boulianne et al, Nature 312:643 (1984); Neuberger et al, Nature 314:26% (1985). D52/D53 Gene Expression as a Marker to Distinguish Different Types of Leukemia The present inventors have further discovered that the relative expression levels of the D52 and D53 genes can be used to distinguish between different types of leukemia. In particular, the inventors have observed that the D52 gene is expressed in leukemia cells that have myelocytic characteristics (such as HL-60 cells) but not in leukemia cells having erythroid characteristics (such as K 562 cells); whereas the inverse is true for D53 gene expression. Thus, the invention further provides a diagnostic method for distinguishing between different types of leukemia, which involves assaying leukemia cells for D52 or D53 gene expression; whereby, the presence of D52 gene expression or the lack of D53 gene expression indicates that the leukemia cells have myelocytic characteristics and the presence of D53 gene expression or the lack of D52 gene expression indicates that the leukemia cells have erythroid characteristics. Preferably, the method is used to distinguish different types of acute myeloid leukemia. As indicated, the method of the invention can be performed by assaying for the presence or absence of either D52 or D53 gene expression. However, preferably, the expression of both genes is assayed. The human (h) D52 gene is described in detail in Byrne, J.A., et al, Cancer Research 55:2896-2903 (1995) and the mD52 gene is described below. The hD53 gene is also described below. Methods for detecting D52 and D53 gene expression in leukemia cells are described in detail above and in the Examples below. As above, D52 and D53 gene expression can be assayed by detecting either the corresponding mRNA or protein. MLN 50, 51, 62, 64 and D53 Nucleic Acid Molecules, Polypeptides and Fragments Thereof Using the information provided herein, such as the nucleotide sequences of MLN 62, 50, 64, 51, D53, or mD52 as set out in Figures 6, 14, 16, 21(A-D) 24(B) and 25(B), respectively (SEQ ID NOS:l, 3, 5, 7, 9 and 11, respectively), an isolated nucleic acid molecule of the present invention may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. By "isolated" nucleic acid molecules(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for purposes of the invention as are recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention. By "isolated" polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention, as are native or recombinant polypeptides which have been partially or substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988). Isolated nucleic acid molecules and polypeptides also include such compounds produced synthetically. As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double- or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the noncoding strand, also referred to as the antisense strand. The MLN 50, 51, 62, 64 genes and the D53 gene were deposited on June 14, 1996, at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852 and given the accession numbers indicated herein. The MLN 50, 51, 62, 64, D53 and mD52 nucleic acid molecules of the present invention are discussed in more detail below. MLN 62 The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the CARTl polypeptide (corresponding to the MLN 62 cDNA clone) whose amino acid sequence is shown Figure 6 (SEQ ID NO:2) or a fragment of the polypeptide. Such isolated nucleic acid molecules include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 85-87 of the nucleotide sequence shown in Figure 6 (SEQ ID NO: 1) and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 85-87 of the nucleotide sequence of Figure 6 (SEQ ID NO:l) but which, due to the degeneracy of the genetic code, still encode the CARTl polypeptide or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above. The invention further provides isolated nucleic acid molecules encoding the CARTl polypeptide having an amino acid sequence as encoded by the cDNA of the clone deposited as ATCC Deposit No. 97610 on June 14, 1996. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 6 (SEQ ED NO:l) or the nucleotide sequence of the CARTl gene contained in the above-described deposited cDNA, or a fragment thereof. Such isolated DNA molecules and fragments thereof are useflil as DNA probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the CARTl gene in human tissues (including breast and lymph node tissues) by Northern blot analysis. Of course, as discussed above, if a DNA molecule includes the ORF whose initiation codon is at position 85-87 of Figure 6 (SEQ ED NO:l), then it is also useful for expressing the CARTl polypeptide or a fragment thereof. MLN SO The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the Lasp-1 polypeptide (corresponding to the MLN 50 cDNA clone) whose amino acid sequence is shown in Figure 14 (SEQ ED NO:4) or a fragment of the polypeptide. Such isolated nucleic acid molecules include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 76-78 of the nucleotide sequence of Figure 14 (SEQ ED NO:3) and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 76-78 of the nucleotide sequence of Figure 14 (SEQ ED NO:3) but which, due to the degeneracy of the genetic code, still encode the Lasp-1 polypeptide. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above. The invention further provides isolated nucleic acid molecules encoding the Lasp-1 polypeptide having an amino acid sequence as encoded by the cDNA of the clone deposited as ATCC Deposit No. 97608 on June 14, 1996. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 14 (SEQ ED NO:3) or the nucleotide sequence of the Lasp-1 gene contained in the above-described deposited cDNA, or a fragment thereof. Such isolated DNA molecules and fragments thereof are useful as DNA probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the Lasp-1 gene in human tissues (including breast and lymph node tissues) by Northern blot analysis. Of course, as discussed above, if a DNA molecule includes the ORF whose initiation codon is at position 76-78 of Figure 14 (SEQ ID NO:3), then it is also useful for expressing the Lasp-1 polypeptide or a fragment thereof. MLN 64 The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the MLN 64 polypeptide whose amino acid sequence is shown Figure 16 (SEQ ED NO:6) or a fragment of the polypeptide. Such isolated nucleic acid molecules include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 169-171 of the nucleotide sequence of Figure 16 (SEQ ID NO:5) and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 169-171 of the nucleotide sequence of Figure 16 (SEQ ID NO: 5) but which, due to the degeneracy of the genetic code, still encode the MLN 64 polypeptide or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate DNA molecules above. The invention further provides isolated nucleic acid molecules encoding the MLN 64 polypeptide having an amino acid sequence as encoded by the cDNA of the clone deposited as ATCC Deposit No. 97609 on June 14, 1996. The invention further provides an isolated DNA molecule having the nucleotide sequence shown in Figure 16 (SEQ ID NO:5) or the nucleotide sequence of the MLN 64 gene contained in the above-described deposited cDNA, or a fragment thereof. Such isolated DNA molecules and fragments thereof are useful as DNA probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the MLN 64 gene in human tissues (including breast and lymph node tissues) by Northern blot analysis. Of course, as discussed above, if a DNA molecule includes the ORF whose initiation codon is at position 169-171 of Figure 16 (SEQ ID NO:5), then it is also useful for expressing the MLN 64 polypeptide or a fragment thereof. MLN 51 The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the MLN 51 polypeptide whose amino acid sequence is shown Figure 21 (A-D) (SEQ ID NO: 8) or a fragment thereof. Such isolated nucleic acid molecules include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 234-236 of the nucleotide sequence of Figure 21(A-D) (SEQ ID NO:7) and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 234-236 of the nucleotide sequence of Figure 21 (A-D) (SEQ ED NO: 7) but which, due to the degeneracy of the genetic code, still encode the MLN 51 polypeptide or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate DNA molecules above. The invention further provides isolated nucleic acid molecules encoding the MLN 51 polypeptide having an amino acid sequence as encoded by the cDNA of the clone deposited as ATCC Deposit No. 97611 on June 14, 1996. The invention further provides an isolated DNA molecule having the nucleotide sequence shown in Figure 21 (A-D) (SEQ ID NO: 7) or the nucleotide sequence of the MLN 51 gene contained in the above-described deposited cDNA, or a fragment thereof. Such isolated DNA molecules and fragments thereof are useful as DNA probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the MLN 51 gene in human tissues (including breast and lymph node tissues) by Northern blot analysis. Of course, as discussed above, if a DNA molecule includes the ORF whose initiation codon is at position 234-236 of Figure 21 (A-D) (SEQ ID NO:7), then it is also useful for expressing the MLN 51 polypeptide or a fragment thereof. D53 The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the D53 polypeptide whose amino acid sequence is shown Figure 24(B) (SEQ ED NO: 10) or a fragment thereof. Such isolated nucleic acid molecules include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 181-183 of the nucleotide sequence of Figure 24(B) (SEQ ID NO:9) and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 181-183 of the nucleotide sequence of Figure 24(B) (SEQ ED NO:9) but which, due to the degeneracy of the genetic code, still encode the D53 polypeptide or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate DNA molecules above. The invention further provides isolated nucleic acid molecules encoding the D53 polypeptide having an amino acid sequence as encoded by the cDNA of the clone deposited as ATCC Deposit No. 97607 on June 14, 1996. The invention further provides an isolated DNA molecule having the nucleotide sequence shown in Figure 24(B) (SEQ ID NO:9) or the nucleotide sequence of the D53 gene contained in the above-described deposited cDNA, or a fragment thereof. Such isolated DNA molecules and fragments thereof are useful as DNA probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the D53 gene in human tissue (including breast and lymph node tissues) by Northern blot analysis. Of course, as discussed above, if a DNA molecule includes the ORF whose initiation codon is at position 181-183 of Figure 24(B) (SEQ ID NO:9), then it is also useful for expressing the D53 polypeptide or a fragment thereof. Murine D52 The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the murine D52 polypeptide whose amino acid sequence is shown Figure 25(B) (SEQ ED NO: 12) or a fragment thereof. Such isolated nucleic acid molecules include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 22-24 of the nucleotide sequence of Figure 25(B) (SEQ ID NO: 11) and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 22-24 of the nucleotide sequence of Figure 25(B) (SEQ ID NO: 11) but which, due to the degeneracy of the genetic code, still encode the D52 polypeptide or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate DNA molecules above. The invention further provides an isolated DNA molecule having the nucleotide sequence shown in Figure 25(B) (SEQ ED NO: 11) or a fragment thereof. Such isolated DNA molecules and fragments thereof are useful as DNA probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the murine or human D52 gene in mouse or human tissue (including breast and lymph node tissues) by Northern blot analysis. Of course, as discussed above, if a DNA molecule includes the ORF whose initiation codon is at position 22-24 of Figure 25(B) (SEQ ID NO:l 1), then it is also useful for expressing the murine D52 polypeptide or a fragment thereof. Fragments, Derivatives and Variants of the Isolated Nucleic Acid Molecules of the Invention By "fragments" of an isolated DNA molecule having the nucleotide sequence shown in Figure 6, 14, 16, 21 (A-D), 24(B), or 25 (B) (SEQ ID NO:l, 3, 5, 7, 9, or 11, respectively) are intended DNA fragments at least 15 bp, preferably at least 20 bp, and more preferably at least 30 bp in length which are useful as DNA probes as discussed above. Of course, larger DNA fragments of about 50-2000 bp in length are also useful as DNA probes according to the present invention as are DNA fragments corresponding to most, if not all, of the nucleotide sequence shown in Figure 6, 14, 16, 21 (A-D), 24(B), or 25(B) (SEQ ID NO: 1, 3, 5, 7, 9, or 11, respectively). By a fragment at least 20 bp in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figure 6, 14, 16, 21(A-D), 24(B), or 25(B) (SEQ DD NO:l, 3, 5, 7, 9, or 11, respectively). As indicated, such fragments are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR). For example, the present inventors have constructed a labeled DNA probe corresponding to the full length human cDNA (nucleotides 1-2004) to detect CARTl gene expression in human tissue using Northern blot analysis {see infra, Example 2). Further, the present inventors have constructed a labeled DNA probe corresponding to a 1.0 kb Bam l fragment to detect Lasp-1 gene expression in human tissues using Northern blot analysis {see infra, Example 3). The present inventors have also constructed a labeled DNA probe corresponding to nucleotides 1 to 2008 of Figure 16 (SEQ ED NO:5) to detect MLN 64 gene expression in human tissues using Northern blot analysis {see infra, Example 4). Still further, a 5' probe of MLN 64 was obtained using an amplified (by PCR) DNA fragment (nucleotides 1-81 of Figure 16 (SEQ ID NO:5)), as was a 3' probe corresponding to anE oRI fragment (nucleotides 60-2073 of Figure 16 (SEQ ID NO:5)). Finally, the present inventors have also labeled the 842 bp insert of clone 116783 (Fig. 1(A)) to isolate the Ul clone (now D53), as well as to detect D53 expression in human tissues using Northern blot analysis {see infra, Example 5). Since the MLN 62, 50, 64, 51 genes and the D53 gene have been deposited and the nucleotide sequences shown in Figures 6, 14, 16, 21 (A-D), 24(B) and 25(B), respectively (SEQ ID NO: 1, 3, 5, 7, 9, or 11, respectively) are provided, generating such DNA fragments of the present invention would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, the DNA fragments of the present invention could be generated synthetically according to known techniques. Preferred nucleic acid molecules of the present invention will encode the mature form of the MLN 62, 50, 64, 51, mD52 or D53 protein and/or additional sequences, such as those encoding the leader sequence, or the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, noncoding sequences, including for example, but not limited to introns and noncoding 5* and 3' sequences such as the transcribed, nontranslated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding, and mRNA stability; and additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl Acad Sci. USA 86: 821-824 (1989), for example, hexa-histidine provides for convenient purification of the fusion protein. The HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:767 (1984). The present invention further relates to variants of the isolated nucleic acid molecules of the present invention, which encode fragments, analogs or derivatives of the MLN 62, 50, 64, 51, mD52 or D53 protein. Variants may occur naturally, such as an allelic variant. Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include those produced by nucleotide substitutions, deletions or additions. Especially preferred among these are silent or conservative substitutions, additions and deletions, which do not alter the properties and activities of the MLN 62, 50, 64, 51, mD52 or D53 protein or fragment thereof. Further embodiments of the invention include isolated nucleic acid molecules that are at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical to the above-described isolated nucleic acid molecules of the present invention. In particular, the invention is directed to isolated nucleic acid molecules at least 90%, 95%, 97%, 98%, or 99% identical to the nucleotide sequences contained in the deposited cDNAs or in Figures 6, 14, 16, 21 (A-D), 24(B) or 25(B) (SEQ ID NO:l, 3, 5, 7, 9 or 11, respectively). By the invention, "% identity" between two nucleic acid sequences can be determined using the "fastA" computer algorithm (Pearson, W.R. & Lipman, D.I, Proc. Natl. Acad Sci. USA 55:2444 (1988)) with the default parameters. Uses of such 95%, 97%, 98%, or 99% identical nucleic acid molecules of the present invention include, inter alia, (1) isolating the MLN 62, 50, 64, 51, mD52, hD52, or D53 gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the MLN 62, 50, 64, 51, mD52, hD52 or D53 gene as described in Verma et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, NY, 1988); and (3) Northern Blot analysis for detecting MLN 62, 50, 64, 51, mD52, hD52 or D53 mRNA expression in specific tissues. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J.U. et al, Science 247:1306-1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, J.U., et al, Science 247:1306-1310 (1990), and the references cited therein. The invention is further related to nucleic acid molecules capable of hybridizing to a nucleic acid molecule having a sequence complementary to or hybridizing directly to one of the deposited cDNAs or the nucleic acid sequence shown in Figure 6, 14, 16, 21(A-D), 24(B) or 25(B) (SEQ ID NO: 1, 3, 5, 7, 9 or 11, respectively) under stringent conditions. By "stringent conditions" is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150 mMNaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg ml denatured, sheared salmon sperm DNA (ssDNA), followed by washing the filters in 0. lx SSC at about 65°C. Examples of variant nucleic acid molecules made according to the present invention are discussed below. The present inventors have cloned and identified a number of MLN 64 gene variants resulting from nucleotide substitutions, deletions and/or insertions. Interestingly, the modifications principally occurred at exon/intron boundaries, suggesting that the MLN 64 variants result from defective splicing processes. These variations of the MLN 64 gene are described in Table VI below and include the following: two substitutions, of a C to T at nucleotide 262 and of an A to G at nucleotide 518, changing Leu to Phe at amino acid 32 and Gin to Arg at amino acid 117, respectively (Table VI, variants A and B); a 99 bp deletion of nucleotides 716 to 814, leading to a 33 amino acid deletion in the MLN 64 protein (i.e., a deletion of amino acids 184-216, giving a 412 amino acid variant protein) (Table VI, variant C); a 51 bp insertion between nucleotides 963-964, generating a stop codon 48 bp downstream of the insertion site and giving rise to a 281 amino acid chimeric C-terminal truncated protein containing 16 aberrant amino acids at the C-terminus (Table VI, variant D); a 657 bp insertion between nucleotides 963-964, generating a 285 amino acid chimeric C-terminal truncated protein containing 20 aberrant amino acids at the C-terminus (Table \ _, variant E); the 99 bp deletion described above and a 13 bp deletion of nucleotides 531-543, generating a frameshift leading to 247 amino acid chimeric C-terminal truncated protein containing the 121 N-terminal amino acids of MLN 64 and 126 aberrant amino acids at the C-terminal part (Table VI, variant F); and a 137 bp deletion of nucleotides 115-251 leading to a loss of the initiating ATG codon, the 13 bp deletion described above and a 199 bp insertion downstream of nucleotide 715 encoding an N-terminal truncated protein containing the 138 C- terminal amino acids of MLN 64 (Table VI, variant G). Based on the above description, generating these seven distinct variants A-G and the polypeptides they encode would be routine for one skilled in the art. For example, as discussed in detail in Example 4, below, the present inventors have cloned these variants from cDNA libraries obtained from metastatic axillary lymph node tissue, an SKBR3 breast cancer cell line, and nontransformed placenta tissue. Moreover, several variants could also be generated by site-directed mutagenesis of the MLN 64 gene whose sequence is shown in Figure 16 (SEQ ID NO:5). In a further aspect, the present invention is directed to polynucleotides having a nucleotide sequence complementary to the nucleotide sequence of any of the polynucleotides discussed above. Expressed Sequence Tags An expressed sequence tag (EST) is a segment of a sequence from a randomly selected cDNA clone that corresponds to a mRNA (Adams, M.D. et al, Science 252:1651-1656 (1991); Adams, M.D. et al, Nature 355:632-634 (1992); Adams, M.D. et al, Nat. Genet. 4:373-380 (1993)). Nine ESTs with at least partial homology to a portion of the CARTl (MLN 62) nucleotide sequence were identified by the present inventors in GenBank (Accession Nos. T64889, T97084, R37445, R61143, T96972, R12544, T40174, R61861 and T41053). The alignment of these ESTs relative to the CARTl nucleotide sequence is provided in Figure 22. Twenty-two ESTs with at least partial homology to a portion of the Lasp- 1 (MLN 50) nucleotide sequence were identified by the present inventors in GenBank (Accession Nos. T15543, T33692, T32123, T34158, F04305, T33826, T32139, T51225, D12116, T61881, T51339, T24771, T10815, T60382, M86141, T34342, T08601, T32161, T34065, Z45434, T08349 and F06105). The alignment of these ESTs relative to the Lasp-1 nucleotide sequence is provided in Figure 14(B). Fourteen ESTs with at least partial homology to a portion of the MLN 64 nucleotide sequence were identified by the present inventors in GenBank (Accession Nos. M85471, T49922, T85470, T85372, R02020, S70803, R02021, R17500, R41043, R36697, R37545, R42594, R48774 and R48877). Three ESTs with at least partial homology to a portion of the MLN 51 nucleotide sequence were identified by the present inventors in GenBank (Accession Nos. Z25173, D19971 and Dl 1736). The alignment of these ESTs relative to the MLN 51 nucleotide sequence is provided in Figure 23. Three ESTs with at least partial homology to a portion of the D53 nucleotide sequence were identified by the present inventors in GenBank (Accession Nos. T89899, T68402 and T93647). Isolated RNA Molecules The present invention further provides isolated RNA molecules which are in vitro transcripts of one of the deposited cDNAs described above, a nucleic acid sequence shown in Figure 6, 14, 16, 21(A-D), 24(B) or 25(B) (SEQ ID NO:l, 3, 5, 7, 9 or 11, respectively) or a fragment thereof. Such RNA molecules are useful as antisense RNA probes for detecting CARTl, Lasp-1, MLN 64, MLN 51, mD52, hD52 or D53 gene expression by in situ hybridization. For example, the present inventors have generated a labeled antisense RNA probe by in vitro transcription of a BglU fragment (corresponding to nucleotides 279-1882 of Figure 6 (SEQ ED NO: 1)) of the CARTl cDNA. The RNA probe was used to detect CARTl gene expression in malignant epithelial cells and invasive carcinomas {see infra, Example 2). The present inventors also generated a labeled antisense RNA probe specific for the human MLN 64 cDNA by in vitro transcription. This RNA probe was used to detect MLN 64 gene expression in malignant epithelial cells and invasive carcinomas (see infra, Example 4). Polypeptides and Fragments Thereof CARTl Polypeptide The invention further provides an isolated CARTl polypeptide having an amino acid sequence as encoded by the cDNA deposited as ATCC Deposit No. 97610, or as shown in Figure 6 (SEQ ED NO:2), or a fragment thereof. The CARTl polypeptide, which the inventors have shown is localized in the nucleus of breast carcinoma cells, is an about 470-residue protein exhibiting three main structural domains. First, a cysteine-rich domain was located at the N-terminal part of the protein (amino add residues 18-57 of Figure 6 (SEQ ED NO:2)) which corresponds to an unusual RING finger motif, presumably involved in protein- protein binding. Second, an original cysteine-rich domain was located at the core of the protein (amino acid residues 83-282 of Figure 6 (SEQ DD NO:2)) and is constituted by three repeats of an HC3HC3 consensus motif, possibly involved in nucleic acid and/or protein-protein binding, that has been designated as the CART motif Third, the C-terminal part of the CARTl protein corresponds to a TRAF domain (amino acid residues 308-470 of Figure 6 (SEQ ID NO:2)) known to be involved in protein/protein interactions. Similar association of RING, CART and TRAF domains has been observed in the art in the human CD40-binding protein and in the mouse tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), both involved in signal transduction mediated by TNF receptor family and, in the developmentally regulated Dictyostelium discoideum DG17 protein. This suggests that, together with CARTl, these structurally related proteins are members of a new protein family and, that CARTl may be involved in TNF-related cytokine signal transduction during breast cancer progression. Thus, since the CARTl DNA sequence is provided in Figure 6 (SEQ ED NO: 1) as are the regions which encode the RING, CART and TRAF domains, it would be well within the purview of the skilled artisan to generate recombinant constructs similar or equivalent to those listed below. As discussed above, the present inventors have discovered that the CARTl polypeptide is a prognostic marker of breast cancer. Thus, this polypeptide and its fragments can be used to generate polyclonal and monoclonal antibodies as discussed above for use in prognostic assays such as immunohistochemistry and RIA on cytosol. For example, the present inventors have substantially purified recombinantly produced CARTl and injected it into mice to raise monoclonal antibodies. Moreover, a polypeptide fragment of CARTl, corresponding to the sequence Q 393 to D 4U of Figure 6 (SEQ ED NO:2), has been injected into rabbits to raise a polyclonal antibody. Lasp-1 Polypeptide The invention further provides an isolated Lasp-1 polypeptide having an amino acid sequence as encoded by the cDNA deposited as ATCC Deposit No. 97608, or as shown in Figure 14 (SEQ ED NO:4), or a fragment thereof. The present inventors have discovered that the Lasp-1 polypeptide is an about 261- residue protein exhibiting two main structural domains. First, one copy of a cysteine-rich LIM/double zinc finger-like motif is located at the N-terminal part of the protein (amino acids 1-51 of Figure 14 (SEQ ID NO:4)). Second, a SH3 (Src homology region 3) domain is located at the C-terminal part of the protein (amino acids 196-261 of Figure 14 (SEQ ED NO:4)). Lasp-1 is the first protein exhibiting associated LIM and SH3 domains and thus constitutes the first member of a new protein family. Thus, since the Lasp-1 DNA sequence is provided in Figure 14 (SEQ ID NO:3) as are the regions which encode the LIM and SH3 domains, it would be well within the purview of the skilled artisan to generate recombinant constructs similar or equivalent to those listed below. As discussed above, the present inventors have discovered that the Lasp-1 polypeptide is a prognostic marker of breast cancer. Thus, this polypeptide and its fragments can be used to generate polyclonal and monoclonal antibodies as discussed above for use in prognostic assays such as immunohistochemistry and RIA on cytosol. MLN 64 Polypeptide The invention further provides an isolated MLN 64 polypeptide having an amino acid sequence as encoded by the cDNA deposited as ATCC Deposit No. 97609, or as shown in Figure 16 (SEQ DD NO:6), or a fragment thereof. The invention also provide polypeptides encoded for by the seven variants A-G discussed above. These variations of the MLN 64 protein are discussed in deta¬ in Example 4, below. The present inventors have discovered that the MLN 64 protein shown in Figure 16 (SEQ ED NO: 6) is an about 445-residue protein exhibiting two potential transmembrane domains (at residues 1-72 and 94-168) and several potential leucine zipper and leucine-rich repeat structures. Amino acid composition analysis showed 11.5% aromatic residues (Phe, Tip and Tyr) and 26% aliphatic residues (Leu, lie, Val and Met). Thus, since the MLN 64 DNA sequence is provided in Figure 16 (SEQ ED NO:5), it would be well within the purview of the skilled artisan to generate recombinant constructs similar or equivalent to those listed below. The present inventors have discovered that the MLN 64 polypeptide is a prognostic marker of breast cancer. Thus, this polypeptide, its fragments, and the polypeptide variants discussed above can be used to generate polyclonal and monoclonal antibodies for use in prognostic assays such as immuno -histochemistry and RIA on cytosol. For example, a polypeptide fragment of the MLN 64 protein, 16 amino acids in length located in the C-terminal part of the MLN 64 protein, was synthesized by the inventors in solid phase using Fmoc chemistry and coupled to ovalbumin through an additional NH2-extra-terminal cysteine residue, using the bifunctional reagent MBS. This synthetic MLN 64 fragment was injected into BALB/c mice periodically until obtention of positive sera. Spleen cells were removed and fused with myeloma cells according to St. Groth & Scheidegger, J. Immunol Meth. 55:1-21 (1980). Culture supematants were screened by ELISA using the unconjugated peptide fragment as antigen. Positive culture media were tested by immunocytofluorescence and Western blot analysis on MLN 64 cDNA transfected COS-1 cells. Several hybridomas, found to secrete monoclonal antibodies specifically recognizing MLN 64 protein, were cloned twice on soft agar. Monoclonal antibodies directed against the synthetic MLN 64 peptide fragment were employed in an immunohistochemical analysis which showed MLN 64 protein staining restricted to transformed epithelial cells {see infra, Example 4). MLN 51 Polypeptide The invention further provides an isolated MLN 51 polypeptide having an amino acid sequence as encoded by the cDNA deposited as ATCC Deposit No. 97611, or as shown in Figure 21 (A-D) (SEQ DD NO:8), or a fragment thereof. The present inventors have discovered that the MLN 51 polypeptide is an about 534-residue protein. Thus, since the MLN 51 DNA sequence is provided in Figure 21(A-D) (SEQ ID NO:7), it would be well within the purview of the skilled artisan to generate recombinant constructs similar or equivalent to those listed below. As discussed above, the present inventors have discovered that the MLN 51 polypeptide is a prognostic marker of breast cancer. Thus, this polypeptide, its fragments, and the polypeptide variants discussed above can be used to generate polyclonal and monoclonal antibodies for use in prognostic assays such as immunohistochemistry and RIA on cytosol. D53 Polypeptide The invention further provides an isolated D53 polypeptide having an amino acid sequence as encoded by the cDNA deposited as ATCC Deposit No. 97607, or as shown in Figure 24(B) (SEQ DD NO: 10), or a fragment thereof. The present inventors have discovered that the D53 polypeptide is about 204 amino acids in length and have identified a single coiled-coil domain in hD53, as well as in the hD52 homolog and mouse D52, towards the N-terminus of each protein, which is predicted to end at Leu 71 in all 3 proteins. This coiled-coil domain overlaps with the leucine zipper predicted in hD52/N8 using helical wheel analysis. The presence of a coiled-coil domain in D52 family proteins indicates that specific protein-protein interactions are required for the functions of these proteins. The present inventors have identified the presence of 2 candidate PEST domains in the three proteins, hD53, hD52 and mD52, indicating that their intracellular abundances may be in part controlled by proteolytic mechanisms. Interestingly, the extent of the N-terminally located PEST domain overlaps that of the coiled- coil domain in both D52 and D53 proteins. It could thus be envisaged that interactions via the coiled-coil domain could mask this PEST domain, in accordance with the hypothesis that PEST sequences may act as conditional proteolytic signals in proteins able to form complexes (Rechsteiner, M., Adv. Enzyme Reg. 27:135-151 (1988)). Also, the sequences of the three proteins contain an uneven distribution of charged amino acids; while approximately the first and last 50 amino acids of each protein exhibits a predominant negative charge, the central portion of each protein exhibits an excess of positively charged residues. Finally, the present inventors have identified similar potential post- translational modification sites in the three proteins. The present inventors have discovered that the D53 polypeptide is a tumor marker in breast cancer. Moreover, relative hD52/hD53 gene expression levels are useful as a marker for distinguishing between different forms of leukemia. Murine D52 Polypeptide The invention further provides an isolated mD52 polypeptide having an amino acid sequence as shown in Figure 25(B) (SEQ ED NO: 12), or a fragment thereof The present inventors have discovered that the mD52 polypeptide is an about 185 amino acid residue protein having domain features as described above. Polypeptide Fragments and Variants Fragments of CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 other than those described above capable of raising both monoclonal and polyclonal antibodies will be readily apparent to one of skill in the art and will generally be at least 10 amino acids, and preferably at least 15 amino acids, in length. For example, the "good antigen" criteria set forth in Van Regenmortel et al, Immunol. Letters 77:95-108 (1988), could be used for selecting fragments of the CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 protein capable of raising monoclonal and polyclonal antibodies. It will be recognized in the art that some amino acid sequences of CART 1 , Lasp-1, MLN 64, MLN 51, mD52 or D53 can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Such areas will usually comprise residues which make up the binding site, or which form tertiary structures which affect the binding site. In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a noncritical region of the protein. Thus, the present invention further includes variations of the CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 protein which show substantial protein activity or which include regions of the CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 protein such as the protein fragments discussed above capable of raising antibodies useful in immunohistochemical or RIA assays. Such mutants include deletions, insertions, inversions, repeats and type-substitutions (e.g., substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity. Typically seen as conservative substitutions are as follows: the replacements, one for another, among the aliphatic amino acids, Ala, Val, Leu and He; interchange of the hydroxyl residues, Ser and Thr; exchange of the acidic residues, Asp and Glu; substitution between the amide residues, Asn and Gin; exchange of the basic residues, Lys and Arg; and replacements among the aromatic residues, Phe, Tyr. As indicated in detail above, further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on a function) can be found in Bowie, J.U. etal, Science 247:1306-1310 (1990). Preferably, such variants will be at least 90%, 95%, 97%, 98% or 99% identical to the CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 polypeptides described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. By the invention, "% identity" between two polypeptides can be determined using the "fastA" computer algorithm with the default parameters (Pearson, W.R. & Lipman, D.I, Proc. Natl. Acad Sci. USA 55:2444 (1988)). The isolated CARTl, Lasp-1, MLN 64, MLN 51, mD52, or D53 polypeptide, or a fragment thereof, are preferably provided in an isolated form, and preferably are substantially purified. Of course, purification methods are known in the art. In preferred embodiment, a recombinantly produced version of the CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 polypeptide is substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). The CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 protein can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be nonglycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, and in some cases as a result of host-mediated processes. Vectors and Hosts The present invention also relates to vectors which include an isolated DNA molecule(s) of the present invention, host cells which are genetically engineered with the vectors, and the production of CARTl, Lasp-1, MLN 64, MLN 51, mD52 or D53 polypeptide(s), or fragments thereof, by recombinant techniques. A DNA molecule, preferably a cDNA, encoding the CARTl, Lasp-1, MLN 51, MLN 64, mD52 or D53 polypeptide or a fragment thereof, may easily be inserted into a suitable vector. Ideally, the vector has suitable restriction sites for ease of insertion, but blunt-end ligation, for example, may also be used, although this may lead to uncertainty over reading frame and direction of insertion. In such an instance, it is a matter of course to test transformants for expression; 1 in 6 of which should have the correct reading frame. The CARTl, Lasp-1, MLN 51, MLN 64, mD52 or D53 polypeptide(s), or fragments thereof, can be expressed in any suitable host cell. The extent of expression may be analyzed by SDS polyacrylamide gel electrophoresis (Laemmelli, etal, Nature 227:680-685 (1970)). Cultures useful for production of such polypeptides include prokaryotic, eukaryotic and yeast expression systems. Preferred systems include E. coli, Streptomyces and Salmonella typhimurium and yeast, mammalian or plant cells. Mammalian hosts include HeLa, COS, and Chinese Hamster Ovary (CHO) cells. Yeast hosts include S. cerevisiae. Insect cells include Drosophila S2 and Spodoptera Sf9 cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art. Vectors capable of directing expression in the above-mentioned host cells are also known in the art. The present inventors have designed the following recombinant DNA expression constructs which encode either the entire CARTl protein or fragments of the CARTl protein corresponding to the individual domains discussed above. Bacterial expression systems are as follows: pGEX-CARTl; pGEX-RENG; pGEX-CART; pGEX-CART-TRAF; and pGEX-TRAF. Yeast expression systems are as follows: pBTMN-CART-TRAF; pBTMN-CART; pBTMN-TRAF; pVP-CART-TRAF; pVP-CART; and pVP-TRAF. Eukaryotic expression systems are as follows: pSG5-CARTl, pAT3-CARTl; pAT4-CARTl; pBC-CARTl; and pCMV-CARTl. For example, by pAT4-CARTl, is intended the pAT4 vector containing the entire CARTl DNA coding sequence as an insert. Similarly, by pBTMN- CART-TRAF, is intended the pBTMN vector containing the DNA sequence encoding the CART and TRAF regions of the CARTl protein. The remaining constructs listed above are to be interpreted in a like-manner. The pGEX, pBTMN, pVP, ρSG5, pAT3, pAT4, pBC and pCMV vectors are known in the art and publicly available. The present inventors have designed the following recombinant DNA expression constructs which encode either the entire Lasp-1 protein or fragments of the Lasp-1 protein. Bacterial expression systems are as follows: pGEX- LASP1; pGEX-L ; and pGEX-SH3. Yeast expression systems are as follows: pBTMN-LASPl; pBTMN-LIM; pBTMN-SH3; pVP-LASPl; pVP-LEM; and pVP-SH3. Eukaryotic expression systems are as follows: pSG5-LASPl; pBC- LASP1; and pCMV-LASPl. The pGEX, pBTMN, pVP, pSG5, pBC and pCMV vectors are known in the art and publicly available. The present inventors have designed the following recombinant DNA expression constructs which encode the MLN 64 protein. Bacterial expression systems include pGEX-MLN 64. Eukaryotic expression systems include pSG5- MLN 64 and pBC-MLN 64. The pGEX, pSG5 and pBC vectors are known and publicly available. Having generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting. Experiments Example 1 Identification of Four Novel Human Genes Amplified and Overexpressed in Breast Carcinoma and Located to the qll-q21.3 Region of Chromosome 17 Introduction Despite earlier detection and a lower size of the primary tumors at the time of diagnosis (Nystrόm, L. etal, Lancet 547:973-978 (1993); Fletcher, S.W. et al, J. Natl. Cancer Inst. 55:1644-1656 (1993)), associated metastases remain the major cause of breast cancer mortality (Frost, P. & Levin, R., Lancet 339: 1458-- 1461 (1992)). Therefore, defining the mechanisms involved in the formation and growth of metastases is still major challenge in breast cancer research (Rusciano, D. 8c Burger, M.M., BioEssays 74:185-194 (1992); Hoskins, K. & Weber, B.L., Curr. Opin. Oncol. 6:554-559 (1994)). The processes leading to the formation of metastases are complex (Fidler, II, Cancer Res. 50:6130-6138 (1990); Liotta, L. etal, Cell 64:327-336 (1991)), and identifying the related molecular events is thus critical for the selection of optimal treatments. The initial steps of transformation characterized by the malignant cell escape from normal cell cycle controls, are driven by the expression of dominant oncogenes and/or the loss of tumor suppressor genes (Hunter, T. & Pines, I, Cell 79:573-582 (1994)). Tumor progression can be considered as the ability of the malignant cells to leave the primary tumoral site and, after migration through lymphatic or blood vessels, to grow at a distance in host tissue and form a secondary tumor (Fidler, II, Cancer Res. 50:6130-6138 (1990); Liotta, L. et al, Cell 64:327-336 (1991)). Progression to metastasis is dependent not only upon transformation but also upon the outcome of a cascade of interactions between the malignant cells and the host cells/tissues. These interactions may reflect molecular modification of synthesis and/or of activity of different gene products both in malignant and host cells. Several genes involved in the control of tumoral progression have been identified and shown to be implicated in cell adhesion, extracellular matrix degradation, immune surveillance, growth factor synthesis and/or angiogenesis (reviewed in, Hart, I.R. & Saini, A., Lancet 339: 1453-1461 (1992); Ponta, H. etal, B.B.A. 7795:1-10 (1994); Bernstein, L.R. & Liotta, L.A., Curr. Opin. Oncol. 6:106-113 (1994); Brattain, M.G et al, Curr. Opin. Oncol. 6:77-81 (1994); Fidler, II & Ellis, L.M., Cell 79:185-188 (1994)). In order to identify and clone genes which could be involved in the cancer progression, we performed a differential screening of a cDNA library established from breast cancer derived metastatic axillary lymph nodes (MLN). In breast cancer, axillary lymph nodes are usually the earliest sites for metastasis formation, and they are routinely removed for diagnostic purposes (Carter, CL. et al, Cancer 65: 181-187 (1989)). Systemic metastases will usually occur later on in the disease, principally in bone, brain and visceres (Rusciano, D. & Burger, M.M., BioEss ys 74: 185-194 (1992)) and, because there is no benefit in terms of survival for the patients, they are rarely removed. Similar differential screening protocols have already permitted the identification of several genes possibly involved in tumor progression, including the stromelysin-3 gene which is overexpressed in most invasive breast carcinomas (Basset, P. et al, Nature 545:699-704 (1990)) and the maspin gene, whose expression is reduced in breast cancer cell lines (Zou, Z. etal, Science 265:526-529 (1994)). In the present study, the screening of the MLN cDNA library was performed using two probes representative of malignant (MLN) and of nonmalignant (fibroadenomas; FA) breast tissues, respectively. Metastatic samples were obtained from patients harboring clinical and histological characteristics associated with a poor prognosis and a high propensity of metastatic spreading. FAs, which are benign tumors, have been selected as control tissues since, although nonmalignant, they are proliferating tissues, thereby minimizing the probability to identify mRNAs characteristic of cellular growth, but unrelated to the malignant process. Here we report the identification of four novel genes, co-localized on the chromosome 17 long arm, and amplified and overexpressed in malignant breast tissues. Materials and Methods Tissues and Cell Cultures Surgical specimens obtained at the Hόpitaux Universitaires de Strasbourg, were frozen in liquid nitrogen for RNA extraction. Adjacent sections were fixed in 10% buffered formalin and paraffin embedded for histological examination. The cell lines (ZR75-1, MCF7, SK-BR-3, BT-20, BT-474, HBL-100, MDA-MB231 and T-47D) are described and available in the American Type Culture Collection (ATCC, Rockville, MD). The lines MCF7, ZR75-1, BT-474 and T-47D are estrogen receptor positive, whereas BT-20, SK-BR-3 and MDA- MB-231 were estrogen receptor negative. Cells were routinely maintained in our laboratory and were cultured at confluency in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. RNA Preparation and Analysis Surgical specimens were homogenized in the guanidinium isothiocyanate lysis buffer and purified by centrifiigation through cesium chloride cushion (Chirgwin, IM. et al, Biochemistry 75:52-94 (1979)). PolyA + RNA was purified using oligodT cellulose chromatography (Aviv, H. & Leder, P., Proc. Natl. Acad. Sci. USA 69:1408-1412 (1977)). RNAs from cultured cell lines were extracted using the single-step procedure of Chomczynski, P. & Sacchi, N., Anal. Biochem. 762:156-159 (1987)). RNAs were fractionated by electrophoresis on 1% agarose, 2.2 M formaldehyde gels (Lehrach, H. etal, Biochemistry 76:4743-4751 (1977)), transferred to nylon membrane (Hybond N, Amersham Co ., Arlington Heights, EL) and immobilized by baking for 2 hrs at 80°C. cDNA Library Construction PolyA * RNA from four independent surgical specimens of breast cancer MLNs were pooled. The cDNA was synthesized using MMLV reverse transcriptase (Superscript™, Gibco BRL, Gaithersburg, MD) and oligodT (Pharmacia Fine Chemicals, Piscataway, NJ) as primer. Second strand synthesis was performed by RNaseH replacement (Gubler, U. & Hoffman, B.I, Gene 25:263-269 (1983)). After blunt-ending using T4 DNA Polymerase I, EcoRI adaptors were added. After ligation, excess of adaptors and molecules less than 300 bp were removed by gel filtration chromatography on Biogel A50m (Bio-Rad, Richmond, CA). Size selected cDNAs were ligated in the EcόRI cloning site of lambda ZAPH (Stratagene Inc., La Jolla, CA). Probe Preparation In order to obtain a MLN specific probe (plus probe), 3 μg of poly A + RNA purified from MLN were subjected to first strand cDNA synthesis and 370 ng of cDNA were obtained by oligodT priming. RNA molecules were removed by NaOH hydrolysis and single-stranded cDNA was hybridized to 7 μg of polyA + RNA purified from a breast FA (19x excess). After hybridization for 24 hrs at 68°C (Hedrick, S.M. et al, Nature 505: 149-153 (1984); Rhyner, T.A. etal, J. Neurosci. Res. 76:167-181 (1986)), single-stranded material (12% of the starting cDNA) was purified by hydroxylapatite chromatography (Bio-Rad, Richmond, CA). The minus probe, derived from a breast FA, was similarly obtained from 5 μg of poly A + RNA which were converted into 560 ng of single- stranded cDNA and hybridized to 7 μg of normal colon and liver (20x excess). After hydroxylapatite chromatography, 14% of the cDNA remained single- stranded. In both cases, single-stranded cDNAs were concentrated and washed with T 10 E j using Centricon 30 (Amicon, Beverly, MA). Twenty ng and 40 ng of plus and minus probes were obtained, respectively. The 32 P-random labeling (Feinberg, AP. 8c Vogelstein, B., Anal. Biochem. 772:195-203 (1983)) of 10 ng of single-stranded cDNA gave 2xl0 9 and 3xl0 9 cpm/μg of plus and minus probes, respectively. cDNA Library Screening One hundred thousand pfu from the MLN library were plated, and nylon filter replica (Biodyne A transfer membrane, Pall Europe Limited, Portsmouth) were hybridized at 42°C in 50% formamide, 5x SSC, 0.4% ficoll, 0.4% polyvinylpyrrolidone, 20 mM sodium phosphate, pH 6.5, 0.5% SDS, 10% dextran sulfate and 100 μg/ml denatured salmon sperm DNA, for 36-48 hrs, with the 32 P- labeled plus or minus probes diluted to 0.5-lxl0 6 cpm/ml. Stringent washings were performed at 60°C in O.lx SSC and 0.1% SDS. Filters were autoradiographed at -80°C for 24-72 hrs. Plaques giving differential signals with the plus and minus probes were picked up and subjected to a secondary screening using the same hybridization conditions. Plasmid Recovery and Southern Blot Analysis Pure plaques were directly recovered as bacterial colonies using the pBluescript/ftλZAPII in vivo excision system (Stratagene Inc., La Jolla, CA). Small scale plasmid extractions were performed (Zhou, C. etal, Biotechniques 8:172- 173 (1990)) and approximately 1/10 of the material (200 ng) was digested with EcoRI and loaded on 2 agarose gels, run in parallel. After electrophoresis, gels were blotted onto nylon membranes (Hybond N + , Amersham Corp.) and membranes were hybridized to the plus and minus probes. Inserts from selected clones were purified from agarose gel and 32 P-labeled by random priming, and used for Northern and Southern blot analyses and cross-hybridizations. Sequencing and Computer Analysis Plasmid templates, prepared as previously described, were treated with RNaseA (10 μg/ml) for 30 min, then precipitated by 0.57 volume of polyethylene glycol NaCl (20%, 2 M), washed with ethanol, vacuum-dried and resuspended at 200 ng/μl in T i( β_. The double-stranded DNA templates were sequenced with Taq polymerase and either pBluescript universal or internal primers, using dye- labeled ddNTPs for detection on an Applied Biosystems 373A automated sequencer. Sequence analyses were performed using the GCG sequence analysis package (Wisconsin package, version 8.0, Genetics Computer Group, Madison, WI). Sequence homologies were identified using the FastA and Blast programs by searching the complete combined GenBank/EMBL databanks (release 84.0/39.0) and in the case of translated sequences, by searching the complete SwissProt database (release 29.0). Genomic DNA Extraction and Southern Blot Analysis Cells were grown in 75 mm 2 flasks at confluency, and washed with lx PBS. After addition of 2 ml of extraction buffer (10 mM Tris-HCl, pH 8.0, 0.1 M N-^EDTA, pH 8.0, 20 μg/ml RNaseA, 0.5% SDS, 100 μg/ml proteinase K), the flasks were incubated at 42 °C for 12 hrs. Genomic DNA was recovered by precipitation with 1 volume of isopropanol. After washing in 70% ethanol, DNA was air-dried and dissolved in T IQ E, at 4°C. For DNA amplification studies, 10 μg of cell line genomic DNA were BamΗl digested until completion. For chromosomal localization, DNA extracted from human/rodent somatic cell hybrids (NIGMS Mapping panel #2; Coriell Cell Repositories, Camden, NJ) digested with BamHl or EcoRI until completion was used. In both cases, BamΗl or £cσRI digested genomic DNA was fractionated on 0.8% agarose gel and blotted onto Hybond N + membranes. Quantitation of MLN gene copy number in breast cell lines was determined by dotblot analysis. Genomic DNA (2.5 μg) was denatured in 0.4M NaOH at 65 °C for 1 hr and 2-fold serial dilutions were spotted onto Hybond >T membranes. Hybridization and washing were performed as described for cDNA library screening. Control probe p53 corresponded to a 2.0 kb BamΗl fragment released from php53B (ATCC No. 57254). RNA loading control suitable for human cells and tissues was an internal (0.7 kb) Pstl fragment of 36B4 (Masiakowski, P. et al, Nucleic Acids Res. 70:7895-7903 (1982)). Gene Mapping Chromosomal assignment of genes MLN 50, 51, 62 and 64 was carried out by in situ hybridization on chromosome preparations obtained from phytohemagglutinin-stimulated human lymphocytes, cultured for 72 hrs. 5-Bromodeoxyuridine (60 μg/ml) was added to the medium for the final 7 hrs of culture to ensure posthybridization chromosomal banding of good quality. cDNA probes were 3 H-labeled by nick-translation to a specific activity of 1.5x10 dpm/ml. The radiolabeled probes were hybridized to metaphases spreads at a final concentration of 25 ng/ml of hybridization solution, as previously described (Mattei, M.G. et al, Human Genet. 69:268-271 (1985)). After the slides were coated with nuclear track emulsion (NTB2; Kodak, Rochester, NY), they were exposed for 19 days at 4°C before development. To avoid any slipping of silver grains during the banding procedure, chromosome spreads were first stained with buffered Giemsa solution, and metaphases were photographed. R-banding was then performed by the fluorochrome-photolysis-Giemsa method, and metaphases were rephotographed before analysis. Results Differential Screening of the MLN cDNA Library Four patients with ductal breast carcinomas were selected according to their age (below 50 years of age), the large size and high histological grade of their primary tumor (Bloom, H.J.G. & Richardson, W.W., Brit. J. Cancer 77:359- 366 (1957)) and the presence of MLN (Table I). Because of the high heterogeneity of breast tumors (Lόnn, U. et al, Intl. J. Cancer 55:40-45 (1994) and refs. therein), RNAs were extracted from metastatic samples coming from the four patients and pooled in relative equal amounts, in order to prepare a representative breast MLN cDNA library. Histological examination of the selected MLN samples revealed above 80% of metastatic tissue. However, in order to avoid dilution of rare differential transcripts, we prepared the enriched plus probe using MLNs exclusively obtained from patient C. This patient had 17 involved lymph nodes (Table I), and, in addition, her primary tumor exhibited two poor prognostic factors which were an estradiol and progesterone receptor negative status (Osborne, C.K. et al, Receptors, in BREAST DISEASES 301-325 (2nd ed., Harris, J.R etal, eds. IB. Lippincott, Philadelphia, PA 1991)) and a c- erbB-2 overexpression (Slamon, D.I etal, Science 244:707-712 (1989); Borg, A. etal, Oncogene 6:137-143 (1991); Toikkanen, S. etal, J. Clin. Oncol. 5:103- 112 (1992); Muss, H.B. et al, N. Engl. J. Med 300: 1260-1266 (1994)). A total of 10 5 recombinants from the MLN cDNA library were differentially screened using two enriched probes. The plus probe was derived from MLN cDNAs and deprived of sequences expressed in a FA. The "minus" probe was derived from FA cDNAs and deprived of sequences expressed in normal liver and colon {see Materials and Methods). Comparison of the patterns obtained with these two probes allowed for the detection of 195 "differential plaques" which were positive with the "plus" probe and negative with the "minus" probe. Twenty four differential plaques were subjected to a second screening and plasmid DNAs recovered from pure plaques were tested for the presence of "differential inserts" by Southern blot analysis {see Materials and Methods). Identified differential inserts were 32 P-labeled and used to reprobe the MLN cDNA library lifts and the Southern blots in order to identify related cDNA clones. The same protocol was used to characterize the remaining "differential plaques" and finally, ten independent families of differential clones were identified. The longest cDNA insert of each family (MLN 4, 10, 19, 50, 51, 62, 64, 70, 74 and 137) were selected for further studies. Expression Analysis of the Ten MLN Genes In order to test the differential expression of the genes corresponding to these clones, Northern blots were prepared using MLN, FA and normal axillary lymph node (NLN) RNAs. Filters were hybridized with the ten 32 P-labeled MLN cDNAs. As shown in Figure 1, all detected mRNAs were preferentially observed in MLN (lanes 1) whereas no signal or only a faint signal was observed in NLN and FA (lanes 2 and 3). The mRNA sizes, detected by the ten probes, varied from 0.5 kb (MLN 70) up to 5 kb (MLN 74) indicating that our screening protocol did not favor a preferential transcript size. Although the expression levels differed, they remained relatively high, even for the least abundant of them (MLN 62) (Figure 1). cDNA and Putative Protein Sequences of the Ten MLN Genes In a first step, cDNAs were partially sequenced on both extremities using universal primers for the pBluescript vector. These partial sequences were compared to the combined GeneBank EMBL DNA databanks. MLN 74, 19, 10 and 4 corresponded to the already known genes fibronectin (Accession Nos. X02761, K00799, K02273, X00307 and X00739; Kornblihtt, A.R. et al, EMBO J. 5:221-226 (1983)), c-erbB-2 (Accession No. Ml 1730; Coussens, L. et al, Science 250:1132-1139 (1985)), nonspecific cross-reacting antigen (NCA, Accession No. Ml 8728; Tawaragi, Y. et al, Biochem. Biophys. Res. Commun. 750:89-96 (1988)) and calcyclin (Accession Nos. M14300 and J02763; Calabretta, B. etal, J. Biol. Chem. 26:12628-12632 (1986)), respectively. Altogether they were the most abundant clones recovered in this screening since, as indicated in Table π, they represented 75% of the differential clones. The relationship of these genes to cancer and, for some of them to metastasis, has been already reported. In a second step, when no sequence homology was initially found, the complete cDNA sequences were established and the putative corresponding protein sequences were compared to those present in the SwissProt databank. MLN 70 (Accession No. X80198) and MLN 137 (Accession No. X80197) showed homologies with proteins from other species and could be classified in the S100 and keratin families (Kligman, D. & Hilt, D.C., Trends Biol. Sci. 75:437-443 (1988); Donato, R., Cell Calcium 72:713-726 (1991); Smack, D.P. et al, J. Amer. Acad Dermatol. 50:85-102 (1994)), respectively. The 30 amino acid long ZF-1 pig cysteine-rich peptide (Accession No. P80171, Sillard, R. etal, Eur. J. Biochem. 277:377-380 (1993)) showed 100% identity to the N-terminal part of the MLN 50 putative protein (Accession No. X82456). In addition, several sequence homologies were found with various expressed sequence tags (ESTs; Adams, M.D. et al, Nature 555:632-634 (1992)) within the 3 ' noncoding regions of the MLN 50 (Accession Nos. T08349, T08601 and M86141, Adams, M.D. et al, Nature 555:632-634 (1992); Adams, M.D. et al, Nat. Genet. 4:373-380 (1993); T10815, Bell, G.I. & Takeda, I, Hum. Mol. Genet. 2:1793-1798 (1993); D12116, Okubo, K. et al, Nat. Genetics 2:173-179 (1992)) and MLN 51 (Accession No. X80199; EST Accession Nos. Z25173 and D19971, Okubo, K. et al, Nat. Genetics 2:173-179 (1992)) cDNA sequences. Surprisingly, we observed 100% homology with part (129 bp) of an 401 bp long EST (Accession No. M85471, Adams, M.D. etal, Nature 555:632-634 (1992)) and the 5' coding region of MLN 64 (Accession No. X80198), suggesting that this EST could correspond to a chimera or to an unspliced RNA. Since most homologies observed for MLN 50, 51 and 64 were restricted to small noncoding DNA sequences and since no homology was found for MLN 62 (Accession No. X80200), we assumed that they belong to new protein families and further characterizations were undertaken. Chromosomal Assignment of MLN 50, 51, 62 and 64 Genes Southern blots were constructed by loading EcoRI or BamHl digest of genomic DNAs from human somatic cell hybrids, corresponding to individual human chromosome in a rodent background. MLN 51 and 64 probes showed an unique hybridization signal on chromosome 17, whereas MLN 50 and 62 probes showed a strong hybridization to chromosome 17 and a faint signal on chromosomes 3 and 16, and on chromosome 5, respectively (Table HI). Since the four probes showed hybridization with chromosome 17, the same Southern blot was reprobed with MLN 19 corresponding to the c-erbB-2 oncogene, previously localized on the chromosome 17 (Fukushige, S.I. et al, Mol. Cell. Biol. 6:955- 958 (1986)). As expected, MLN 19 showed a hybridization restricted to this chromosome (Table HI). In order to define the precise location of the four new genes on chromosome 17, we carried out chromosomal in situ hybridization. Using MLN 50, 100 metaphase cells were examined. 276 silver grains were associated with the chromosomes and 83 of these (30%) were located on chromosome 17. The distribution of grains was not random: 65/83 (78.3%) of them mapped to the qll-q21 region of the long arm of chromosome 17 (Fig. 2(A)). Two secondary sites were detected, at 3p22-3p21.3 (36/276, 13% of total grains) and at 16ql2.1 (26/276, 9.4% of total grains). Using MLN 51, 100 metaphase cells were examined. 176 silver grains were associated with the chromosomes and 60 of these (34.1%) were located on chromosome 17. The distribution of grains was not random: 49/60 (81.6%) of them mapped to the ql2-q21.3 region of the long arm of chromosome 17 (Fig. 2(A)). Using MLN 62, 150 metaphase cells were examined. 204 silver grains were associated with the chromosomes and two sites of hybridization were detectable. 20.1% were located on chromosome 17 and 82.9% of them mapped to the ql l-ql2 region of the long arm (Fig. 2(A)). 16.6% were located on chromosome 5. The distribution of grains was not random: 79.4% mapped to the (q3 l-q32) region of chromosome 5 long arm. Using MLN 64, 150 metaphase cells were examined. 247 silver grains were associated with chromosomes and 64 of these (25.9%) were located on chromosome 17. The distribution of grains was not random: 73.4% of them mapped to the ql2-q21 region of the long arm of chromosome 17 with a maximum in the q21.1 band (Fig. 2(A)). These results are in good agreement with the findings previously obtained by Southern blot hybridization and suggest that, along the long arm of the chromosome 17, MLN 50 and 62 and MLN 51 and 64 are centromeric and telomeric to MLN 19 {c-erbB-2), respectively (Fig. 2(B)). Amplification and Expression of MLN 50, 51, 62 and 64 Genes Five of the cDNA clones isolated in this study corresponded to genes located on the chromosome 17, namely MLN 50, 51, 62, 64 and 19. Moreover, they are all localized on the long arm of chromosome 17 in the ql l-q21.3 region. Since it is known that c-erbB-2 overexpression in breast carcinomas is mostly dependent on gene amplification (Slamon, D.I et al, Science 255:177-182 (1987); van de Vijver, M. et al, Mol. Cell. Biol 7:2019-2023 (1987)), we looked for MLN 50, 51, 62 and 64 gene amplification. Each of them showed amplification in 10-20% of sporadic breast carcinomas (data not shown). Nevertheless, amplification does not always correlate with gene overexpression. Then, in order to study the relationship between MLN gene amplification and expression, we have performed genomic DNA and RNA analyses of a panel of human breast cancer cell lines, including MCF7, TO-47D, BT-474, SKBR-3, MDA-MB-231, BT-20 and ZR-75-1, and the immortalized breast epithelial cell line HBL-100. MLN amplification and expression patterns were compared to those of c-erbB-2 and of p53, a gene located on the short arm of chromosome 17 and frequently mutated or lost but never amplified in breast carcinoma (Baker, S.I etal, Science 244:217-221 (1989)). Hybridization of Southern blots containing & BamHl digest of genomic DNAs extracted from these cell lines showed that the c-erbB-2, MLN 50, 51 and 64 genes were amplified in some cell lines, whereas the MLN 62 and p53 genes were not (Table IV). Moreover, in order to quantify the level of amplification, dot blots containing serial dilutions of cell genomic DNAs were performed. As summarized in Table TV, MLN 64 and c-erbB-2 genes were found to be co-amplified in SK-BR-3 (8 and 16 copies, respectively) and BT-474 (16 and 32 copies, respectively). MLN 50 gene was only amplified in BT-474 (8 copies) and MLN 51 gene in SK-BR-3 (4 copies). Northern blots containing RNAs extracted from the same cell lines were hybridized to the MLN cDNA probes (Fig. 3). MLN 64 and 19 {c-erbB-2) genes were overexpressed in SK-BR-3 and BT-474, MLN 50 gene in BT-474 and MLN 51 gene in SK-BR-3. These results clearly showed that, in cell lines, MLN 50, 51 and 64 overexpression were related to their gene amplification. Overexpression above basal level was observed for MLN 62 in SK-BR-3 and BT-20, and for p53 in MCF7 and HBL-100, independently of gene amplification. Amplification patterns observed in breast cancer cell lines suggested that MLN 50 (co-amplified with c-erbB-2, but not with MLN 62) and MLN 64 (co- amplified with c-erZ>B-2 in two cell lines, whereas MLN 51 was only in one cell line) should be located closest to c-er_B-2 than MLN 62 and 51, respectively. Thus, according to their chromosomal assignments and amplification patterns, the five locus framework order cen-MLN 62-MLN 50-c-er >B-2-MLN 64-MLN 51 -tel could be proposed (Fig. 2(B)). Discussion In the present study, we report the identification of cDNAs by differential screening of a breast cancer MLN cDNA library with two subtracted cDNA probes, representative of malignant (MLN) and nonmalignant (FA) breast tissues. The identified cDNAs corresponded to ten distinct genes expressed in MLNs, but not in normal lymph nodes or FAs. 75% of these cDNAs corresponded to known genes, namely the c-erbB-2, NCA, fibronectin and calcyclin genes, which have been previously shown to be involved in metastatic processes. c-erbB- 2 overexpression has been demonstrated in 15-30% of breast carcinomas and has been associated with shorter survival, particularly in patients with invaded lymph nodes (Slamon, D.I et al, Science 244:707-712 (1989); Borg, A. et al, Oncogene 6:137-143 (1991); Toikkanen S. et al, J. Clin. Oncol. 5:103-112 (1992); Muss, H.B. et al, N. Engl J. Med. 500:1260-1266 (1994)). NCA belongs to the carcinoembryonic antigen (CEA) family. CEA expression is elevated in 50-80% of patients with metastatic breast cancer and is used as a circulating marker to detect disease recurrence (Loprinzi, C. et al, J. Clin. Oncol. 4:46-56 (1986)). A modulation of fibronectin expression by alternative splicing has been reported in malignant tumors (Carnemolla, B. et al, J. Cell Biol. 705:1139-1148 (1989); Humphries, M.I, Semin. Cancer Biol. 4:293-299 (1993)). Calcyclin, a member of the SI 00 Ca * * binding protein family, is a cell cycle related protein and has been shown to be overexpressed in highly metastatic human melanoma cell lines (Weterman, M.A. et al, Cancer Res. 52:1291-1296 (1992)). About half of the last 25% of identified cDNAs corresponded to two novel members of the SI 00 and keratin protein families, respectively. Finally, the remaining differential clones (MLN 50, 51, 62 and 64) corresponded to cDNAs which did not belong to any previously characterized gene or protein family. The four genes corresponding to these cDNAs were co-localized to the ql l-q21.3 region of the chromosome 17 long arm. Several genes implicated in breast cancer progression have already been assigned to the same portion of this chromosome, notably the oncogene c-erbB-2 in ql2 (Fukushige, S.I. et al, Mol Cell. Biol. 6:955-958 (1986)) and the recently cloned tumor suppressor gene BRCA1 in q21 (Hall, J.M. et al, Science 250:1684-1689 (1990); Mil , Y. et al, Science 266:66-71 (1994) and refs. therein). According to their chromosomal assignments, we mapped the four novel genes proximal (MLN 62 and 50) and distal (MLN 64 and 51) to the c-erbB-2 gene, and, most probably, proximal to the BRCA1 gene. In vivo, the four MLN genes showed amplification in 10-20% of breast carcinomas. Moreover, in breast cancer cell lines, MLN 64 exhibited an amplification pattern identical to that of c-erbB-2 showing a clear amplification in BT-474 and SK-BR-3. However, MLN 50 and 51 gene amplification was restricted to BT-474 and SK-BR-3, respectively, and, any cell lines showed MLN 62 amplification. Altogether, these results support the concept that c-erbB-2 amplicon nature and size are variable from one malignant cell line to another (Muleris, M. etal, Genes Chrom. Cancer 70:160-170 (1994)), exemplifying the breast cancer heterogeneity (Lonn, U. et al, Intl. J. Cancer 55:40-45 (1994) and refs. therein). Finally, in breast cancer cell lines, MLN 50, 51 and 64 gene overexpression was correlated with gene amplification. It is assumed that DNA amplification plays a crucial role in tumor progression by allowing cancer cells to upregulate numerous genes (Kallioniemi, A. etal, Proc. Natl. Acad Sci. USA 97:2156-2160 (1994); Lonn, U. et al, Intl. J. Cancer 55:40-45 (1994)). Frequency of gene amplification as well as gene copy number increase during breast cancer progression, notably in patients who do not respond to treatment, suggesting that overexpression of the amplified target genes confers a selective advantage to malignant cells (Schimke, R.T., J. Biol. Chem. 265:5989-5992 (1988); Lonn, U. et al, Intl. J. Cancer 55:40-45 (1994); Guan, X.Y. et al, Nat. Genet. 5:155-161 (1994)). Recently, amplified loci, distinct from those of currently known oncogenes, have been mapped, using comparative genomic hybridization (Kallioniemi, A. et al, Proc. Natl. Acad Sci. USA 97:2156-2160 (1994); Muleris, M. et al, Genes Chrom. Cancer 70: 160-170 (1994)), suggesting the presence of unknown genes whose expression contributes to breast cancer. As we report here, the use of differential screening could be an efficient methodology for the identification of such unknown genes, since it allows for the direct cloning of amplified and overexpressed genes. Although amplification involves large regions of chromosomal DNA, it is known to target oncogenes (Schwab, M. & Amler, L., Genes Chrom. Cancer 7:181-193 (1990)). The correlation between amplification and overexpression is necessary to identify the targeted gene. Thus, within the 17ql2 amplicon, c-erbB-2 is often co- amplified with c-erbA but c-erbA overexpression was never observed (van de Vijver, M. et al, Mol. Cell. Biol. 7:2019-2023 (1987)). A similar finding was observed within the 1 lql3 amplicon where the cyclinD/PRADl gene is linked to int-2 and hst-1 two fibroblast growth factor related genes and only PRADl is overexpressed in the carcinomas (Lammie, G.A. et al, Oncogene 6:439-444 (1991)). In this context, the fact that the four novel genes identified in the present study are not only amplified but also overexpressed, suggests that they may contribute to the genesis and/or the progression of breast tumors. Table I: Clinical and Histological Characteristics of the Breast Carcinomas Patient Age (yrs.) Tumor size (cm) Histological Number of grade involved lymph nodes Biophys. Res. Co mun. 750:89-96 (1988) MLN 19 28 3.0 c-et_B-2 (Coussens, L. et al Science 230: 1132- 1139 (1985) Table III: Chromosomal Assignment of MLN 50, 51, 62, and 64 Genes " Mapping panel #2 from NIGMS, Coriell Cell Repositories (Camden, N.J., U.S.A.) - no, + weak, + + strong signals Table IV: MLN 50, 51, 62 and 64 Gene Amplification Among Breast Cancer Cell Lines MLN 19 (c-e/*B-2) MLN 64 MLN 62 MLN 50 (8) MLN 51 (4) p53 • no amplification could be detected; estimated copy numbers are indicated in parenthesis. Example 2 CARTl, a Gene Expressed in Human Breast Carcinoma, Encodes a Novel Member of the Tumor Necrosis Factor Receptor- Associated Protein Family Introduction Human CARTl cDNA corresponds to the MLN 62 cDNA clone discussed above in Example 1. The clone was identified through a differential screening performed by using two subtractive probes, respectively representative of metastatic and nonmalignant breast tissues and was mapped on chromosome 17, at the ql l-ql2 locus, a locus which includes the oncogene c-er_»B-2 whose overexpression is correlated with a shorter overall and disease free survival for breast cancer patients (Slamon, D.J. et al, Science 235:177-182 (1987); Muss, H.B. et al, N. Engl. J. Med 330:1260-1266 (1994)). In this example, we investigated the CARTl gene expression in a panel of normal and malignant human tissues and characterized the CARTl cDNA protein and gene organization. CARTl was specifically expressed in epithelial breast cancer cells. The amino acid sequence of CARTl reveals structural domains similar to those present in TNF receptor associated proteins, suggesting that CARTl is implicated in signal transduction for TNF-related cytokines. Materials and Methods Tissues Collection Depending on subsequent analysis, tissues were either immediately frozen in liquid nitrogen (RNA extraction), or fixed in formaldehyde and paraffin embedded {in situ hybridization). Frozen tissues were stored at -80 °C whereas paraffin-embedded tissues were stored at 4°C. The mean age of the 39 patients included in the present study was 55 years. The main characteristics of the breast carcinomas were as followed: SBR grade I (13%), grade II (38%), grade III (49%); estradiol receptor positive (25%), negative (75%); lymph nodes without invasion (39%), with invasion (61%). RNA Isolation and Analysis Total RNA prepared by a single-step method using guanidinium isothiocyanate (Chomczynski, P. & Sacchi, N., Anal. Biochem. 162:156-159 (1987)) was fractionated by agarose gel electrophoresis (1%) in the presence of formaldehyde. After the transfer, RNA was immobilized by heating (12 hr, 80°C). Filters (Hybond N; Amersham Corp.) were acidified (10 min, 5% CH 3 COOH) and stained (10 min, 0.004% methylene blue, 0.5M CH 3 COONa, pH 5.0) prior to hybridization. A CARTl probe corresponding to the full-length human cDNA (nucleotides 1 to 2004), cloned into pBluescript II SK vector (Stratagene) was 32 P-labeled using random priming (~10 6 cpm ng DNA) (Feinberg, A.P. & Vo Vogelstein, B., Anal. Biochem. 132:6 (1983)). Filters were prehybridized for 2 hrs at 42°C in 50% formamide, 5x SSC, 0.1% SDS, 0.5% PVP, 0.5% Ficoll, 50 mM sodium pyrophosphate, 1% glycine and 500 μg/ml ssDNA. Hybridization was for 18 hrs under stringent conditions (50% formamide, 5x SSC, 0.1% SDS, 0.1% PVP, 0.1% Ficoll, 20 mM sodium pyrophosphate, 10% dextran sulfate, 100 μg/ml ssDNA; 42 °C). Filters were washed for 30 min in 2x SSC, 0.1% SDS at room temperature, followed by 30 min in O.lx SSC, 0.1% SDS at 55 °C. In Situ Hybridization In situ hybridization was performed using a 35 S-labeled antisense RNA probe (5x10* cpm/μg), obtained after in vitro transcription of a BglU fragment (nucleotides 279-1882) of the human CARTl cDNA. Formaldehyde-fixed paraffin-embedded tissue sections (6 μm thick) were deparaffined in LMR, rehydrated and digested with proteinase K (1 μg/ml; 30 min, 37°C). Hybridization was for 18 hrs, followed by RNase treatment (20 μg/ml; 30 min, 37°C) and stringently washed twice (2x SSC, 50% formamide; 60°C, 2 hrs). Autoradiography was for 2 to 4 weeks using NTB2 emulsion (Kodak). After exposure, the slides were developed and counterstained using toluidine blue. 35 S- labeled sense transcript from CARTl was tested in parallel as a negative control. CARTl Genomic DNA Cloning Fifty μg of human genomic DNA was partially digested with Sau3A. After size selection on a 10-30% sucrose gradient, inserts (16-20 kb) were subcloned at the BamHl replacement site in lambda EMBL 301 (Lathe, R. et al, Gene 57:193-201 (1987)). 2.5xl0 6 recombinant clones were obtained and the library was amplified once. One million pfu were analyzed for the presence of genomic CARTl DNA, using the full-length CARTl cDNA probe. Thirty clones gave a positive signal. After a second screening, four of these clones were subcloned into pBluescript II SK- vector (Stratagene), sequenced and positioned with respect to the CARTl cDNA sequence. Sequencing Reactions CART cDNA clones and genomic subclones prepared as described (Zhou, C. et al, Biotechniques 5:172-173 (1990)) were further purified with RNaseA treatment (10 μg/ml; 30 min, 37°C) followed by PEG/NaCl precipitation (0.57 vol.; 20%, 2 M) and ethanol washing. Vacuum dried pellets were resuspended at 200 ng/μl in TE. Double-stranded DNA templates were then sequenced with Taq polymerase, using either pBluescript universal primers and/or internal primers, and dye-labeled dNTPs for detection on an Applied Biosystems 373 A automated sequencer. Computer Analysis Sequence analysis were performed using the GCG sequence analysis package (Wisconsin Package, version 8, Genetic Computer Group). The CARTl cDNA sequence and its deduced putative protein were used to search the complete combined GenBank/EMBL databases and the complete SwissProt database respectively, with BLAST (Altschul, S.F. et al, J. Mol. Biol. 215:403- 410 (1990)) and FastA (Pearson, W.R. & Lipman, D.J., Proc. Natl. Acad Sci. USA -"5:2444-2448 (1988)) programs. The RING finger motif and consensus sequences of CARTl protein were further identified by the Motifs program in the PROSITE dictionary (release 12). The sequence alignments were obtained automatically by using the program PileUp (Feng, D.F. & Doolittle, R.F., J. Mol. Evol. 25:351-360 (1987)). Results Expression of the CARTl Gene Using Northern blot analysis, we have studied CARTl gene expression in benign (16 fibroadenomas) and malignant (39 carcinomas and 5 metastatic axillary lymph nodes) human breast tissues. Hybridization with a CARTl cDNA probe gave a positive signal corresponding to CARTl transcripts with an apparent molecular weight of 2 kb, in 4 carcinomas and 2 metastases (Fig. 4, lanes 7, 11, 13 and 17, and data not shown). The fibroadenomas did not show CARTl expression above the basal level (Fig. 4, lanes 1-6). No CARTl transcripts were observed in normal human axillary lymph node, skin, lung, stomach, colon, liver kidney and placenta (data not shown). In situ hybridization, using an antisense CARTl RNA probe, was performed on primary breast carcinomas and axillary lymph node metastases. CARTl was expressed in malignant epithelial cells (Fig. 5(C)) and invasive carcinomas (Fig. 5(B)), whereas tumoral stromal cells were negative. CARTl transcripts were homogeneously distributed among the positive areas. Normal epithelial cells did not express the CARTl gene, even when located at the proximity of invasive carcinomatous areas (Fig. 5(A) and data not shown). A similar pattern of CARTl gene expression was observed in metastatic axillary lymph nodes from breast cancer patients with expression limited to cancer cells whereas noninvolved lymph node areas were negative (Fig. 5(D) and data not shown). Determination of Human CARTl cDNA and Putative Protein Sequences The complete CARTl cDNA sequence has been established from three independent cDNA clones. Both sense and antisense strands have been sequenced. The longest cDNA clone contained 2004 bp, a size consistent with the previously observed 2 kb transcript suggesting that this cDNA corresponded to a full-length CARTl cDNA (Fig. 6) (SEQ ID NO:l). The first ATG codon (at nucleotide position 85) had the most favorable context for initiation of translation (Kozak, M., Nucl Acids Res. 75:8125-8149 (1987)), and a classical AATAAA poly(A) addition signal sequence (Wahle, E. & Keller, W., Annu. Rev. Biochem. 67:419-440 (1992)) was located 18 bp upstream of the poly(A) stretch. Thus, the open reading frame was predicted to encode a 470-residue protein (Fig. 6) (SEQ DD NO:2), with a molecular weight of 53 KD and a pHi of 8. The putative protein showed several consensus sequences, and notably two potential nuclear localization signals (NLS), a monopartite KPKRR (residues 11-15 of Fig. 6, SEQ ID NO:2) (Dang, CN. & Lee. W.M.F., J. Biol Chem. 264:18019-18023 (1989)) and a bipartite RR-X n -KRRLK (residues 123-140 of Fig. 6, SEQ ID ΝO:2) (Dingwall, C. & Laskey, R.A., Trends Biochem. Sci. 76:478-480 (1991)). The molecule also contained potential sites (reviewed in, Kemp, B.E. & Pearson, R.B., Trends Biochem. Sci. 75:342-346 (1990)) specific of N-glycosylation (NGS, residues 355-357 of Fig. 6, SEQ ID NO:2), phosphorylation by casein kinase I (EELS, residues 300-303; SVGS, residues 303-306; ECFS, residues 331-334; all of Fig. 6, SEQ ID NO:2) and casein kinase II (SEE, residues 86-88; SRRD, residues 122-125; SGE, residues 149-151; SHE, residues 155-157; TSE, residues 185-187; TKE, residues 199-201; SGE, residues 357-359; SLLD, residues 389- 392; SLDE, residues 426-429; SHQD, residues 441-444; all of Fig. 6, SEQ ID NO:2), proline-dependent phosphorylation (FSPA, residues 333-336 of Fig. 6, SEQ ID NO:2) and cAMP-dependent phosphorylation (RRVT, residues 384-387 of Fig. 6, SEQ ED NO: 62). Moreover, two cystein-rich (C-rich) regions were identified, one located at the N-terminal part of the protein (residues 18-57) and the other at the core of the molecule (residues 83-282). Finally, the C-terminal part of the CARTl protein corresponded to the recently described TRAF domain (Rothe, M. et al, Cell 75:681-692 (1994)) (Fig. 6). CARTl Contains an Unusual N-terminal RING Finger Motif The N-terminal C-rich structure of the putative CARTl protein contained a CX 2 CXι 2 CX,HX 2 CX 2 CX n CX > (C3HC3D) motif (residues 18-57 of Fig. 6, SEQ ID NO:2) reminiscent of the C3HC4 consensus sequence (Freemont, P.S. et al, Cell 64:483-484 (1991); Fig. 7). This sequence, located either at the N- or at the C-terminal part of proteins, could potentially give rise to two zinc fingers and has been named the RING finger motif (Freemont, P.S., Ann. N.Y. Acad Sci. 684:174-192 (1993) and refs. therein). The proteins which share such a structure often exhibit DNA or RNA binding properties, and have been reported to be implicated during development such as DG17 (Driscoll, D.M. & Williams, J.G., Mol. Cell Biol. 7:4482-4489 (1987)) and SU(z)2 (Van Lohuizen, M. et al, Nature 555:353-355 (1991)), gene transcription such as RPT-1 (Patarca, R. et al, Proc. Natl. Acad Sci. USA 55:2733-2737 (1988)), SS-A/Ro (Chan, E.K.L. et al, J. Clin. Invest. 57:68-76 (1991)), XNF7 (Reddy, B. etal.,Dev. Biol. 148:107-116 (1991)) and RING1 (Lovering, R et al, Proc. Natl. Acad Sci USA 90:2112-2116 (1993)), DNA repair such as RAD- 18 (Jones, J.S. et al, Nucl Acids Res. 76:7119-7131 (1988)), cell transformation such as MEL-18 (Tagawa, M. etal, J. Biol. Chem. 265:20021-20026 (1990); Goebl, M.G., Cell 66:623 (1991)), tumor suppression such as BRCA1 (MM, Y. et al, Science 266:66-71 (1994)), or signal transduction such as CD40-binding protein (CD40-bp) (Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994)) and TRAF2 (Rothe, M. et al, Cell 75:681-692 (1994)). The distribution of C- and H-residues is highly conserved in all these RING fingers (Fig. 7). However, CARTl contained an aspartic acid (D-) residue instead of the last C-residue of the C3HC4 motif (Fig. 7). In order to confirm the presence of this D-residue, and since D-codon sequence lead to an Avail restriction site (Fig. 8(A)), an AvdΑ digestion was performed on the full- length CARTl cDNA. Gel electrophoresis showed the presence of four bands (253, 428, 531 and 792 bp, respectively), a pattern consistent with the presence of a D-codon (Fig. 8(B)). However, since the CARTl cDNA was cloned from a cDNA library established using malignant tissues, we could not exclude the possibility that the D-residue resulted from an alteration occurring during carcinogenesis (Bishop, J.M., Cell 64:235-348 (1991)). Thus, in order to identify the physiological residue, we sequenced CARTl DNA from a normal leukocyte genomic library {see Materials and Methods). This analysis confirmed the presence of a D-residue, and consequently the C3HC3D motif. Data bank library analysis did not reveal any other protein sharing an identical RING finger motif. Identification and Characterization of a Novel C-rich Motif, the CART Motif The second C-rich region expanded from residues 83 to 282 and constituted almost half of the protein (Fig. 6) (SEQ ID NO:2). It contained 23 C- and 12 H-residues, corresponding to 96% and 67% of the remaining C- and H- residues, respectively. A careful examination of spacing of these C/H residues allowed the detection of an ordonnance giving rise to three HX 3 CX 6 CX 3 CX 11 . j2 HX 4 CX 6 CX M CX u (HC3HC3) repeats. The most N-terminal of them (residues 101-154) contained the potential bipartite NLS (Figs. 6 and 9). Homologies between these repeats were not restricted to the C/H residues and to the spacer sizes. Alignment of the three CARTl HC3HC3 motifs showed around 50% similarity and 30% identity with each other (Fig. 9). Homology searches in the protein database revealed the presence of one copy of an analogous motif (residues 193-250) in the Dictyostelium discoideum DG 17 protein (Fig. 9) (SEQ ID NO:28) (Driscoll, D.M. & Williams, J.G., Mol. Cell. Biol. 7:4482-4489 (1987)), and of two copies in the human CD40-bp (Fig. 9) (residues 134-189 and 190-248, SEQ ID NOS:24 and 25, respectively) (Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994)) and in the mouse TRAF2 (Fig. 9) (residues 124-176 and 177-238, SEQ ED NOS:26 and 27) (Rothe, M. etal, Cell 75:681-692 (1994)). It should be noted that the sequences of the two N-terminal CARTl HC3HC3 motifs were most similar to those of the N-terminal motifs of CD40-bp (50% and 40%, respectively) and of TRAF2 (52% and 46%, respectively). The C-terminal CARTl HC3HC3 motif however was most similar to the C-terminal motifs of CD40-bp (58%) and of TRAF2 (55%), and to that of DG17 (51%) (Fig. 9). From these comparisons, the consensus sequence was proposed for this novel motif that we named the CART motif for "C-rich motif Associated to RENG and TRAF domains" {see, infra) (Fig. 9). CARTl Contains a C-terminal TRAF Domain The TRAF domain, recently identified in the TNF receptor-associated factors 1 (TRAFl) and 2 (TRAF2), is involved in TNF signal transduction pathway. TRAF domains encompass the 230 C-terminal residues of these proteins and share 53% identity (Rothe, M. et al, Cell 75:681-692 (1994)). The TRAF motif was also reported in the CD40-bp which associates with the cytoplasmic tail of CD40, another member of the TNF receptor family (Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994)). The C-terminal part of CARTl (residues 267- 470) showed two degrees of homology with the TRAF domain. Thus, residues 267 to 307 showed a weak homology (12-23% identity). From structural predictions, this N-terminal part of CARTl TRAF domain is supposed to give rise to an alpha helix (Chou, P.Y. & Fasman, G.D., Annu. Rev. Biochem. 47:251-276 (1978)). Such a structure, already proposed for the corresponding regions of TRAF1, TRAF2 and CD40-bp is supposed to be involved in protein/protein interactions (Rothe, M. et al, Cell 75:681-692 (1994); Hu, H.M. etal, J. Biol. Chem. 269:30069-30072 (1994)). The C-terminal part of CARTl TRAF domain (residues 308-470) showed high degree of similarity and identity with the corresponding part of TRAFl (60% and 42%), TRAF2 (69% and 47%) and CD40-bp (62% and 43%), thus defining a "restricted TRAF domain" (Fig. 10). Finally, since DG17 already contained a N-terminal RING finger and a CART motif, we looked for the presence of a restricted TRAF domain in its C-terminal part. We observed 55% similarity and 30% identity between the last 150 residues of CARTl and DG17 (data not shown). However, the protozoan DG17 protein showed numerous mismatches with the restricted TRAF consensus motif derived from human and mouse proteins (Fig. 10), suggesting that DG17 contains a primitive TRAF domain. CARTl Gene Organization Two independent clones have been selected from a screening of a human leukocyte genomic library using the full-length CARTl cDNA probe. These clones contained 3 and 3.2 kb BamH fragments which have been subcloned and partially sequenced in order to map splicing sites. The human CARTl gene was found to be split into 7 exons (Fig. 11 and Table V (exon/intron Nos. 1-6 corresponding to SEQ ED NOS:52-57, respectively). Comparison of the intron/exon boundaries showed that each corresponded to a canonical splice consensus sequence (Breathnach, R. & Chambon, P., Annu. Rev. Biochem. 50:349-383 (1981)). The total length of the CARTl gene is approximately 5.5 kb (Fig. 11). Analysis of the genomic structure of the RING finger domain revealed that it is encoded by two exons separated by the presence of an intronic sequence located between nucleotides 226-227 (Fig. 4). Thus, the C3HC2 and the CD parts of the C3HC3D motif are encoded by exons 1 and 2, respectively (Fig. 11). The three CART motifs were encoded by three separate exons of 161 (exon 4) (SEQ ED NO:55), 161 (exon 5) (SEQ ID NO:56) and 156 (exon 6) (SEQ ID NO:57) bp, respectively (Fig. 11 and Table V). In addition to their similar size, the three exons exhibited about 40% identity with each other, suggesting they have arisen by duplication of an ancestral exon. Finally, the α-helix and the restricted TRAF domain were encoded by exon 7 which also encoded for the 3 ' untranslated region. CARTl Protein Subcellular Localization - CARTl subcellular localization was performed on paraffin-embedded sections from a human invasive breast carcinoma using a rabbit polyclonal antibody. The antibody specificity was established by Western blot analysis of CARTl recombinant protein (data not shown). Consistent with our findings using in situ hybridization, CARTl immunoperoxidase staining (brown staining) was observed in malignant epthelial cells. Moreover, CARTl protein appeared to be located in the nucleus showing that almost one of the CARTl nuclear localization signals was functional. The intensity of staining was variable from one cell to another, even within a given area of the section. Discussion We characterized a cDNA and corresponding putative protein encoded by a novel gene that we call the CARTl gene (identified as MLN 62 in Example 1) by screening a breast cancer metastatic lymph node cDNA library. CARTl was overexpressed in 10% of primary breast carcinomas and 50% of metastatic axillary lymph nodes, whereas the corresponding nonmalignant tissues did not. CARTl transcripts were specifically detected in malignant epithelial cells and homogeneously distributed throughout the carcinomatous areas. No CARTl expression was observed in a panel of normal human tissues including skin, lung, stomach, colon, liver, kidney and placenta. This expression pattern, restricted to some malignant tissues, suggests that CARTl is involved in processes leading to the formation and/or progression of primary carcinomas and metastases. The putative CARTl protein sequence, deduced from the cDNA open reading frame, exhibited several structural domains. The CARTl N-terminal part contained a C- rich domain characterized by the presence of a RING finger (Freemont, P.S., Ann. N.Y. Acad Sci. 654: 174-192 (1993)). The RING finger protein family presently comprises more than 70 members involved in the regulation of cell proliferation and differentiation (reviewed in, Freemont, P.S., Ann. N.Y. Acad Sci. 654:174- 192 (1993)). Interestingly, one of the recently identified members of the family is the tumor suppressor gene BRCA1, responsible for about 50% of inherited breast cancers (Mϋd, Y. et al, Science 266:66-71 (1994)). RING finger motif is assumed to fold into two zinc fingers and to be involved in protein/nucleic acid interaction(s) (Schwabe, J.W.R & Klug, A, Nature Struc. Biol. 7:345-349 (1994) and refs. therein). In CARTl RING finger, the last C-residue is substituted by a D-residue giving rise to a C3HC3D motif instead of the usual C3HC4 motif. Since aspartic acid has already been described as a potential zinc coordinating residue (Vallee, B.L. & Auld, D.S., Biochem. 29:5647-5659 (1990)), we assume that the C3HC3D motif may efficiently bind metal atoms through the zinc finger structure. Consistent with this hypothesis, aspartic acid has already been reported to be functional in another type of zinc finger motif, the LIM domain (Sanchez- Garcia, I. & Rabbits, T.H., Trends Genet. 9:315-320 (1994) and refs. therein). CARTl RING finger is encoded by two exons coding for the C3HC2 and CD part of the C3HC3D motif, respectively, a genomic organization slightly different from that previously described for the consensus MEL-1 8 RING finger which results from two exons encoding the C3H and C4 putative zinc finger, respectively (Asano, H. et al, DNA Sequence 5:369-377 (1993)). CARTl also contained an original C-rich region, located more centrally within the protein and composed of three repeats of an HC3HC3 motif corresponding to a novel protein signature and that we designated the CART motif. These three repeats were encoded by distinct exons homologous with each other, suggesting that they derived from an ancestral exon. CART motifs were only found, in variable copy numbers, in three RING finger proteins, the human CD40-bp (two copies), the mouse TRAF2 (two copies) and the Dictyostelium discoideum DG17 protein (one copy) (Hu, H.M. et al, J. Biol. Chem. 269:30069- 30072 (1994); Rothe, M. et al, Cell 75:681-692 (1994); Driscoll, D.M. & Williams, J.G., Mol. Cell. Biol. 7:4482-4489 (1987)). The corresponding C-rich regions of CD40-bp, TRAF2 and DG17 have been previously reported to be partially arranged in pattern resembling either the CHC3H2 "B box" motif or the C2H2Xenopuslaevis transcription factor HI A motif (Freemont, P.S., Ann. N.Y. Acad Sci. 654:174-192 (1993); Hu, H.M. etal,J. Biol Chem. 269:30069-30072 (1994); Rothe, M. et al, Cell 75:681-692 (1994); Driscoll, D.M. & Williams, J.G., Mol. Cell. Biol. 7:4482-4489 (1987)). The CART motif, as defined in the present study, encompasses almost the totality of the C-rich region observed in CARTl, CD40-bp, TRAF2 and DG17. The function of the CART domain remains to be determined. Preliminary protein studies (C.R., unpublished results) indicate that the correct folding of the CART motif is depending on the presence of zinc, supporting the hypothesis that CART corresponds to a novel zinc binding motif presumably involved in nucleic acid binding (Schwabe, J.W.R. & Klug, A., Nature Struc. Biol. 7:345-349 (1994); Schmiedeskamp, M. & Klevit, R.E., Curr. Opin. Struc. Biol. 4:28-35 (1994)). The C-terminal part of CARTl corresponded to a TRAF domain previously identified in TRAFl, TRAF2 and CD40-bp. This motif is involved in protein/protein interaction and TRAF2 and CD40-bp have been reported to specifically interact with the cytoplasmic domain of two members of the TNF- receptor family, TNF-R2 and CD40, respectively (Rothe, M. et al, Cell 75:681- 692 (1994); Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994)). The TRAF domain is composed of two structural domains, a N-terminally located domain which corresponds to a weakly conserved alpha helix and a C-terminally located domain which is highly conserved and corresponds to what we called the "restricted TRAF domain," since it includes only part of the previously described TRAF domains (Rothe, M. et al, Cell 75:681-692 (1994); Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994)). Both structural motifs were encoded by the same exon of the CARTl gene. Homology was also observed with the C- terminal part of the protozoan DG17 protein which, although less conserved, could be considered as a TRAF domain. Thus, CARTl shared a protein organization similar to that of the human CD40-bp, the mouse TRAF2 and protozoan DG17, including a N-terminal RING finger, one to three central CART motifs and a C-terminal TRAF domain (Fig. 12). These results suggest that these structurally related proteins belong to the same protein family and may exhibit analogous function. DG17 is expressed during Dictyostelium discoideum aggregation which occurs under stress conditions in order to permit cell survival through a differentiated multicellular organism. The precise function of DG17 function remains unknown (Driscoll, D.M. & WilUams, J.G., Mol. Cell. Biol. 7:4482-4489 (1987)). However, both CD40-bp and TRAF2 have been previously shown to be involved in TNF-related cytokine signal transduction (Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994); Rothe, M. et al, Cell 75:681-692 (1994)). In contrast to growth factor receptors, cytokine receptors generally do not contain kinase activity in their cytoplasmic region, and their signal transduction mechanisms remain elusive (reviewed in, Taga, T. & Kishimoto, T., FASEB J. 6:3387-3396 (1993)). To date, the TNF and TNF receptor families contain 8 and 12 members, respectively. The lack of sequence homology among TNF-receptor cytoplasmic domains, required for signal transduction, suggests the existence of specific signaling pathway for each receptor (reviewed in, Smith, CA. et al, Cell 65:959-962 (1994)). Recently, it has been proposed that signal transduction through CD40 and TNF-R2 involved the interaction of their cytoplasmic domain with two cytoplasmic proteins, CD40-bp and TRAF2, respectively (Rothe, M. etal, Cell 75:681-692 (1994); Hu, H.M. et al, J. Biol. Chem. 269:30069-30072 (1994)). Thus, CD40-bp and TRAF2 could be latent cytoplasmic transcription factors, which would be translocated to the nucleus under receptor activation by their respective ligands. A similar system has already been proposed for the protein family of signal transducers and activators of transcription (STAT) involved in gene activation pathways triggered by interferons (Darnell, J.E. et al, Science 264:1415-1421 (1994)). This system implies a direct signal transduction pathway through STAT migration from cytoplasm to nucleus, presumably triggered by STAT phosphorylation following receptor activation (Ihle, J.N. et al, Trends Biochem. Sci. 79:222-227 (1994); Darnell, J.E. et al, Science 264:1415-1421 (1994)). From all these observations, it is tempting to speculate that CARTl, which not only shares a structural arrangement of RING, CART and TRAF domains identical to that observed in two TNF receptor associated proteins, but also exhibits putative NLS and phosphorylation sites, may exert similar function for TNF-related cytokine signal transduction. TNF ligand family members have been shown to induce pleiotropic biological effects, including cell differentiation, proliferation, activation or death, all processes involved during carcinogenesis and tumor progression (Smith, CA. etal., Cell 65:959-962 (1994), and refs. therein). In breast carcinomas, p55 and p75 TNF receptors have been shown to be expressed in malignant tissues, and a dramatic increase of the secretion of their corresponding TNFα ligand has been associated with metastatic step of the disease (Pusztai, L. et al, Brit. J. Cancer 70:289-292 (1994), and refs. therein). Our observation of CARTl overexpression in breast carcinomas suggests that, CARTl may be involved in signal transduction pathway either involving p55/p75 or another member of the TNF-receptor family. The nature of TNF receptor as well as the nature of protein(s) which may interact with CARTl are now under characterization. Table V ExonΔntron Organization of the CARTl Gene Exa ple 3 Lasp-1 (MLN 50), Encodes the First Member of a New Protein Family Characterized by the Association of LIM and SH3 Domains Introduction In Example 1 above, we describe the isolation of MLN 50 (Lasp-1) cDNA from a breast cancer derived metastatic lymph node cDNA library by differential hybridization using malignant (metastatic lymph node) versus nonmalignant (fϊbroadenoma and normal lymph node) breast tissue. Chromosomal mapping allowed us to map the Lasp-1 gene to the ql2-q21 region of the chromosome 17 long arm. This region is known to be altered in 20 to 30% of breast cancers leading to the amplification of the proto-oncogene c-erbB-2 (Fukushige, S.I. et al,Mol Cell. Biol. 6:955-958 (1986); Slamon, D.J. etal, Science 244:707-712 (1989)). In breast cancer cell lines, we found that Lasp-1 RNA overexpression was correlated with its gene amplification and to c-erbB-2 amplification/overexpression suggesting that Lasp-1 and c-erbB-2 belong to the same amplicon. In the present example, we determined the frequency of Lasp-1 overexpresion in human breast cancer and characterized the encoded protein. Materials and Methods Tissue and Cell Cultures Surgical specimens obtained at the Hopitaux Universitaires de Strasbourg, were frozen in liquid nitrogen for RNA extraction. Adjacent sections were fixed in 10% buffered formalin and paraffin embedded for histological examination. The cell lines (SK-BR-3, BT-474, MCF-7) are available from the American Type Culture Collection (ATCC, Rockville, MD). Cells were routinely maintained in our laboratory and cultured at confluency in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum (SK-Br-3) and with 10 μg/ml of insulin (MCF-7), and in RPMI supplemented with 10% fetal calf serum and 10 μg/ml of insulin (BT-474). RNA Preparation and Analysis Surgical specimens were homogenized in the guanidinium isothiocyanate lysis buffer and purified by centrifiigation through cesium chloride cushion (Chirgwin, J.M. etal, Biochem. 75:52-94 (1979)). RNAs from cultured cell lines were extracted using the single-step procedure of Chomczynski, P. & Sacchi, N., Anal. Biochem. 162:156-159 (1987). RNAs were fractionated by electrophoresis on 1% agarose, 2.2 M formaldehyde gels (Lehrach, H. et al, Biochem. 16:4743- 4751 (1977)), transferred to nylon membrane (Hybond N, Amersham Corp.) and immobilized by baking for 2 hrs at 80 °C Probe Preparation and Hybridization Lasp-1 probe corresponded to a 1.0 kb BamHl fragment released from MLN 50 subcloned into pBluescript. The RNA loading control probe 36B4 was an internal 0.7 kb Pstl fragment (Masiakowski, P. et al, Nucleic Acids Res. 70:7895-7903 (1982)). Northern blots were hybridized at 42°C in 50% formamide, 5x SSC, 0.4% ficoll, 0.4% polyvinylpyrrolidone, 20 mM sodium phosphate pH 6.5, 0.5% SDS, 10% dextran sulfate and 100 μg/ml denatured salmon sperm DNA, for 36-48 hrs with the 32 P-labeled probe diluted to 0.5-1.10 6 cpm/ml. Stringent washings were performed at 60°C in 0. lx SSC and 0.1% SDS. Blots were autoradiographed at -80°C for 24 hrs. Sequence Analysis Sequence analyses were performed using the GCG sequence analysis package (Wiskonsin package version 8.0, Genetics computer Group, Madison, WE). The Lasp-1 cDNA and amino acid sequences were used to search the complete combined GenBank EMBL database and the complete SwissProt database with BLAST (Altschul, S.F. et al, J. Mol. Biol. 275:403-410 (1990)) and FastA (Pearson, W.R 8c Lipman, D.J., Proc. Natl. Acad Sci. USA 55:2444- 2448 (1988)) programs, respectively. The LIM motif and consensus sequences of Lasp-1 were further identified by the motif program in the PROSITE dictionary (release 12). The sequence alignments were obtained automatically by using the program PileUp (Feng, D.F. & Doolittle, R.F., J. Mol Evol. 25:351-360 (1987)). Results and Discussion To determine Lasp-1 mRNA distribution we carried out Northern blot analysis using the cDNA as a probe. A single 4.0 kb mRNA band was detected at low level in all the human tissue and cell lines studied (Fig. 13 and data not shown). Lasp-1 mRNA overexpression was found in 8% (5/61) primary breast cancers (Fig. 13(A), lane 8) and in 40% (2/5) breast cancer derived metastatic lymph nodes (Fig. 13(A), lanes 1 and 2). No expression (0/15) above the basal level was found in nonmalignant breast tissues (Fig. 13(A), lanes 13-17, fibroadenomas; lane 18, hyperplastic breast) nor in normal adult tissues (Fig. 13(B), lanes 1-6 and data not shown). By comparison with c-erbB-2 overexpression, Lasp-1 was found to be coexpressed in most (Fig. 13(A), lanes 1, 2 and 8; Fig. 13(B), lane 8) but not in all (Fig. 13(A), lane 12; Fig. 13(B), lane 7) human breast cancer and cell lines. These results suggest that Lasp-1 is quite ubiquitous at the RNA level, with an increased expression in some breast cancer tissue and derived metastatic lymph nodes which is probably caused by gene amplification centered around the c-er_»B-2 locus. The complete Lasp-1 cDNA sequence was established from four independent cDNA clones. Both sense and antisense strands were sequenced. The longest cDNA clone contained 3848 bp, a size consistent with the transcript size suggesting that this clone should correspond to the full length cDNA (Fig. 14(A)) (SEQ ED NO:3). At the nucleotide level, sequence homologies were found with 22 expressed sequences tags (ESTs) (Weinstock et al, Curr. Opin. Biotech. 5:599-603 (1994), and refs. therein). Some of these sequences are redundant and they were mostly located on the 3* untranslated end of the molecule (Fig. 14(B)). Most of these ESTs were established from different human cDNA libraries established using normal tissues (fetal brain, white blood cells, prostate gland, liver, pancreatic islet cells and fetal spleen). The presence of Lasp-1 transcripts in all these samples is in good agreement with our finding of ubiquitous expression of Lasp-1 mRNA (Fig. 13 and data not shown). The first ATG codon (nucleotide position 76 of Fig. 14(A) (SEQ ID NO:3)) had a favorable context for initiation of translation (Kozak, M., Nucl Acids Res. 75:8125-8149 (1987)), and a classical AATAAA ρoly(A) addition signal sequence (Wahle, E. & Keller, W., Annu. Rev. Biochem. 67:419-440 (1992)) was located 13 bp upstream of the poly(A) stretch (Fig. 14(A) (SEQ ID NO:3)). The deduced open reading frame encoded a 261 amino acid protein, with a molecular weight of 30 KD and a pHi of 6.5 (Fig. 14(A) (SEQ ID NO:4)). The protein showed several consensus sequences: an amidation site (GGKR, residues 203-206 of Fig. 14(A), SEQ ID NO:4), several phosphorylation sites by cAMP and cGMP dependent protein kinase (RRDS, residues 141-144 of Fig. 14(A), SEQ ID NO:4), casein kinase π (SGGE, 139-136; SAAD, 213-216; SFQD, 221- 224; all of Fig. 14(A), SEQ ID NO:4), protein kinase C (TEK, 14-16; TCK, 33- 35; SYR, 150-152; all of Fig. 14(A), SEQ ID NO:4)) and tyrosine kinase (KKGYEKKPY, 38-45; KDSQDGSSY, 137-144; all of Fig. 14(A), SEQ ID NO:4). Moreover, a cystein rich region was identified as a LIM (Sanchez-Garcia, I. & Rabbits, T.H., Trends Genet. 9:315-320 (1994)) domain in the N-terminal part and a SH3 (Musacchio et al, FEBSLett. 507:55-61 (1992)) domain at the C-terminal portion of the protein. The deduced primary sequence of Lasp-1 contains two likely tyrosine phosphorylation sites (underlined in Fig. 14); these residues are followed by short tripeptides demonstrating homology to the predicted SH2 binding motif (Songyang et al, Cell 72:767-778 (1993)). A Single LIM Domain is Present at the N-part of Lasp-1 The LIM domain is an arrangement of seven cysteine and histidine residues (C-X 2 -C-X 16/23 -H-X 2 -C-X 2 -C-X 2 -C-X 1& 1 -C-X 2/3 -C/D/H) present in a number of invertebrate and vertebrate proteins. The generic name was given for the product of the three firstly identified LIM genes (lin-11, lsl-1 and mec-3). The family of LIM containing proteins is continuously increasing and could be subdivided in distinct groups (Sanchez-Garcia, I. & Rabbits, T.H., Trends Genet. 9:315-320 (1994)). One group designated LIM-HD, includes protein having two LIM domains associated with a homeodomain (lin-11, lsl-1, mec-3). Another group designated LIM-only, includes proteins exhibiting a single (CRDP), two (CRP, TSF3, RBTN1, RBTN2, RBTN3) or three (zyxin) LIM domains. Recently, a new group designated LIM-K, including proteins having two LIM domains associated with a kinase domain, had been described (Sanchez-Garcia, I. & Rabbits, T.H., Trends Genet. 9:315-320 (1994); Mizuno et al, Oncogene 9: 1605-1612 (1994)). The LIM domain defines a zinc binding structure and zinc binding is necessary for the proper folding of the domain. Sequence alignments of LIM proteins with Lasp-1 showed a best score alignment with the C. elegans YLZ4 putative protein (Accession No. P34417). Although the overall homology is low (36% identity and 55% similarity), it is high within the LIM domain (66% identity and 80% similarity). The protein YLZ4 was identified in the whole sequencing of the C. elegans chromosome EQ (Wilson, R. et al, Nature 565:32-38 (1994)). The LIM domain of YLZ4 does perfectly fit the LIM consensus, the first two cysteines are spaced by four instead of two residues, leading to a gap in the alignment (Fig. 15(A)). Among other LIM containing proteins besides the LIM consensus sequence, additive homologies were found in the human cysteine-rich protein-CRP (Liebhaber, et al, Nucl Acids Res. 75:3871- 3879 (1990)), the rat cysteine-rich intestinal protein CREP and the physiological function of these proteins is not yet known, although a role for CRIP in intestinal zinc absorption has been suggested and CRP was identified as a binding partner for a LIM-only protein zyxin. The interaction between these two proteins, believed to have regulatory or signaling functions in focal adhesion plaques (Crawford et al, J. Cell Biol. 776: 1381-1393 (1992); Crawford et al, J. Cell Biol. 124:117-127 (1994); Sadler et al, J. Cell Biol. 779:1573-1587 (1992)), is mediated by sequence-specific interactions between their LIM domains (Shmeichel & Beckerle, Cell 79:211-219 (1994)). The LIM domain can be considered as a protein/protein modular binding interface similarly to SH2 and SH3 domains (Shmeichel & Beckerle, Cell 79:211-219 (1994)). Our findings showing a strong conservation for Lasp-1 LIM domain across a wide range of different species mammals, nematodes and plant suggest an important function for this domain. Lasp-1 Contains a SH3 Domain at the C-terminal Part The SH3 (src homology region 3) is a small protein domain of 60 amino acids, first identified as a conserved sequence in the N-terminal noncatalytic part of the src protein tyrosine kinase (Sadowski et al, Mol. Cell. Biol. 6:4396-4408 (1986); Mayer et al, Nature 332:272-275 (1988)). A number of proteins involved in the tyrosine kinases signal transduction pathway contain SH3 domains (Schlessinger, Curr. Opin. Genet. Develop. 4:25-30 (1994)), this domain could also been found in proteins of unrelated functions such as cytoskeleton associated proteins (Musacchio et al, FEBS Lett. 507:55-61 (1992)). The function of the SH3 domain remains unclear; however, SH3 containing proteins are usually located close to the plasmic membrane suggesting a role for this domain in the targeting of protein to this cellular compartment (Musacchio et al, FEBS Lett. 507:55-61 (1992)). Direct evidences of the adaptor molecule Grb2, SH3 domain targeting properties, were provided (Bar-Sagi et al, Cell 74:83-91 (1993)). Hints to the function were achieved by the resolution of several different SH3 domains, showing that the overall structure is conserved and independently folded. Also, several protein ligands for the SH3 domains of oncogenic tyrosine kinases have been isolated, leading to the definition of specific proline-rich regions required for the binding to SH3 domains (Alexandropoulos et al, 92:3110-3114 (1995) and refs. therein). Sequence alignment revealed homology of the Lasp-1 C-terminal part with several SH3 containing proteins (Fig. 15(B)), including in the SH3 domain of EMSl (Schuuring et al, Oncogene 7:355-361 (1992)) a human homolog of the src tyrosine kinase substrate cortactin (Wu et al., Mol Cell Biol. 77:5113-5124 (1991)). The strongest conservation was found with the YLZ3 putative protein of C. elegans (Accession No. P34416), the overall homology is low (23% identity and 40% similarity) but significant within the SH3 domain (57% identity and 74% similarity). This protein was deduced from the whole C. elegans chromosome Q sequencing. Interestingly, on the F42H1O.3 cosmid the gene encoding YLZ3 lies next to the gene encoding YLZ4 which contained a LiM domain strongly homolog with that of Lasp-1 (Fig. 15(A)). This may reflect modular evolution processes leading to join in the same protein functional domains separated in proteins from primitive organisms. In conclusion, Lasp-1 carries a LIM domain and a SH3 domain. These domains are involved in protein/protein interactions occurring in different cellular processes including development, transcription, transformation and cell signaling. L M domains have been shown to be associated with two distinct functional domains, the homeo and kinase domains. SH3 domains are often found in association with SH2, pleckstrin homology (PH) and kinase domains. A link between LIM and SH3 domains was found by the interaction of the cytosquelettal protein paxillin (LIM only protein) with SH2 and SH3 domains of vinculin and the focal adhesion kinase (ppl25 f,k ). To date Lasp-1 is the first protein containing both domains and could represent the first member of a new protein family of adaptor molecules involved in cell signaling. The ubiquitous expression of Lasp-1 in human adult tissues suggests a basic cellular function for this protein, moreover its overexpression though genetic amplification in 10 to 15% of human breast cancer suggests that Lasp-1 could be implicated in carcinogenesis or tumor progression. Example 4 MLN 64, a Gene Co-Expressed with the c-erbB-2 Oncogene in Malignant Cells and Tissues Introduction In Example 1 above, we describe isolating human MLN 64 cDNA from a metastatic breast cancer cDNA library. This clone was identified through a differential screening performed by using two subtractive probes, respectively representative of metastatic and nonmalignant breast tissues, in order to identify new genes susceptible to be specifically involved in breast cancer. We mapped MLN 64 at the ql2-q21 region of the long arm of chromosome 17 with a maximum in the q21.1 band {see, supra, Example 1). This region already includes two genes known to be involved in breast cancer disease, the oncogene c-erbB-2 (Slamon, D.J. et al, Science 255:177-182 (1987)) in ql2 and the tumor suppressor gene BRCAI (Hall, J.M. et al, Science 250: 1684-1689 (1990); Brown & Solomon, Curr. Opin. Genet. Dev. 4:439-445 (1994), and refs. therein) in q21. oerbB-2 overexpression is correlated with a shorter overall and disease free survival for breast cancer patients (Muss, H.B. et al, N. Engl. J. Med 500:1260-1266 (1994), and refs. therein). Moreover, c-er_>B-2 overexpression has been shown to be dependent of gene amplification during carcinogenesis (van de Vijver, M. et al, Mol. Cell Biol. 7:201-223 (1987)). We established in Example 1 that the MLN 64 gene was co-amplified with the c-erbB-2 gene in SKBR3 and BT474 breast cancer cell lines. It is assumed that DNA amplification plays a crucial role in tumor progression by allowing cancer cells to upregulate numerous genes (Lonn, U. etal, Intl. J. Cancer 55:40-45 (1994); Kallioniemi, A. etal, Proc. Natl. Acad Sci. USA 97:2156-2160 (1994)), and notably oncogenes. Frequency of gene amplification as well as gene copy number increase during breast cancer progression, notably in patients who do not respond to treatment, suggesting that overexpression of the amplified target genes confers a selective advantage to malignant cells (Schwab, M. & Amler, L., Genes. Chrom. Cancer 7: 181-193 (1990); Lonn, U. et al, Intl. J. Cancer 58:40-45 (1994); Guan, X.Y. et al, Nat. Genet. 5:155-161 (1994)). BRCAI is responsive for about half of the inherited forms of breast carcinomas, suggesting that other tumor suppressor gene(s) could be implicated (Milci, Y. et al, Science 266:66-71 (1994)). BRCAI has been shown to exhibit various possible disease-causing alterations including frameshifts and nonsense mutations (Castilla et al, Nat. Genet. 5:387-391 (1994); Friedman et al, Nat. Genet. 5:399-404 (1994); Simard etal, Nat. Genet. 5:392-398 (1994)). Finally, in sporadic primary breast carcinomas, various sites of DNA mutation, deletion or amplification have been reported in the ql2-q21 region of the chromosome 17 (Kirchweger et al, Intl. J. Cancer 56: 13-19 (1994); Futreal et al, Science 266:120-122 (1994); Guan, X.Y. et al, Nat. Genet. 5:155-161 (1994)). In this context, MLN 64, which is located in ql2-q21 region of the chromosome 17 and amplified and overexpressed in breast cancer cell lines, may be involved in molecular processes leading to breast cancer development and/or progression. In the present Example, we characterized the MLN 64 cDNA, protein and gene organization, and investigated the MLN 64 gene expression in a panel of normal and malignant human tissues. Materials and Methods Tissue and Cell Line Collections Depending on subsequent analysis, tissues were either immediately frozen in liquid nitrogen (RNA extraction), or fixed in formaldehyde and paraffin embedded {in situ hybridization and immunohistology). Frozen tissues were stored at -80°C whereas paraffin-embedded tissues were stored at 4°C The mean age of the 39 patients included in the present study was 55 years. The main characteristics of the breast carcinomas were as followed: SBR grade I (13%), grade H (38%), grade m (49%); estradiol receptor positive (25%), negative (75%); lymph nodes without invasion (39%), with invasion (61%). RNA Isolation and Analysis Total RNA prepared by a single-step method using guanidinium isothiocyanate (Chomczynski, P. & Sacchi, N., Anal. Biochem. 762:156-159 (1987)) was fractionated by agarose gel electrophoresis (1%) in the presence of formaldehyde. After transfer, RNA was immobilized by heating (12 hrs, 80 °C). Filters (Hybond N; Amersham Corp.) were acidified (10 min, 5% CH 3 COOH) and stained (10 min, 0.004% methylene blue, 0.5M CH 3 COONa, pH 5.0) prior to hybridization. The MLN 64 probe described in Example 1 corresponding to the fiill- length human cDNA (nucleotides 1-2008), cloned into pBluescript II SK-vector (Stratagene) was 32 P-labeled using random priming (-10 6 cpm/ng DNA) (Feinberg, A.P. & Vogelstein, B., Anal. Biochem. 137:266-267 (1984)). Filters were prehybridized for 2 hrs at 42°C in 50% formamide, 5x SSC, 0.1% SDS, 0.5% PVP, 0.5% Ficoll, 50 mM sodium pyrophosphate, 1% glycine, 500 μg/ml of ssDNA. Hybridization was for 18 hrs under stringent conditions (50% formamide, 5x SSC, 0.1% SDS, 0.1% PVP, 0.1% Ficoll, 20 mM sodium pyrophosphate, 10% dextran sulfate, 100 μg/ml ssDNA; 42 °C). Filters were washed 30 min in 2x SSC, 0.1% SDS at room temperature, followed by 30 min in 0.1% SSC, 0.1% SDS at 55 °C After dehybridization, filters were rehybridized with a c-erbB-2 specific probe. The 36B4 probe (Masiakowski, P. et al, Nucleic Acids Res. 70:7895-7903 (1982)) was used as positive internal control. Autoradiography was for 2 days for hybridizations of MLN 64 and c-erbB-2 whereas 36B4 hybridization was exposed for 16 hrs. Genomic DNA Isolation and Analysis Genomic DNAs (10 mg) from human leucocytes and from monkey, pig, rabbit, rat, hamster, mouse, chicken, fly and worm were digested with EcoRl or Taql, fractionated by agarose gel electrophoresis (0.8%), and transferred to nylon membranes (Hybond N + , Amersham Corp.). The hybridization conditions for Southern blots were identical to those previously described for Northern blots. Preparation of Monoclonal Antibodies and Immunohistochemistry The synthetic peptide PC94 corresponding to 16 AA (amino acid(s)) located in the C-terminal part of the putative MLN64 protein (FIG. 16) was synthesized in solid phase using Fmoc chemistry (Model 431 A peptide synthesizer, Applied Biosystems, Inc., Foster City, CA), verified by amino acid analysis (Model 420A-920A-130A analyzer system; Applied Biosystems, Inc.) and coupled to ovalbumin (Sigma Chemical Co., St. Louis, MO) through an additional NH2- extraterminal cysteine residue, using the bifiinctional reagent MBS (Aldrich Chemical Co., Milwaukee, WI). Two 8-weeks-old female BALB/c mice were injected intraperitoneally with 100 μg of coupled antigen every two weeks until obtention of positive antisera. Four days before the fusion, the mice received a booster injection of antigen (100 μg), and then 10 μg intravenous and 10 μg intraperitoneal route every day until spleen removal. The spleen cells were fused with Sp2/0-Agl4 myeloma cells according to St. Groth & Scheidegger, J. Immunol. Meth. 55: 1-21 (1980). Culture supematants were screened by ELISA using the unconjugated peptide as antigen. Positive culture media were then tested by immunocytofluorescence and Western blot analysis on MLN64 cDNA transfected COS-1 cells. Five hybridomas, found to secrete antibodies specifically recognizing MLN 64, were cloned twice on soft agar. They all corresponded to IgGl, k subclass of immunoglobulins (Isotyping kit, Amersham Corp.). Immunohistochemical analysis was performed as previously described (Rio, M.C et al, Proc. Natl. Acad Sci. USA 54:9243-9247 (1987)) using paraffin-embedded tissue sections. Hybridoma supernatant was diluted 2-fold and a peroxidase-antiperoxidase system (DAKO, Carpinteria, CA) was used for the revelation. In Situ Hybridization In situ hybridization was performed using a 35 S-labeled antisense RNA probe (5x10 s cpm μg) specific of the human MLN 64 cDNA. Formaldehyde-fixed paraffin-embedded tissue sections (6 μm thick) were deparaffined in LMR, rehydrated and digested with proteinase K (1 μg/ml; 30 min, 37°C). Hybridization was for 18 hrs, followed by RNase treatment (20 μg/ml; 30 min, 37°C) and stringently washed twice (2x SSC, 50% formamide; 60°C, 2 hrs). Autoradiography was for 2 to 4 weeks using NTB2 emulsion (Kodak). After exposure, the slides were developed and counterstained using toluidine-blue. 35 S- labeled sense transcript from MLN 64 was tested in parallel as a negative control. MLN 64 Genomic DNA Cloning Fifty μg of human genomic DNA was partially digested with Sau3A. After size selection on a 10-30% sucrose gradient, inserts (16-20 kb) were subcloned at the BamHl replacement site in lambda EMBL 301 (Lathe, R. et al, Gene 57: 13-201 (1987)). 2.5xl0 6 recombinant clones were obtained and the library was amplified once. One million pfu were analyzed in duplicate for the presence of genomic MLN 64 DNA, using a 5' and a 3 1 end specific MLN 64 probes. The 5' probe was obtained using amplified DNA fragment (nucleotides 1 to 81) and the 3' probe corresponded to an EcoRl fragment encompassing MLN 64 XYZbp (nucleotides 60 to 2073). Ten and 18 clones gave a positive signal with the 5 1 and 3 1 probe, respectively. After a second screening, 4 clones, hybridizing with the two probes, were subcloned into pBluescript 11 SK- vector (Stratagene), sequenced and positioned with respect to the MLN 64 cDNA sequence. RT-PCR - Sequencing Reactions MLN 64 cDNA clones and genomic subclones prepared as described (Zhou, C et al, Biotechniques 5:172-173 (1990)) were further purified with RNaseA treatment (10 μg ml; 30 min, 37°C) followed by PEG/NaCl precipitation (0.57 vol., 20%, 2 M) and ethanol washing. Vacuum dried pellets were resuspended at 200 ng/μl in TE. Double-stranded DNA templates were then sequenced with Taq polymerase, using either pBluescript universal primers and/or internal primers, and dye-labeled ddNTPs for detection on an Applied Biosystems 373 A automated sequencer. Computer Analysis Sequence analyses were performed using the GCG sequence analysis package (Wisconsin Package, version 8, Genetic Computer Group). The MLN 64 cDNA sequence and its deduced protein were used to search the complete combined GenBank/EMBL databases and the complete SwissProt database respectively, with BLAST (Altschul, S.F. et al, J. Mol Biol. 275:403-410 (1990)) and FastA (Pearson, W.R. 8c Lipman, D.J., Proc. Natl. Acad Sci. USA 55:2444-2448 (1988)) programs. Results Determination of Human MLN 64 cDNA and Putative Protein Sequences The complete MLN 64 cDNA sequence has been established from six independent cDNAs, coming from a tissular cDNA library constructed using human metastatic axillary lymph nodes (Example 1). For each clone, both sense and antisense strands have been sequenced. The full-length MLN 64 cDNA contained 2073 bp (Fig. 16) (SEQ DD NO:5). The first ATG codon (nucleotides 169-171) had the most favorable context for initiation of translation (Kozak, M., Nucl. Acids Res. 75:8125-8148 (1987)), and an AATTAAA ρoly(A) addition signal sequence (nucleotides 2050-2056 of SEQ ED NO:5) (Wahle, E. & Keller, W., Annu. Rev. Biochem. 67:41-40 (1992)) was located 24 bp upstream of the poly(A) stretch. Thus, the open reading frame encodes a 445 amino acid (AA) protein (Fig. 16) (SEQ ID NO:6), with a molecular weight of 50 KD and a pHi of 8.2. DNA database searches reveal homology with various human expressed sequence tags (ESTs) identified in libraries established using either adult (heart), postnatal (brain) or embryo (placenta, liver, spleen and brain). Moreover, 75% homology was observed with the cDNA sequence (606 bp) of the clone plO.15, recently identified through differential screening of a rat osteosarcoma cell line cDNA library (Waye & Li, J. Cell Biochem. 54:273-280 (1994)), suggesting that MLN 64 could correspond to the human homolog of the rat pi 0.15. Surprisingly, protein alignment revealed that the homology between the two putative proteins was restricted to the last 21 C-terminal AA of MLN 64 which were identical to 21 AA located at the core of the plO.15 protein (Waye & Li, J. Cell Biochem. 54:273-280 (1994)). A careful examination of both putative proteins has been performed and showed that they result from different open reading frames including only 21 codons in common (Waye & Li, J. Cell Biochem. 54:273-280 (1994)). MLN 64 exhibited 29% identity and 55% similarity with the Caenorhabditis elegans U 12964 putative protein of unknown function (Waterston R-, direct submission). The putative MLN 64 protein analysis showed potential sites (reviewed in, Kemp, B.E. & Pearson, R.B., Trends Biochem. Sci. 15:342-346 (1990)) specific of N-glycosylation (NESD, residues 219-222; NKTV, residues 311-314; both of Fig. 16, SEQ ID NO:6), phosphorylation by casein kinase II (SFFD, residues 94-97; SPPE, residues 209- 212; SDNE, residues 217-220; SDEE, residues 221-224; SAQE, residues 232- 235; SPRD, residues 343-346; TMFE, residues 426429; all of Fig. 16, SEQ ED NO:6), protein kinase C (SPR, residues 343-345; SAK, residues 370-372; THK, residues 375-377; all of Fig. 16, SEQ DD NO:6), amidation (AGKK, residues 226- 229; Fig. 6, SEQ ID NO:6). Moreover, structural analysis revealed two potential transmembrane domains (residues 1-72 and 94-168 of Fig. 16, SEQ ID NO:6). MLN 64 amino acid composition showed 11.5% of aromatic residues (Phe, Tip and Tyr) and 26% of aliphatic residues (Leu, Be, Val and Met). A careful examination of spacing of these aliphatic residues has been performed in order to detect a possible ordonnance of them. The Leu residues are principally distributed in the 200 N-terminal AA (37 Leu), between AA285 and AA328 (7Leu/43AA) and AA406 and AA441 (7Leu/35AA). No consensus leucine zipper (reviewed in, Busch & Sassone-Corsi, Trends Genet. 6:36-40 (1990)) nor leucine-rich repeats (Kobe & Deisenhofer, Trends Biochem. Sci. 77:415-421 (1994)) could be drawn. MLN 64 variants The tissular cDNA library was constructed using metastatic axillary lymph nodes coming from four distinct patients. Six independent MLN 64 cDNAs have been cloned from this library and sequenced. We observed a high degree of variability between their sequences. Thus, we observed two substitutions, of a C to T (nucleotide 262) and A to G (nucleotide 518), changing Leu to Phe (AA32) and Gin to Arg (AA117), respectively (Table VI, variants A and B). Another cDNA presented a 99 bp deletion (nucleotides 716-814) leading to the deletion of 33 AA (AA184-AA216) and to a 412 AA putative protein (Table VI, variant C). Finally, one clone exhibited a 51 bp insertion (between nucleotides 963-964) generating a stop codon 48 bp downstream of the insertion site and giving rise to a 281 AA chimeric C-terminal truncated protein containing 16 aberrant AAs at its C-terminal part (Table VI, variant D). These results showed that, at least 4 modifications occur in the MLN 64 open reading frame. Since genes exhibiting genetic and epigenetic DNA alterations leading to protein modifications and presumably to loss of function could play a role in transformation and/or cancer progression (Joensen et al. , Amer. J. Pathol 745: 867-874 ( 1993); Katagiri et al. , Cytogenet. Cell Genet. 68:39-44 (1995)) and in order to avoid the possibility that the observed variations result from cDNA library artifacts, we decided to redone MLN 64 cDNAs from a second library established using SKBR3 breast cancer cell line (unpublished data). Twenty-five new MLN 64 cDNAs were cloned and MLN 64 specific primers were designed in order to identify, using PCR, the presence of insertion/deletion variants identical to those previously isolated from the tissular library. Among the 25 clones, 6 showed modified sizes consistent with already identified deletion/insertion events whereas the 19 remaining clones showed a size identical to that of the wild type MLN 64 cDNA (data not shown). Sequence analyses of the 6 variant clones showed that they all contained a C at nucleotide 262 position and an A to G substitution at nucleotide 518 position (Table VI, variant B), suggesting that single nucleotide variations observed in the MLN 64 clones isolated from the tissular library could correspond to individual polymoφhism since the library was established using tissues from 4 patients. Four clones presented a 99 bp deletion (nucleotides 716-814), a modification previously observed in cDNAs cloned from the metastatic library (Table VI, variant C). In addition to the 99 bp deletion, one clone exhibited a 13 bp deletion (nucleotides 531-543) generating a frameshift and giving rise to a 247 AA chimeric C-terminal truncated protein containing the 121 N-terminal AAs of MLN 64 and 126 aberrant AAs at the C-terminal part (Table VI, variant F). A 657 bp insertion (between nucleotides 963 and 964) was observed in another clone which results in a 285 AA C-truncated protein (Table VI, variant E). The remaining clone showed three modifications, a 137 bp deletion (nucleotides 115-251) leading to the loss of the initiating ATG codon, the already described 13 bp deletion (nucleotides 531-543) and a 199 bp insertion (downstream nucleotide 715). Since the first potential ATG codon is located at nucleotides 1087 to 1089, this clone could possibly encode a N-terminal truncated protein containing the 138 C-terminal AA of the MLN 64 (Table VI, variant G). Thus, in addition to the variants previously observed in the tissular cDNA library, we observed 3 novel MLN 64 variants in the cellular cDNA hbrary. All studied clones presented a polyA+ excluding the possibility that insertions could correspond to unspliced pre-messenger RNAs. The identification of 2 identical variants (Table VI, variants B and C) isolated from the 2 distinct libraries, showed that they are not due to cDNA library artefacts but to cDNA modifications specific of the MLN 64 gene. The putative nonsense protein sequences present in variants D, E and F showed no homology with already known protein sequences contained in databases. In order to determine if these variants were specific of malignancy and since MLN 64 was expressed in placenta {see, infra), we used a human cDNA placenta library (J.M. Gamier, unpublished data) to search for variants using the same PCR protocol as for the previously described SKBR3 library screening. Nine independent clones have been identified and checked for alternative splicing events. The incidence of variants was lower than in transformed tissues since only one variant corresponding to the insertion of 199 bp, already identified in malignant tissue, was found. MLN 64 Gene Organization A human leukocyte genomic library was screened using two probes corresponding to nucleotides 1-81 (Fig. 16; SEQ ID NO:5) obtained by PCR amplification and to the almost full-length MLN 64 cDNA (nucleotides 60-2073), respectively {see Materials and Methods). One hundred and six clones were hybridized, leading to the obtention of positive signal with one of the two probes. No clones showed simultaneous hybridization with both probes. Four clones hybridized with the smallest probe. They all contained a 6 kb insert which was sequenced using internal primers in order to determine the exon/intron boundaries. Four other clones hybridized to the longest probe. BamH digestion of the inserts gave two fragments (3.5 and 6 kb) which were subcloned and sequenced using various primers in order to map splicing sites. The sizes of the introns were estimated by sequencing or PCR amplification of genomic subclones using primers located within the cDNA and at exon boundaries. The human MLN 64 gene whose total length was approximately 20 kb, was found to be split into 15 exons (Fig. 17 and Table VII (exon/intron Nos. 1-14 corresponding to SEQ DD NOS:58- 71)). Exon 1 and part of exons 2 and 15 contain 5' and 3' untranslated regions of the MLN 64 gene. Translated cDNA sequence starts at nucleotide 55 of exon 2. Intron/exon boundaries analysis showed that the 5' splice donor sequences related to exons 2 (SEQ ID NO:59), 3 (SEQ ID NO:60), 4 (SEQ ED NO:61), 6 (SEQ ID NO:63), 9 (SEQ ID NO:66) and 13 (SEQ DD NO:70), and the 3' splice acceptor sequences related to exons 2 (SEQ DD NO:59), 3 (SEQ DD NO:60), 6 (SEQ DD NO:63), 11 (SEQ DD NO:68) and 12 (SEQ DD NO:69) did not correspond to the canonical splice consensus sequence (Breathnach, R. & Chambon, P., Annu. Rev. Biochem. 50:349-383 (1981)) (Table Vπ). The cDNA modifications leading to the protein variants were all distributed from exon 2 to intron 9. Single nucleotide substitutions were observed in exon 2 and 4 (Fig. 17, a and c). The 137 bp and 13 bp deletions occurred at the 5' end of the exon 2 (Fig. 17, b) and at the 3' end of the exon 4 (Fig. 17, d), respectively. The 99 bp deletion concerned the entire exon 7 (Fig. 17, f). The 199 bp insertion corresponded to the 5' end of the intron 6 (Fig. 17, e), and the 51 bp or 657 bp insertions to the 5' end or to the entire intron 9 (Fig. 17, g and h). Thus, the deletion/insertion events occurred at the boundaries of intron I/exon 2 (SEQ DD NO:58/SEQ DD NO:59), exon 4/intron 4 (SEQ ID NO:61, exon 6/intron 6 (SEQ ED NO:63), intron 6/exon 7 (SEQ ID NO:63/SEQ ID NO:64) and exon 9/intron 9 (SEQ ED NO: 66), presumably due to the low degree of conservation of these splicing sites (Table VII). Moreover, we looked for the conservation of MLN 64 gene, using a zooblot containing either EcoRl or BamHl digested genomic DNAs from worms, fly, hamster, mouse, rat, pig and human. MLN 64 cDNA hybridization gave faint and strong signals with invertebrates and vertebrates, respectively (data not shown), indicating that MLN 64 is well conserved throughout evolution suggesting an important function for this protein. MLN 64 is Overexpressed in Human Malignant Tissues Northern blot hybridization with the MLN 64 cDNA probe {see Materials and Methods) gave a positive signal corresponding to MLN 64 transcripts with an apparent molecular weight of 2 kb (Fig. 18, lanes 11, 12, 17, 18 and data not shown). Moreover, a longer transcript of 3 kb was also detected in samples which contain the higher amount of the 2 kb transcripts (Fig. 18, lanes 7, 17, 18 and data not shown). After longer autoradiography, two additional species of mRNA became visible. Polyadenylated RNA extracted from BT474 cell line exhibited identical pattern of hybridization (data not shown). Using Northern blot analysis, MLN 64 overexpression was observed in malignant tumors of breast (14/93 cases), brain (2/3 cases), lung (2/23 cases) whereas colon (4 cases), intestine (1 case), skin (5 cases), thyroid (2 cases) and head and neck (25 cases) were negative ((Fig. 18, lanes 7, 11, 12, and data not shown). Moreover, metastatic lymph nodes derived from breast (2/6 cases), liver (l_ cases) and head and neck (1/16 cases) cancers expressed MLN 64, whereas those from skin (7 cases), lymphoma (3 cases) and kidney (1 case) cancers were MLN 64 negative (Fig. 18, lanes 17, 18, and data not shown). Three liver metastases derived from breast cancer (1/1 case) and colon cancer (2/7 cases) also expressed the MLN 64 whereas one skin and one epiploon metastases derived from breast and ovary cancer, respectively, did not (data not shown). No MLN 64 transcripts were observed in normal human breast, axillary lymph node, stomach, colon, liver and kidney, whereas faint signal was observed in skin, lung, head and neck epidermoid tissues and placenta (Fig. 18, lanes 15 and 16 and data not shown). Moreover, the breast fibroadenomas (13 cases studied), which are benign tumors, did not show MLN 64 expression above the basal level (Fig. 18, lanes 1-6). Altogether, these results showed that MLN 64 could be overexpressed in the primary tumors or metastases of a wide panel of tissues including breast, colon, liver, lung, brain and head and neck. Nevertheless, the level of MLN 64 overexpression observed in carcinomas of breast origin was 3-5 fold higher than in cancer of other tissues. Since in breast cancer cell lines, the MLN 64 overexpression was always correlated with those of the erbB-2 oncogene, successive hybridizations of the same filters with a c-erbB-2 cDNA probe have been performed. In all MLN 64 positive malignant tissues, we observed an overexpression of the erbB-2 oncogene (Fig. 18, lanes 6, 10, 11, 16 and 17, and data not shown). Thus, as in cell lines, the two genes were co-expressed in vivo. MLN 64 Expression is Restricted to Malignant Epithelial Cells In situ hybridization, using an antisense MLN 64 RNA probe, was performed on primary breast carcinomas and axillary lymph node metastases. MLN 64 was expressed in the malignant epithelial cells, in in situ (Fig. 19) and invasive (Fig. 19) carcinomas, whereas tumor stromal cells were negative. MLN 64 transcripts were homogeneously distributed among the positive areas. Normal epithelial cells did not express the MLN 64 gene, even when located at the proximity of invasive carcinomatous areas (Fig. 19 and data not shown). A similar pattern of MLN 64 gene expression was observed in metastatic axillary lymph nodes from breast cancer patients with expression limited to cancer cells whereas noninvolved lymph node areas were negative (Fig. 19 and data not shown). Using monoclonal antibody directed against a MLN 64 synthetic peptide {see Materials and Methods), breast carcinoma immunohistochemical analysis showed MLN 64 staining restricted to the transformed epithelial cells. Moreover, the MLN 64 protein showed a particular distribution with cytoplasmic condensation sites, suggesting an organite localization for MLN 64 (Fig. 20). Identical pattern was observed using the BT474 breast cancer cell line (Fig. 20). Discussion In the present Example, we characterized the MLN 64 cDNA and its corresponding protein. In Example 1 above, MLN 64 cDNA was identified by differential screening of a breast cancer metastatic lymph node cDNA hbrary. The MLN 64 protein which contains 445 AA, showed two potential transmembrane domains and several potential leucine zipper and leucine-rich repeat structures previously identified in a number of diverse proteins involved in protein-protein interaction and signal transduction (Busch & Sassone-Corsi, Genet. 6:36-40 (1990); Kobe & Deisenhofer, Trends. Biochem. Sci. 77:415-421 (1994)). Although the MLN 64 cDNA presented a high degree of homology with the rat plO.15 cDNA, no homology was observed between the two predicted proteins with the exception of 21 AA (Waye & Li, J. Cell. Biochem. 54:273-280 (1994)). The highest degree of homology was for the Caenorhάbditis elegans U 12964 putative protein of unknown function. MLN 64 gene contains 15 exons and the coding region encompasses from the 3' end of the exon 2 to the 5' end of the exon 15. In Example 1 above, we observed that no obvious rearrangements, insertions or deletions affected the MLN 64 gene in a panel of breast cancer cell lines. In these cell lines, the MLN 64 gene expression was always correlated with MLN 64 gene amplification. In the present Example, in breast cancer cell and/or tissue, we identified and characterized 7 distinct MLN 64 cDNAs, resulting from nucleotide substitutions, deletions and/or insertions. Interestingly, the cDNA modifications principally occurred at exon/intron boundaries, suggesting that the MLN 64 variants result from defective splicing processes. Consistently, almost all the concerned splicing site sequences were defective (Breathnach, R. & Chambon, P., Annu. Rev. Biochem. 50:349-383 (1981)). Two variants lead to AA substitution and 5 variants encode N- or C- truncated MLN 64 proteins. In addition, 3 of them lead to chimeric proteins containing additive nonsense protein sequences of 16, 20 and 126 AA, respectively. Using RT-PCR, 1 MLN 64 mRNA containing the intron 6 sequence has been detected in placenta, showing that, at least in this case, MLN 64 alternative splicing was not a transformation specific event. It remains to be seen, using antibodies directed against appropriate epitopes, if all MLN 64 variant RNAs are effectively translated, specifically in cancerous-tissues and/or naturally occurring. In both physiological and/or pathological conditions, alternative splicing have been reported to occur in transcription of a panel of genes including those coding for the oestradiol receptor (Miksicek, Semin. Cancer Biol 5:369- 379 (1994) and refs. therein), the ubiquitous cell surface glycoprotein CD44 (Arch etal, Science 57:682-685 (1992); Joensen et al, Amer. J. Pathol 745:867-874 (1993)), the metalloprotease/disintegrin-like protein MDC (Katagiri et al, Cytogenet. Cell Genet. 65:39-44 (1995)) and the tumor suppressor p53 (Han & Kulesz-Martin, Nucl. Acids Res. 20:179-181 (1992)). Although the biological significance of these variants was not always well established, their presence in transformed tissues is usually associated with a poor prognosis and a high metastatic potentiality (Miksicek, Semin. Cancer Biol. 5:369-379 (1994). Using Northern blots, we observed two major messenger sizes at 2 kb consistent with the wild type ARNm, and at 3 kb, only observed in the tissues highly expressing the 2 kb mRNA. Human normal skin, lung, head and neck and placenta expressed MLN 64 at a low level, whereas breast, lymph nodes, stomach, colon, liver, kidney and breast fibroadenomas did not. Interestingly, skin, lung and head and neck are all epidermoid tissues, suggesting that MLN 64 protein could play a physiological role in tissues of this origin. MLN 64 was overexpressed in breast, colon, brain, liver, lung, and head and neck primary malignant tumors and/or metastases, the highest level of expression being observed in breast malignant tissues. Thus, MLN 64 which is observed in a wide panel of transformed tissues, should be involved in basic process occurring in carcinogenesis and or tumoral progression. In both breast primary tumor and metastasis, MLN 64 transcripts were homogeneously distributed throughout the carcinomatous areas, whereas normal tissues were negative. Moreover, MLN 64 is expressed in in situ tumors, suggesting that it may be involved in precocious events leading to tumor invasion. Monoclonal antibody, directed against a C-terminally located MLN 64 synthetic peptide, permitted us to localize the MLN 64 protein in vesicle-like structures in the cytoplasm of the malignant epithelial cells. Using Western blot, MLN 64 was found in both BT474 cell and culture medium extracts. Thus, despite the absence of a hydrophobic secretion signal at the N-terminal part of the molecule, the MLN 64 is probably translocated across the endoplasmic reticulum membrane via a nonclassical mechanism. The MLN 64 positive bundles also contain F-actine, suggesting that MLN 64 is related to the cytoskeleton of the transformed cells, possibly to podosomes. Podosomes are close contact cell-adhesive structures regarded as a key structure in invasive processes. We showed in Example 1 that, in breast cancer cell lines, MLN 64 overexpression is correlated with MLN 64 gene amplification and with oncogene erbB-2 amplification suggesting that both genes, which are co-localized in ql2- q21 on the long arm of the chromosome 17, belong to the same amplicon. Consistently, we have now observed, in vivo, a coexpression of the two genes. erbB-2 amplification is one of the most common genetic alteration occurring in breast carcinomas (reviewed in, Devilee & Comelisse Biochim. Biophys. Acta 775:113-130 (1994) and refs. therein) and is associated with a poor prognosis (Slamon, D.J. et al, Science 244:707-712 (1989); Muss, H.B. etal, N. Engl. J. Med. 550:1260-1266 (1994)). It is currently admitted that gene amplification/overexpression confers a preferential growth to the cells and concerned the oncogenes (Schwab, M. & Amler, L., Genes. Chrom. Cancer 7:181-193 (1990); Kallioniemi, A. et al, Proc. Natl. Acad Sci. USA 97:2156- 2160 (1994)), whereas, the variants resulting in dramatic modification of the protein permit a growth of the cells by inactivation of proteins including tumor suppressor genes (Kulesz-Martin et al, Mol. Cell Biol 74:1698-1708 (1994); Katagiri et al, Cytogenet. Cell Genet. 65:39-44 (1995)). In this context, it may be paradoxical that the MLN 64 gene which is amplified showed numerous variant species. What could be the efficiency of amplification if the product of the target amplified gene is defective? Whatever the mechanism(s), since genes showing amplification leading to overexpression or alternative splicing leading to defective proteins (Miksicek, Semin. Cancer Biol. 5:369-379 (1994)) are most often strongly related to cancerous processes, our results suggest that MLN 64 may participate in carcinogenesis and/or tumor progression. Since it has recently been proposed that the oncogenic properties of erbB-2 could be increased by the overexpression of downstream signaling molecules possibly co-localized on the chromosome 17, such as GRB7, it is tempting to speculate that MLN 64 could be involved in the erbB-2 signaling pathway. Table VI: MLN64 Variants in SKBR3 and Metastatic Breast Cancer cDNA Libraries Table VII: Exon/intron Organization of the MLN 64 Gene. Exon Sequences Are Indicated in Capital Letters, and Intron Sequences in Small Letters. EXON INTRON Example 5 Definition of the D52 Gene/Protein Family through Cloning of a D52 Homolog, D53 Introduction The human D52 (hD52) cDNA was cloned through differential screening of a breast carcinoma cDNA library (Byrne, J. A et al. , Cancer Res. 55:2896-2903 (1995)). The hD52 gene is overexpressed in approximately 40% of human breast carcinomas, where it is specifically expressed in the cancer cells. The hD52 locus has been mapped to chromosome 8q21, a region which is frequently amplified in breast carcinoma (Kallioniemi, A etal, Proc. Natl. Acad Sci. USA 97:2156-2160 (1994); Muleris, M. etal, Genes Chrom. Cancer 70: 160-170 (1994)), in cancers of the prostate (Cher, ML. etal, Genes Chrom. Cancer 77:153-162 (1995)) and bladder (Kallioniemi, A. et al, Genes Chrom. Cancer 12: 213-219 (1995)), and in osteosarcoma (Tarkkanen, M. et al, Cancer Res. 55:1334-1338 (1995)). Accordingly, we noted hD52 gene amplification in the breast carcinoma cell line SK-BR-3 (Byrne, J.A et al, Cancer Res. 55:2896-2903 (1995)), which has been previously reported to harbor a chromosome 8q21 amplification (Kallioniemi, A. et al, Proc. Natl. Acad Sci. USA 97:2156-2160 (1994)). The predicted hD52 amino acid sequence is highly novel, possessing very little homology with sequences thus far reported (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). Using the differential display technique (Liang, P. & Pardee, A.B., Science 257:967-971 (1992)), a hD52 cDNA (known as N8) was also recently cloned through its differential expression between normal and tumorous lung- derived cell lines. Comparing the hD52 protein sequence with translated nucleotide sequences in genetic databases identified several expressed sequence tag (EST) sequences which when translated, showed 48 to 67% identity with 24 to 40 amino acid regions of the hD52 sequence. These sequences derived from human cDNA clones isolated from adult liver and fetal liver/spleen cDNA libraries by the Washington University-Merck EST project. Two such cDNA clones were provided by the IMAGE consortium at the Lawrence Livermore National Laboratory (Livermore, California), and the insert of one was used to screen a breast carcinoma cDNA library. This allowed us to isolate a 1347 bp cDNA whose coding sequence predicts a 204 amino acid protein which is 52% identical to hD52. On the basis of this homology and similarities existing between putative domains in the 2 proteins, we have called this novel gene D53, and propose that this represents a second member of the D52 gene/protein family. Materials and Methods cDNA Library Screening Two cDNAs (clones 83289 and 116783, corresponding to GenBank Accession Nos. T68402 and T89899, respectively) were gifts from the IMAGE consortium at the Lawrence Livermore National Laboratory (Livermore, California). The random-primed 32 P-labeled insert of clone 116783 was used to screen 500,000 plaque forming units pfus) from a breast carcinoma cDNA library (Byrne, J.A etal, Cancer Res. 55:2896-2903 (1995)) which had been transferred to duplicate nylon filters (Hybond N, Amersham Coφ.). Screening was performed basically as previously described (Basset, P. et al, Nature 348:699-704 (1990)), with identified λZAP II clones being replated at densities allowing the isolation of pure plaques, and submitted to secondary screening. Clone inserts were rescued in the form of pBluescript SK- plasmids using the in vivo excision system, according to the manufacturer's instructions (Stratagene). For the isolation of mD52 cDNAs, a CDNA library was used which was constructed by C. Tomasetto (IGBMC, Illkirch, France) using polyA + RNA isolated from apoptotic mouse mammary gland. OligodT-primed cDNAs were ligated with the ZAP-cDNA linker-adaptor, and cloned into the Uni-ZAp™ XR vector according to the manufacturer's protocol (Stratagene). A total of 850,000 pfus were screened using an EcoRI restriction fragment from the hD52 cDNA (containing 91 bp of S'-UTR and 491 bp of coding sequence (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)) at reduced stringency, with final filter washes being performed using 2x SSC and 0.1% SDS at room temperature for 30 min. A single clone (FI) was identified. After purification and insert rescue using in vivo excision, the 3 ^-labeled FI insert was used to rescreen the same cDNA hbrary filters using the same conditions, in order to identify a full-length cDNA (clone Cl). DNA Sequencing Mini-preparations of plasmid DNA which had been further purified by NaCl and polyethylene glycol 6000 precipitation were sequenced with Taq polymerase and either T3 and/or T7 universal primers, or internal primers, and dye-labeled ddNTPs for detection on an Applied Biosystems 373 A automated sequencer. Sequence Analyses Nucleic acid and amino acid sequence analyses were performed using the following programs available in the GCG sequence analysis package: BLAST and FastA, for sequence homology searches; gap, for further sequence alignments; Isoelectric, for the calculation of pl values; Motifs, for the identification of recognized protein motifs; and Pepcoil, for the identification of coiled-coil domains, according to the algorithm of Lupas, A. et al, Science 252: 1162-1164 (1991). PEST sequences were assigned using the PEST-FIND algorithm (Rogers, S. et al, Science 254:364-368 (1986)), which was a gift from Dr. Martin Rechsteiner, University of Utah, USA. Other predictions of secondary structure were performed using the MSEQ (Black, S.D. & Glorioso, J.C, BioTech. 4:448- 460 (1986)), PHD (Rost, B. & Sander, C, Proteins 79:55-72 (1994)) and PSA (Stultz, CM. et al, Prot. Sci. 2:305-314 (1993)) software. Chromosomal Localization Chromosomal localization of the hD53 gene was performed using chromosome preparations obtained from phytohemagglutinin stimulated lymphocytes. Cells were cultured for 72 hrs, with 60 μg/ml 5-bromodeoxyuridine having been added during the final 7 hrs of culture to ensure a posthybridization chromosomal banding of good quality. For the mD52 gene, in situ hybridization experiments were carried out using metaphase spreads from a WMP strain male mouse, in which all autosomes except 19 were in the form of metacentric Robertsonian translocations. The 116783 (hD53) clone containing an insert of 842 bp in a modified pT7T3D plasmid vector (Pharmacia), and the Cl (mD52) clone containing an insert of 2051 bp in pBluescript SK- (Stratagene), were 3 H- labeled using nick-translation to final specific activities of 8x10 7 dpm/μg, and hybridized to metaphase spreads at final concentrations of 200 ng/ml (116783) and 100 ng/ml (Cl) of hybridization solution as described (Mattei, M.G. et al, Human Genet. 69:268-271 (1985)). Autoradiography was performed using NTB2 emulsion (Kodak) for 21 days (116783) and 20 days (Cl) at 4°C To avoid any slippage of silver grains during the banding procedure, chromosome spreads were first stained with buffered Giemsa solution and the metaphases were photographed. R-banding was performed using the fluorochrome-photolysis- Giemsa method and metaphases were rephotographed before analysis. Cell Culture BT-20, BT-474 and MCF7 breast carcinoma cell lines, and the leukemic cell lines HL-60 and K-562 are as described in the American Type Culture Collection catalogue (7th ed.). Cell culture media were for BT-20, MEM supplemented with 10% fetal calf serum (FCS), 2 mM pyruvate, 2 mM glutamine, 10 μg/ml insulin and 1% nonessential amino acids; for BT-474, RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, and 10 μg/ml insulin; for MCF7, DMEM supplemented with 10% FCS, and 0.6 μg/ml insulin; for HL-60, RPMI 1640 supplemented with 10% FCS; and for K-562, RPMI 1640 supplemented with 10% heat-inactivated FCS and 2 mM glutamine. All cells were cultured in the presence of antibiotics (0.1 mg/ml streptomycin, 500 U/ml penicillin and 40 μg/ml gentamycin) at 37°C with 5% CO 2 /95% air in a humidified incubator. For experiments in which breast carcinoma cell lines were cultured in the estradiol supplemented or depleted media, cells were seeded into four 75 cm 2 flasks at low density. These were cultured for one day before normal growth media were replaced (3 flasks) or not (one flask) by phenol red-free DMEM supplemented with 0.6 μg/ml insulin and 10% FCS which had been treated with dextran-coated charcoal to deplete endogenous steroids. Cells were cultured for 2 days in steroid-depleted media before this was supplemented (2 flasks), or not (one flask), with 10 " * M or 10 "9 M estradiol. Cell culture was continued for 3 days, at which point cells were approaching confluency. For experiments in which HL-60 and K-562 cells were induced to differentiate using 12-O-tetradecanoylphorbol- 13 -acetate (TPA), cells were diluted to a density of 2x10 5 cells/ml and 10 ml volumes were seeded into 85 mm diameter culture dishes. At the start of each experiment, one culture dish was immediately harvested for RNA extraction. Media were then supplemented, or not, with 16 nM or 160 nM TPA, and cells were cultured for periods of up to 48 hrs before harvest for RNA extraction. RNA Extraction and Northern Blot Analyses Human surgical specimens were obtained from the Hόpitaux Universitaires de Strasbourg, being frozen and stored in liquid nitrogen. Total RNA was isolated from tissues and cultured cells as previously described (Rasmussen, U.B. et al, CancerRes. 55:4096-4101 (1993)). Northern analyses were performed with 10 μg of total RNA which were electrophoresed through 1.0% denaturing agarose gels and transferred to nylon filters (Hybond N, Amersham Coφ.) using 20x SSC. Northern hybridizations were performed using 3 _ abeled inserts from the 116783 hD53 cDNA and the hD52 cDNA (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). To verify the effectiveness of estrogen treatments in breast carcinoma cell lines, and of TPA treatments in leukemic cell lines, we also rehybridized filters with 3 _ abeled cDNA inserts corresponding to the estrogen- inducible gene pS2 (Rio, M.C. et al, Proc. Natl. Acad Sci. USA 54:9243-9247 (1987)), and the transferrin receptor gene (Kϋhn, L.C et al, Cell 57:95-103 (1984)), in these respective cases. All filters were rehybridized with a 32 P-labeled internal Pstl fragment of the 36B4 cDNA (Masiakowski, P. et al, Nucl. Acids Res. 70:7895-7903 (1982)), representing a ubiquitously expressed gene. Hybridizations and washing steps were performed essentially as described (Basset, P. et al, Nature 545:699-704 (1990)). Results Isolation and Sequencing of the Human D53 cDNA The existence of a hD52 homolog was originally predicted from 3 EST sequences (GenBank Accession Nos. T68402, T89899 and T93647) which when translated, showed 24-40 amino acid regions which were 48-67% identical with regions between amino acids 130-180 of hD52. These ESTs derived from human cDNA clones isolated from adult liver and fetal liver/spleen cDNA libraries by the Washington University-Merck EST project, and 2 of these cDNA clones (clones 83289 and 116783, corresponding to GenBank Accession Nos. T68402 and T89899, respectively) were kindly provided by the IMAGE consortium at the Lawrence Livermore National Laboratory. Sequencing of clones 83289 and 116783 in both directions indicated that they consist of 1626 bp and 842 bp, respectively (Fig. 24(A)). Within their regions of overlap (714 bp), their sequences were identical, except for a deletion of 100 bp in clone 83289 (corresponding to nucleotides 567-666, Fig. 24(B)), and a single T/G polymoφhism at nucleotides 254 and 371 of clones 83289 and 116783, respectively (nucleotide 865, Fig. 24(B)). Clones 83289 and 116783 were found to possess open reading frames extending from their 5'-ends, encoding 60 and 99 amino acids, respectively, and terminating with the same stop codon (Fig. 24(A)). However, because of the sequence deletion present in the 83289 clone, the first 18 amino acids of the 83289 amino acid sequence are frame-shifted with respect to those encoded by the corresponding DNA sequence of the 116783 clone. Thus, the first methionine residue present in the 116783 amino acid sequence (Met 128 , Fig. 24(B), which is present in a moderately favorable context for translation initiation) is no longer in- frame in the 83289 amino acid sequence. For this reason, and also because the lengths of these apparently partial length cDNA clones did not correspond with the observed transcript size of 1.5 kb {see, infra), a breast carcinoma cDNA library was screened with the 116783 clone insert in order to isolate additional clones. The shorter 116783 clone was chosen for screening, because of the presence of an Alu sequence in the extended 832893'-UTR (Fig. 24(A)). Of the 14 positive clones thus identified, 11 remained positive upon secondary screening, and of these, 2 (Ul and SI) possessed additional sequences at their 5' ends with respect to the 116783 sequence. The insert of the longest clone, Ul, was sequenced in both directions. This indicated that the Ul clone possessed 494 additional bp with respect to the 5' extent of clone 116783, and that this sequence included a strong Kozak consensus sequence (nucleotides 175-184; Fig. 24(B); SEQ DD NO:9). Thus the Ul sequence was noted to consist of 180 bp of 5-UTR, a coding sequence of 615 bp and a 3'-UTR of 552 bp, including a 22 bp polyA sequence. The hD52 and Ul coding sequences were found to be well conserved (62% identical) over much of their lengths, but the predicted 5'-UTRs were poorly conserved. It should be noted that as for hD52 (Byrne, J.A. etal, Cancer Res. 55:2896-2903 (1995)), there is no in-frame stop codon present in the Ul 5'UTR sequence. However, if the reading frame is continued in a 5' direction from the proposed hD52 and Ul translation initiation sites, the resulting protein sequences encoded show no homology to each other. This contrasts with the protein sequences encoded after the proposed initiation of translation sites {see, infra), where 60% identity/78% conservation of homology is observed between the first 170 amino acids of hD52 and the corresponding region of Ul. We thus decided to term the novel gene corresponding to the Ul cDNA D53, which is predicted to encode a protein of 204 amino acids (Fig. 24(B); SEQ DD NO: 10) having a molecular mass of 22.5 KD. Isolation and Sequencing of a Mouse D52 cDNA In order to further define the D52 family and the degree to which these sequences may be conserved during evolution, a mouse homolog of the hD52 cDNA was cloned from an apoptotic mouse mammary gland cDNA hbrary. The identity of the initially isolated 735 bp murine FI cDNA (Fig. 25(A)) as a D52 homolog was shown by a high level of homology noted between its incomplete coding sequence and that of hD52 (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). Of four longer cDNAs subsequently identified using the FI cDNA, the longest (Cl, 2051 bp; Fig. 25(B); SEQ ID NO: 11) appeared to contain a full- length, 558 bp coding sequence when compared with that of hD52. The predicted hD52 and mD52 coding sequences are 82% identical, with the latter encoding a protein of 185 amino acids (Fig. 25(B); SEQ ED NO: 12). The remaining 1482 bp of the Cl cDNA represents 3'-UTR sequence, which is approximately 69% identical to the corresponding region of the hD52 3'-UTR (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). This homology ends at the polyadenylation signal, whose sequence and position is conserved in the hD52 sequence, and where its use gives rise to a minor 2.2 kb hD52 transcript (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). The Cl cDNA thus appears to represent a mouse homolog to this minor hD52 transcript, its structure having apparently been conserved between hD52 and mD52 genes. Domain Features Commonly Identified in D52 Protein Family Members The identity of the Ul cDNA as a D52 homolog (termed hD53) was confirmed upon aligning the predicted hD53 amino acid sequence (SEQ ED NO: 10) with those of hD52 (SEQ DD NO:50) and mD52 (SEQ ED NO:12), as shown in Figure 26(A). The 204 amino acids of hD53 are 52% identical/66% conserved with respect to hD52, and human and murine D52 homologs are 86% identical/91% conserved. The hD53, mD52 and hD52 sequences were further examined using a number of sequence analysis programs in order to further evaluate the significance of these homologies. Due to the previous identification of a central region displaying 7-amino acid periodicities of apolar amino acids in hD52 (Byrne, J.A. etal, Cancer Res. 55:2896-2903 (1995)), a program was used which statistically compares query sequences with known coiled-coil domains (Lupas, A. et al, Science 252:1162-1164 (1991)). Coiled-coil domains are amphipathic (α-helical domains characterized by hydrophobic residues at positions a and d of an abcdefg heptad repeat pattern, and frequently also by charged residues at positions e and g (reviewed in, Adamson, J.G. et al, Curr. Opin. Biotechnol. 4:428-437 (1993)). Coiled-coil structures, which represent protein dimerization domains, are formed between 2 coiled-coil domains which adopt a supercoil structure such that their nonpolar faces are continually adjacent, and both hydrophobic and ionic interactions are important for their formation and stability (Adamson, J.G. et al, Curr. Opin. Biotechnol. 4:428-437 (1993)). Putative coiled-coil domains of 40-50 amino acids were identified towards the N- terminus of hD53, mD52 and hD52 sequences, and are predicted to comprise amino acids 22-71 in hD53 (SEQ DD NO:10) and hD52 (SEQ ED NO:51), and amino acids 29-71 in mD52 (SEQ ID NO: 12), as shown in Figure 26(B). It can be noted that not all a and d positions of the heptad repeats in these predicted coiled-coil domains are occupied by hydrophobic residues (Fig. 26(B)). This reflects the fact that certain deviations from the previously mentioned sequence characteristics of coiled-coil domains are not incompatible with the formation of coiled-coil structures (Lupas, A. etal, Science 252:1162-1164 (1991); Adamson, J.G. et al, Curr. Opin. Biotechnol. 4:428-437 (1993)). Visual inspection of these 3 amino acid sequences followed by computated analysis identified a second domain type predicted to be present in each protein, this being the PEST domain (Rogers, S. et al, Science 254:364-368 (1986)). PEST domains are considered to be proteolytic signals, having been identified in proteins known to have short intracellular half-lives (Rechsteiner, M., Semin. Cell Biol. 7:433-440 (1990)). They are enriched in Pro, Glu, Asp, Ser and Thr residues, and are flanked by Lys, Arg or His residues, although in the absence of these, the N- or C-terminus protein end is also a permitted flank (Rogers, S. et al. , Science 254:364-368 (1986)). PEST domains can be objectively found and assessed using an algorithm which assigns a so-called PEST score, giving a measure of the strength of a particular PEST sequence's candidature. We used this algorithm to identify PEST signals, and their sequences and associated PEST scores are listed in Table VD3 (hD52 (AA10-40) (SEQ ID NO:72); mD52 (AA10- 40) (SEQ ED NO:12); hD53 (AA1-37) (SEQ ED NO:10); hD52 (AA152-179) (SEQ ID NO:73); mD52 (AA152-185) (SEQ ED NO: 12); hD53 (AA169-190) (SEQ ED NO: 10)). Almost all putative PEST signals identified have associated PEST scores of greater than zero, which is considered to define a PEST sequence (Rechsteiner, M., Semin. Cell Biol 7:433-440 (1990)), with only the C-terminally located PEST domain of hD53 representing a weaker PEST candidate. A third feature which is common between the 3 sequences is an uneven distribution of charged amino acids within these. All 3 proteins are predominantly acidic, with pis of 4.70, 4.75, and 5.58 for mD52, hD52 and hD53, respectively. However, while approximately the first and last 50 amino acids of each protein exhibits a predominant negative charge (due in part to the presence of PEST domains), the central portion of each protein exhibits an excess of positively charged residues, with the most frequently occurring charged amino acid residue being Lys in all cases (Fig. 26(A)). Finally, mD52, hD52 and hD53 proteins possess sites for similar potential posttranslational modifications, although the frequency and positions of these sites are not identical in the 3 sequences. All 3 proteins may be subject to N-glycosylation, since in both mD52 and hD52, Asn 167 is a potential glycosylation site, with Asn 163 being a second potential site in mD52, whereas Asn a potential site in hD53. A number of potential phosphorylation sites were originally noted in hD52 (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)), and a similar analysis of the potential phosphorylation sites present in mD52 and hD53 reveals that hD53 includes a greater density of potential phosphorylation sites (14 potential sites) than either mD52 or hD52 (8 and 9 potential sites, respectively). Moreover, the distribution of these sites in hD53 differs from the pattern observed in mD52 and hD52, which is largely conserved between these 2 molecules. Of 14 potential phosphorylation sites in hD53, 4 are also found in both mD52 and hD52, and the remainder are distinct to hD53 (Fig. 26(A)). Most interestingly, Tyr 130 of WD53, which is located within a 13 amino acid insertion with respect to the aligned mD52 and hD52 sequences, is predicted to be phosphorylated by tyrosine kinase, whereas no such site exists in either mD52 or hD52. Homologies Between D52 Protein Family Members and Other Amino Acid Sequences In contrast to the degree of homology present between hD53 and h/mD52, the predicted hD53 amino acid sequence (Fig. 24(B); SEQ DD NO: 10) shows relatively little homology with sequences of described proteins, as initially observed for hD52 (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). Homology can be identified between the coiled-coil domain of hD53 and similar domains of other proteins, such as yeast ZEP1 (Sym, M. et al, Cell 72:365-378 (1993)). Lower levels of a ino acid sequence identity are observed between more extensive regions of hD53, and proteins of the cytoskeleton, or other homologous proteins. For example, weak homology (20% identity, 34% conservation) was noted over 172 amino acids of MD53 with moesin from the pig (Lankes, W.T. et al, Biochim. Biophys. Acta 7276:479-482 (1993)), the human (Lankes, W.T. & Furthmayr, H, Proc. Natl. Acad Sci. USA 55:8297-8301 (1991)) and the mouse (Sato, N., J. Cell Sci. 705:131-143 (1992)). Somewhat higher levels of sequence identity (31-36% identity, 45-51% homology) were noted between amino acids 139-177, and histone HI sequences from maize (Razafimahatratra, P. et al, Nucl. Acids Res. 19:1491 (1991)) and wheat (Yang, P. et al, Nucl. Acids Res. 79:5077 (1991)). Recently, we noted a significantly higher degree of homology between h/mD52 and hD53 sequences and that of the putative protein F13E6.1 encoded between nucleotides 5567-6670 of the Caenorhabditis elegans chromosome X cosmid F13E6 (EMBL Accession No. Z68105; Wilson, R. et al, Nature 368:32- 38 (1994)). At 257 amino acids in length, the putative F13E6.1 protein is somewhat longer than D52 and D53, with 42 amino acids (amino acids 121-167) corresponding to predicted exon 4 of the F13E6.1 gene not being present in D52 or D53 sequences. F13E6.1 is most similar to hD52, where aligning the 2 sequences using the programme gap indicates 36.2% identity/45.4% conservation of homology over the 185 amino acids of hD52. The existence of transcripts deriving from this or a similar gene is indicated by EST sequences deriving from cDNA clones from Caenorhabditis elegans (GenBank Accession Nos. D73047, D73326, D76021 and D76362) and the parasitic nematode Strongyloides stercoralis (GenBank Accession No. N21784). In summary, it is possible that a D52 homolog or ancestral gene exists in nematodes. Chromosomal Localizations ofD52 and D 53 Genes Previous gene mapping studies have indicated a single hD52 locus at chromosome 8q21 (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). Thus in the present study we similarly determined the chromosomal localizations for hD52 and mD52, in order to determine whether human gene members of the proposed D52 family are clustered on chromosome 8q, and whether this/these loci may be syntenically conserved in other species. In the 100 metaphase cells examined after in situ hybridization using the hD53 116783 probe, there were 172 silver grains associated with chromosomes, and 57 of these grains (33.1%) were located on chromosome 6. The distribution of grains on this chromosome was not random, 40/57 (70.2%) of these mapping to the q22-q23 region (Fig. 27(A)). These results allow us to map the hD53 locus to the 6q22-q23 bands of the human genome, thus demonstrating that independent loci on separate chromosomes exist for the hD52 and hD53 genes. Using the mD52 Cl probe, 153 silver grains were associated with chromosomes in the 100 metaphase cells examined after in situ hybridization. Forty-one of these grains (26.8%) were located on chromosome 3. The distribution of grains on this chromosome was not random, 35/41 (85.3%) of these mapping to the A1-A2 region (Fig. 27(B)). A secondary hybridization peak was detectable on chromosome 8, since 30 of the total grains were located on this chromosome (19.6%), and the distribution of grains on this chromosome was not random, 23/30 of these mapping to the C band. Thus, we were able to define 2 mD52 loci, on chromosome 3A1-3A2, and chromosome 8C of the mouse genome, a result which was somewhat unexpected given the existence of a single hD52 locus. The mouse chromosome 3A1-3 A2 region has been reported to be syntenic with regions of human chromosome 8q (O'Brien, S.J. et al, Report of the Committee on Comparative Gene Mapping, in HUMAN GENE MAPPING 846 (1993); Lyon, M.F. & Kirby, M.C, Mouse Genome 95:23-66 (1995)), including band 8q22 adjacent to the hD52 gene at 8q21. This suggests that the chromosome 3 Al-3 A2 locus is the major mD52 locus, and corresponds with the distribution of silver grains between the 2 sites, 22.9% of all grains associated with chromosomes being found at chromosome 3A1-A.2, compared with 15.0% associated with chromosome 8C The significance of the dual mouse D52 loci is currently unknown. The chromosome 8C locus may represent a mD52 pseudogene, or another highly mD52-homologous gene. While it is currently not possible to distinguish between these possibilities, it would appear from the existence of a single hD52 locus that either secondary loci do not exist in the human, or that they are co-localized with the primary hD52 locus at human chromosome 8q21. Comparative Expression Patterns ofhD52 and hD53 in Human Breast Tissues and Breast Cancer Cell Lines The expression pattern of hD53 was evaluated in normal adult human tissues, breast carcinomas and fibroadenomas, and a number of cell lines using Northern blot analysis. A single 1.5 kb hD53 transcript was detected in all samples positive for hD53 expression (Fig. 28 and data not shown). Of those normal tissues examined, the hD53 transcript was detected in kidney and very weakly in skin, but not in liver, stomach, colon, kidney or placenta. In breast tumors, the hD53 transcript was detected in 4/9 carcinomas and in 1/3 fibroadenomas, hD53 transcript levels being noted to be similar in these 5 tumors (data not shown). All tissue and tumor samples in which the hD53 transcript was detected also contained detectable levels of hD52 transcripts. However, the hD53 gene appeared to be less widely expressed than hD52 at the level of sensitivity offered by Northern blot analysis, since only a proportion of those tissues expressing hD52 transcripts showed detectable levels of hD53 (data not shown). Initial results from Northern blot analyses of hD53 expression in breast carcinoma cell lines indicated that hD52 transcript levels were higher in estrogen receptor-positive cell lines than in those considered not to express the estrogen receptor (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). Thus, we undertook to examine whether hD52 and/or hD53 transcript levels could be influenced by the presence/absence of estradiol in growth media. Hybridization of hD52 and hD53 probes with RNA samples from human breast carcinoma cell lines indicated that mRNAs corresponding to both genes were detectable in MCF7 and BT-474 cells (which express the estrogen receptor), and in BT-20 cells (which do not express the estrogen receptor) (Fig. 28). However the relative transcript levels for hD52 and hD53 were not identical in these cell lines, hD52 being relatively strongly expressed in BT-474 cells, and relatively weakly expressed in BT-20 cells, whereas the inverse was true for hD53. In MCF7 cells, removal of estrogen from the culture medium coincided with reduced hD53 and hD52 transcript levels, whereas supplementation of the media to estradiol concentrations of 10 '9 /10 "8 M restored control hD52 or hD53 transcript levels (Fig. 28). In the BT-474 cell line, culturing cells for 5 days in steroid-depleted media did not alter hD52 transcript levels, and estradiol supplementation of depleted media to 10 "9 or 10 " * M coincided with decreased hD52 transcript levels. The hD53 transcript levels were altered in BT-474 cells in a different way, in that these decreased in cells cultured in estrogen-depleted media, and were not restored by subsequent estradiol supplementation (Fig. 28). In BT-20 cells, the presence or absence of estradiol resulted in no appreciable changes in hD52 or hD53 transcript levels compared with 36B4 MRNA levels noted in the same samples (Fig. 28). The effectiveness of estradiol deprivation and supplementation was assessed through rehybridizing the same blots with a probe to human pS2, a gene whose transcription is directly controlled by estrogen in MCF7 cells (Brown, A.M.C. etal, Proc. Natl. Acad. Sci. USA 57:6344-6348 (1984)). Levels of pS2 MRNA have been shown to increase for up to 3 days of estradiol treatment, by which time the magnitude of induction is as much as 30-fold (Westley, B. etal, J. Biol. Chem. 259:10030-10035 (1984)). Accordingly, in MCF7 and BT-474 cells, pS2 transcript levels were either low or undetected in steroid depleted media, whereas estradiol treatments resulted in inductions of pS2 gene expression. However, pS2 MRNA was undetected in estrogen receptor-negative BT-20 cells, in agreement with previous findings (May, F.E.B. & Westley, B.R., J. Biol. Chem. 265:12901-12908 (1988)). Reduction in hD52 or hD53 MRNA Levels Upon Induction of Differentiation in Leukemic Cell Lines Initial results from Northern blot analyses had previously indicated that hD52 transcripts were detectable in HL-60 myelocytic leukemia cells, but not in K-562 proerythroblastic leukemia cells (Byrne, J.A. et al, Cancer Res. 55:2896- 2903 (1995)), and we thus decided to examine the expression of hD53 in these same cell lines. In cells cultured under normal conditions {see Materials and Methods), we noted reciprocal patterns of expression for the hD52 and hD53 genes in these cell lines, in that hD52 transcripts were detected in HL-60 cells, but not in K-562 cells, whereas hD53 transcripts were detected in K-562 cells, but not in HL-60 cells (Fig. 29(A) and (B)). The proliferative and differentiation responses of HL-60 cells and K-562 cells to chemical agents such as TPA have been thoroughly characterized (reviewed in, Harris, P. & Ralph, P., J. Leuk. Biol. 57:407-422 (1985); Sutherland, J.A. et al, J. Biol. Resp. Modif. 5:250-262 (1986)), with TPA promoting differentiation along monocyte/macrophage pathway in both cell lines. Culturing cells in the presence of 16 nM or 160 nM TPA resulted in decreased hD52 transcript levels in treated HL-60 cells (Fig. 29(A)), and decreased hD53 transcript levels in treated K-562 cells (Fig. 29(B)), after periods of 18-24 hrs. As a molecular control for the efficacy of TPA treatments, filters were rehybridized with a transferrin receptor cDNA insert (Kuhn, L.C. et al, Cell 57:95-103 (1984)), since reduced transferrin receptor transcript levels have been reported for both HL-60 cells (Ho, P.T.C. et al, Cancer Res. 49:1989-1995 (1989)) and K-562 cells (Schonhom, J.E., J. Biol. Chem. 270:3698-3705 (1995)) after TPA treatment. The kinetics with which decreased transferrin receptor transcript levels were noted in TPA-treated cells (Fig. 29(A) and (B)) are in good agreement with those previously reported (Ho, P.T.C. et al, Cancer Res. 49:1989-1995 (1989); Schonhom, J.E., J. Biol. Chem. 270:3698-3705 (1995)). Interestingly, parallel decreases (with respect to both their magnitudes and kinetics) were observed for transfeπin receptor and hD52 or hD53 transcripts in HL-60 cells (Fig. 29(A)) and K-562 cells (Fig. 29(B)), respectively. Discussion We report the cloning of a novel human cDNA termed hD53, and of the mouse D52 cDNA homolog, due to the clear similarity between these sequences and ΛD52 (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). The high conservation of homology between h/mD52 and hD53 sequences, combined with the low levels of homology existing between these sequences and those of other characterized proteins, lead us to propose the existence of the novel D52 gene/protein family. The fact that mD52 and hD52 sequences are 86% identical/91% conserved, combined with the possible existence of a D52 homolog or ancestral gene in nematodes, suggest basic cellular functions for D52 family proteins, which are as yet unknown. However, the results of sequence analyses and of further experiments presented here have allowed us to form hypotheses regarding their functions. A central hD52 region of approximately 110 amino acids displaying 7-amino acid periodicities of apolar amino acids was previously identified by virtue of low levels of homology with cytoskeletal protein regions (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)). Using the so-called Lupas algorithm (Lupas, A etal, Science 252:1162-1164 (1991)), we have now identified a single coiled- coil domain in hD52, mD52 and hD53 towards the N-terminus of each protein, and which is predicted to end at Leu 71 in all 3 proteins. This coiled-coil domain overlaps with the leucine zipper predicted in hD52/N8 using helical wheel analysis. The presence of a coiled-coil domain in D52 family proteins indicates that specific protein-protein interactions are required for the functions) of these proteins. Similarly, the presence of 2 candidate PEST domains in D52 proteins indicates that their intracellular abundances may be in part controlled by proteolytic mechanisms. Interestingly, the extent of the N-terminally located PEST domain overlaps that of the coiled-coil domain in both D52 and D53 proteins. It could thus be envisaged that interactions via the coiled-coil domain could mask this PEST domain, in accordance with the hypothesis that PEST sequences may act as conditional proteolytic signals in proteins able to form complexes (Rechsteiner, M., Adv. Enzyme Reg. 27:135-151 (1988)). At present, the cellular distribution pattern of hD53 transcripts in tissues is unknown and thus the significance of hD52 and hD53 co-expression in tissues cannot be evaluated. However, the results obtained for hD52 and hD53 expression in breast carcinoma cell lines indicate that the 2 genes may be expressed in the same cell type, with co-expression of hD52 and hD53 transcripts being demonstrated in 3/5 cell lines examined (BT-20, BT-474 and MCF7). In a remaining 2 cell lines (HBL100 and ZR-75-1), only hD52 transcripts were detectable (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995); Byrne, J.A., unpublished results), and thus hD52 may be more frequently or abundantly expressed than hD53 in breast carcinoma cells. Since neither hD52 nor hD53 transcripts were detected in HFL1 fibroblasts (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995); Byrne, J.A., unpublished results), we thus currently hypothesize that hD53, like hD52 (Byrne, J.A. et al, Cancer Res. 55:2896-2903 (1995)), represents an epithelially-derived marker. Estradiol stimulation/deprivation experiments performed in MCF7 cells indicate that the hD52 and hD53 transcript levels normally measured in MCF7 cells cultured with FCS are dependent upon estradiol. At present, the mechanism by which estradiol induces the accumulation of hD52 and hD53 transcripts in MCF7 cells is unknown. It is possible that fluctuations in hD52/hD53 transcript levels may be secondary to the mitogenic effects of estrogen on MCF7 cells, and not directly produced by estradiol per se. However, estradiol stimulation/deprivation experiments performed in a second estrogen receptor- positive breast carcinoma cell line, BT-474, gave different results from those observed in MCF7 cells. The hD52 transcript level present in BT-474 cells cultured with FCS was not estrogen dependent, and indeed supplementing steroid- depleted media with 10 "9 M and 10 " * M estradiol resulted in significantly decreased hD52 transcript levels. Such differing effects in 2 estrogen receptor-positive breast carcinoma cell lines may indicate multiple mechanisms by which the estradiol-estrogen receptor complex may influence hD52 gene expression in breast carcinoma cells, or the existence of different, cell-specific factors in BT-474 and MCF7 cells which cooperate with the receptor complex in this process (Parker, M.G., Curr. Opin. Cell Biol. 5:499-504 (1993); Cavailles, V. et al, Proc. Natl. Acad Sci. USA 97:10009-10013 (1994)). Furthermore, estradiol deprivation/supplementation had different effects on hD52 and hD53 transcript levels in BT-474 cells. Decreased hD53 transcript levels were observed in cells cultured for 5 days in steroid-depleted media, whether or not this media had been subsequently supplemented with estradiol for the last 3 days of culture. We inteφret these results as indicating that the absence of factor(s) in the steroid- depleted media resulted in decreased hD53 transcript levels, and that in this case the factor was not estradiol. While hD52 and hD53 were found to be co-expressed in 3/5 breast carcinoma cell lines, corresponding findings in leukemic cells confirm that co- expression of these genes is not obligatory. HL-60 cells are myelocytic leukemia cells, and can be induced to differentiate along granulocytic or macrophage pathways (Harris, P. & Ralph, P., J. Leuk. Biol. 57:407-422 (1985)), whereas K- 562 leukemia cells have erythroid characteristics, and can be induced to express features characteristic of granulocytic, macrophagic and megakaryocytic differentiation (Sutherland, J.A. et al, J. Biol. Resp. Modif. 5:250-262 (1986)). The present study has provided another molecular distinction between these 2 cell lines, since hD52 transcripts were detected in HL-60 cells but not in K-562 cells, whereas hD53 transcripts were detected in K-562 cells but not in HL-60 cells. This suggests that hD52/hD53 gene expression status may find future use as a marker to distinguish between different forms of leukemia. Treatment of HL-60 and K-562 cells with TPA was found to have similar effects in reducing hD52 and hD53 transcript levels, respectively. This provides a second example of similar regulation of gene expression for these 2 different genes, this time in 2 different cell lines, and could be considered further proof of a functional relationship between the hD52 and hD53 genes. The mechanism by which hD52 and hD53 transcript levels are reduced in HL-60 and K-562 cells by TPA treatment is currently unknown. It is possible that reduced hD52 or hD53 transcript levels arise as an indirect consequence of TPA treatment, which is known to result in a marked cessation of proliferation, and an induction of macrophagic differentiation in both HL-60 and K-562 cells. However, the fact that hD52/hD53 and transferrin receptor transcript levels decreased in parallel fashions in TPA-treated cells indicates that a common stimulus might be responsible for these events. In summary, we have demonstrated the existence of a new gene/protein family, the D52 family, which is presently comprised of D52 and D53. The presence of an acidic coiled-coil domain in both D52 and D53 proteins indicates that specific protein-protein interactions may form an important component of D52 and D53 function. This, combined with the fact that hD52 and hD53 transcripts are coexpressed in some human cell lines, leads us to speculate that hD52 and hD53 may be able to interact in vivo. However, our observations in HL-60 and K-562 cell lines, where the 2 genes were not co-expressed judging from Northern blot data, indicate that if indeed hD52 and MD53 are cellular partners, that this partnership is not obligatory. Other partners may exist for each of these proteins, and it is tempting to speculate that under certain conditions, the formation of homodimers may be favored. TABLE Vm Candidate PEST Domains Identified in hD52, mD52 and hD53 Amino Acid Sequences Sequence Amino PEST domain sequence PEST acids score ETDPVPEEGEDVAATTSATETLSEEEQEE E' 15.8 KTEP VAEEGEDA VTMLSAPEALTEEEQEE E 11.8 MEAQAQGLLETEPLQGTDEDAVASADFSSMLSEEEK 5.8 KPAGGDFGEVLNSAANASATTTEPLPEK 0.6 _g » AGGDFGEV NSTANAT_rπ_rrTEPPPEQ TESP* 9.0 t VGGTNPNGGSFEEVLSSTAH -6-0 Tositively charged amino acids and protein termini are underlined, whereas PEDS residues are shown in bold. Amino acid residues are indicated using the one letter code. Example 6 Two Distinct Amplified Regions Involved at 17qll-q21 in Human Primary Breast Cancer Introduction Gene amplification has been shown to play an important part in the pathogenesis and prognosis of various solid tumors including breast cancer, probably because overexpression of the amplified target gene confers a selective advantage. The first technique to detect gene amplification was cytogenetic analysis. Thus amplification of several chromosomal regions, visualized as either extrachromosomal double minutes (dmin) or integrated homogeneously staining regions (hsrs) are among the major visible cytogenetic abnormalities found in breast tumors (Gebhart, E. et al, Breast Cancer Res. Treat. 5:125-138 (1986); Dutrillaux, B. et al, Cytogenet. 49:203-217 (1990)). Other techniques such as comparative genomic hybridization (CGH) and a novel strategy based upon chromosome microdissection and fluorescence in situ hybridization have also been applied to broad searches for regions of increased DNA copy number in tumor cells (Guan, X.Y. etal, Nat. Genet. 5:155-161 (1994); Muleris, M. et al, Genes Chrom. Cancer 70:160-170 (1994)). These different techniques have revealed some 20 amplified chromosomal regions in breast tumors. These amplified regions results in 5- to 100-fold amplification of a small number of genes, few of which are thought to contribute in a dominant manner to the malignant phenotype. Positional cloning efforts begin to identify the critical gene(s) in each amplified region. To date, genes documented to be amplified in breast cancers include, FGFR1 (8pl2), MYC (8p24), FGFR2 (10q26), CCN 7, GSTP1 and EMSl (llql3), IGFR and FES (15q24-q25), and ERBB2 ( 17q 12-q21) (reviewed in, Brieche, I. & Lidereau, R, Genes Chrom. Cancer 74:227-251 (1995)). DΝA amplification at segment ql l-q21 of chromosome 17 seems one of the most commonly amplified region in human breast carcinomas. FISH, CGH and chromosome microdissection shown a high increase in DNA-sequence copy-number of this region (Kallioniemi, O. et al, Proc. Natl. Acad Sci. USA 59:5321-5325 (1992); Guan, X.Y. et al, Nat. Genet. 5:155-161 (1994); Muleris, M. etal, Genes Chrom. Cancer 70:160- 170 (1994)). Amplification of 17ql2 was originally discovered in breast carcinoma using a probe to the ERBB2 gene (Slamon, D.J. et al, Science 255:177-182 (1987)). Quickly other tumor types followed including cancers of the ovary, stomach and bladder, and less frequently lung and colon carcinomas. Interestingly, the presence of amplification at 17ql2-q21 has been related to be a clinical relevance in breast cancer, where independent studies have shown association with an increased risk of relapse (Slamon, D.J. et al, Science 255:177- 182 (1987); Ravdin, P.M. & Chamness, G.C., Gene 759:19-27 (1995)). To date, only one gene, ERBB2, has been proposed to be responsible for the emergence of this amplicon. The ERBB2 proto-oncogene belongs to the ERBB family, the first identified member of which {ERBB1) encodes the EGF (epidermal growth factor) receptor (Dougall, W.C. et al, Oncogene 9:2109-2123 (1994)). ERBB2 amplification is associated with overexpression of its product. This gene is a good candidate for a role in breast cancer because of its transforming potency (DiFiore, P.P. et al, Science 257:178-182 (1987)) and that transgenic mice carrying the ERBB2 gene show altered mammary cell proliferation and high incidence of mammary adenocarcinomas (Muller, W.J. et al, Cell 54:105-115 (1988)). All these initial reports emphasized a potential role for the ERBB2 proto- oncogene at 17ql2-q21 in human breast carcinomas. However, four novel genes (called MLN 50, 51, 62 and 64) from this chromosomal region have recently been identified by a differential screening of a cDNA library established from breast cancer-derived metastatic axillary lymph nodes (Tomasetto, C. et al, Genomics 28(3)367-376 (1995)). MLN 51 and MLN 64 genes showed little homology with others already described. MLN 62 gene (also known as CARTl or TRAF4) is a novel member of the tumor necrosis factor receptor-associated protein family (Regnier, etal, Journal of Biological Chemistry 270 tV5 :25715-25721 (1995)), while MLN 50 gene (also named Lasp-1) defines a new LIM protein subfamily characterized by the association of LIM motif and a domain of region 3 Src homology (SH3) at the N- and C-terminal parts of the protein, respectively (Tomasetto, C et al, Genomics 28(3): 367-376 (1995)). These four genes have been found amplified and overexpressed in breast cancer cell lines. Therefore, amplification of 17ql l-q21 DNA sequences may be more complex than firstly suspected, and the number and the identity of target gene(s) remain open questions. In the present study we have investigated a large series of primary breast tumors for amplification OΪERBB2 gene and the four novel genes. We report that 25.5% of the breast tumors show amplification of one or more of these genes. Preliminary mapping of the amplicons suggests the involvement of two distinct amplified regions at 17ql l-q21 in human primary breast cancer. Moreover, we suggest three genes (MLN 62, ERBB2 and MLN 64) as likely targets of the amplification event at these two chromosomal regions. Materials and Methods Tumor and Blood Samples Samples were obtained from 98 primary breast tumors surgically removed from patients at the Centre Rene Huguenin (France); none of the patients had undergone radiotherapy or chemotherapy. Immediately following surgery, the tumor samples were placed in liquid nitrogen and stored at -70 °C until extraction of high-molecular-weight DNA and RNA. A blood sample was also taken from each patient. DNA Probes A pMAC117 probe (a 0.8 Kb Accl fragment DNA fragment from a genomic clone of ERBB2) was used to detect ERBB2 (ATCC No. 53408). The four novel clones (MLN 50, 51, 62 and 64) were described in detail in Tomasetto et al. (1995). These five probes were previously positioned and ordered by in situ hybridization (Tomasetto, C etal, Genomics 28(3):367 -376 (1995). For Southem-blot analysis, the control probes used were the human β- globin (Wilson, J.T. et al, Nucl. Acids Res. 5:563-581 (1978)) and the MOS proto-oncogene (ATCC No. 41004). For Northern-blot analysis, the control probe used was a 0.7-kb Pstl fragment of the 36B4 cDNA, as described by Masiakowski, P. etal, Nucl. Acids Res. 70:7895 (1982). DNA Analysis DNA was extracted from tumor tissue and blood leucocytes, according to standard methods (Maniatis, T. et al, MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed., Cold Spring Harbor, NY (1989)). Ten μg of 7αg_ -restricted DNAs were separated by electrophoresis in agarose gel (leucocyte and tumor DNA samples from each patient were run in adjacent lanes), and blotted onto nylon membrane filters (Hybond N + , Amersham Corp.), according to standard techniques. The membrane filters were hybridized with nick-translated 32 P-labeled probes, washed, and autoradiographed at -70 °C for an appropriate period. Determination of DNA Amplification Restriction enzyme-digested tumor DNAs were compared with matching lymphocyte DNA in the same agarose gels. Blots of these gels were first hybridized with ERBB2 and the four MLN probes. Rehybridization of the same blots with the MOS and the β-globin probes provided a control for the amount of DNA transferred onto the nylon membranes. The proto-oncogene and control gene autoradiographs were first scored by visual inspection and then determined by densitometry. Only the signals with an intensity of two copies or more were considered to represent amplification. Amplification level was quantified by serial dilutions of tumor DNA to obtain a Southern hybridization signal similar to that obtained with leucocyte DNA samples. RNA Analysis RNA was extracted from normal and tumoral breast tissue by using the LiCl/urea method (Auffray, C. & Rougeon, F., Eur. J. Biochem. 707:303-314 (1980)). Ten micrograms of RNA was fractionated by electrophoresis on 1.2% agarose gels containing 6% formaldehyde, and analyzed by blot hybridization after transfer onto nylon membrane filters (Hybond N, Amersham Corp.). The same filters were first hybridized with ERBB2 and the four MLN nick-translated 32 P- labeled probes in 50% formamide at 42 °C Membranes were washed under stringent conditions in O.lx SSPE, 0.1% SDS at 50°C and subjected to autoradiography for various periods at -80 °C Membranes were also rehybridized with a 36B4 cDNA probe corresponding to a ubiquitous RNA. The signal obtained was used to check the amount of RNA loaded on the gel in each experiment. The 36B4 signal also showed that the RNA samples were not extensively degraded. Evaluation of RNA Overexpression Relative intensities of the mRNA bands were assessed by visual examination and confirmed by means of densitometry taking the ubiquitous 36B4 bands into account. Increase in expression of at least 2-fold relative normal breast tissues expression were scored as positive. Overexpression was quantified by serial dilution of tumor RNA to obtain a Northern hybridization signal similar to that obtained with normal breast tissue. Results Normal DNA (peripheral blood lymphocytes) and autologous tumor DNA from 98 breast cancer patients were screened on Southern blots for amplification of 5 different genes (ERBB2, MLN 50, 51, 62 and 64) located at 17ql l-q21. Amplification occurred in at least one locus in 25 of the 98 tumors (25.5%). Densitometrical analysis revealed that amplification levels varied not only from case to case but in some tumors also from gene to gene. Amplification ranged from 2- to more than 30-fold. 17qll-q21 Amplicon Maps in Breast Carcinomas The 25 amplified tumors were subdivided into three groups on the basis of pattern and level of amplification: A, tumors with amplification of all genes with similar amplification levels; B, amplification of all genes with varied amplification levels; and C, amplification of some of these genes. Figure 30 shows examples of the most common patterns of genetic changes. Figure 31 summarizes data in the form of amplification maps. The group A (5 cases) corresponds to the existence of a single but large amplicon at 17qll-q21. For these five tumors, amplification levels were always low (2-5x), suggesting polysomies of the entire long arm of chromosome 17. This first group is not of great interest to identify the candidate genes responsible for the emergence of amplicons. The two other groups (groups B and C; 12 and 18 cases, respectively) show that the size and the amplification level varied from tumor to tumor. Tumors T0084, T0284 and Tl 191 had the smallest amplicon involving only MLN 62. With the exception of these three tumors, the amplicons in all the other 17 tumors included ERBB2 and MLN 64. Interestingly, ERBB2 and MLN 64 were always coamplified to similar levels. In 3 cases (T0109, T1273, T1512), these are the only genes amplified at 17qll-q21. In 5 others tumors (T0391, T0183, T0309, T0559 and T0588) the amplicons were discontinuous between MLN 62 and the two loci ERBB2 and MLN 64. In these tumors MLN 50 showed no evidence of amplification. Our finding suggests the existence of two distinct amplified regions at 17ql l-ql2 and 17ql2-q21 in human primary breast cancer, one includes MLN 62 locus and the other ERBB2 and MLN 64 loci, respectively. Expression ofERBB2 and the Four MLN Genes in Breast Carcinomas Whether the amplification of ERBB2 and the four MLN genes contributed to an elevated expression was determined by comparison of RNA expression with DNA amplification. This was performed on a total of 20 tumor samples for which total RNA was available; 10 samples among the 25 tumors amplified in at least one locus and 10 unamplified tumors. Figure 32 shows examples of some overexpressed tumors, evaluated by Northern blot analysis. No gross alteration in the size of the mRNA was detected in any samples. We observed a perfect overlap between RNA overexpression and DNA amplification. Amplified tumors were always overexpressed for amplified genes, and the five genes were never overexpressed in the 10 unamplified tumor DNA specimens. Despite the technical difficulty of obtaining quantitative data from Northern blot analyses, a correlation seems observed between levels of RNA and the degree of DNA amplification. The tumors with high amplified levels showed higher mRNA levels, irrespectively of analyzed genes. Discussion There are various approaches to search genes whose amplification may be responsible for tumorigenesis. Cytogenetic analysis, CGH and chromosome microdissection have allowed the localization of distinct amplified chromosomal regions which might harbor genes contributing to tumorigenesis. Studies using pulsed field electrophoresis have shown that amplicons in human tumor cells usually comprise large regions of genomic DNA which can be up to several megabases in length and contain several genes (Brookes, S. et al, Genes Chrom. Cancer 6.222-231 (1993)). Fine-scale molecular mapping of amplified regions is needed to locate such genes precisely. Thus, coamplification of genes located in a limited chromosomal region have been described in human tumors. Examples include the complex coamplification of multiple genes from 1 lql3 in human breast cancer (Karlseder, J. etal, Genes Chrom. Cancer 9:42-48 (1994)) as well as from 12ql3-ql4 in human malignant gliomas (Reifenberger, G. et al, Cancer Res. 54:4299-4303 (1994)). Several authors observed amplification of the ERBB2 gene from 17ql l- q21 in human breast cancer (Slamon, D.J. etal, Science 255:177-182 (1987); Ah, LU. et al, Oncogene Res. 5:139-146 (1988); Borg, A. etal, Oncogene 6:137- 143 (1991); Paterson, M.C. et al, Cancer Res. 57:556-567 (1991)). As four novel genes from this chromosomal segment have recently been identified and three of them have been found amplified and overexpressed in breast cancer cell lines (Tomasetto, C. et al, Genomics 28(3): 367-376 (1995)), we decided to further characterize the 17q 11 -q21 region in breast cancer biopsies by studying amplification of these four novel genes, in addition to the ERBB2 gene in a large series of tumor DNAs. The aim was to identify the genes within this amphfication, to determine their frequency and their level of amplification, and thereby to more precisely define the actual driver gene(s) in this amplicon(s). Twenty-five (25.5%) of 98 tumors showed at least one of the five genes amplified. Amplification of these five genes is systematically accompanied by mRNA overexpression. However, it is also known that some tumors with single- copy of an oncogene may overexpress the corresponding mRNA. In the present study, we also examined the expression at RNA level of ERBB 2 and the four MLN genes in 10 tumors of the breast, which do not show amplification. We did not observed any unamplified tumor overexpressed for these 5 tested genes. So, it seem that the four MLN genes, like ERBB2 gene, could not be activated by mechanisms other than gene amplification in breast carcinoma such as, for example, alteration of the regulatory sequence of the genes. In the majority of the altered tumors, amplification encompassed not all the tested loci. The two genes most frequently amplified on 17ql l-q21 in our series were ERBB2 and MLN 64 (22.5%) which were systematically coamplified and overexpressed at similar levels. The invariable coamplification of ERBB2 and MLN 64 seen in our study indicates that both genes are likely to be located in close proximity to each other at 17q 12-q21. In consequence, the amplification and consequent overexpression of MLN 64 as well as ERBB2 gene could be of pathogenetic significance for breast neoplastic growth. A third gene, MLN 62, can be regarded as the possible target selected for a second amplicon. This gene is located centromeric to MLN 64 and ERBB2 genes at 17ql l-12. Although MLN 62 gene was less frequently amplified (17.5%) than MLN 64 and ERBB2 genes, it has been found with high levels of amplification in most tumors which showed two distinct amplified regions at 17ql l-q21 and was the only amplified and overexpressed gene in three tumors (T0084, T0284 and T1191). These findings suggest that in some tumors amplification of MLN 62 may provide a selective growth advantage. Even if the amplicons observed in our breast tumor series frequently contained MLN 50 and MLN 51 , the amplification maps suggest that these two genes are not the target genes of the amplification, they were invariably coamplified with MLN 64 and ERBB2 and never showed the highest amplification level in individual tumors. Four other ERBB2 neighboring genes have previously been observed coamplified with ERBB2 in 10-50% OΪERBB2 amplified tumors, including THRA1 (van de Nijver, M. et al, Mol. Cell Biol. 7:2019-2023 (1987)), RARA (Keith, W.Ν. et al, Eur. J. Cancer 29a: 1469-1475 (1993)), GRB-7 (Stein, D. et al, EMBO J. 75:1331-1340 (1994)) and TOP2A (Smith, K. et al, Oncogene 5:933-938 (1993)). These four genes were never amplified alone without ERBB2 amplification. Our data, together with these other results therefore suggest that MLN 50 and MLN 51, as well as THRA1, RARA, GRB-7 and TOP2A, are just incidentally included in some 17ql2-q21 amplicons. To date, little is known about the physiological and pathological functions of MLN 62 and MLN 64. If MLN 64 gene showed little homology with others described, MLN 62/CART1/TRAF4 encodes a protein exhibiting 3 domains also observed in the CD40-binding protein and in the tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), both involved in signal transduction mediated by the TNF receptor family. So, MLN 62/CART1/TRAF4 gene may be involved in TNF-related cytokine signal transduction in breast carcinoma. In conclusion, the present study shows that DNA amplification is frequently observed in two different regions at 17q 11 -q21 in human breast cancer. This suggests that several genes in these two regions are involved in the initiation and/or progression of human breast cancer. Our preliminary mapping of these 17ql l-q21 amplicons in 25 amplified breast tumors shows that they consistently include either MLN 62/CART1/TRAF4 (17ql l-ql2) or MLN 64 and ERBB2 (17ql2-q21). The two new genes are good candidates for a role in breast cancer because, lϊke ERBB2, their amplification leads to their overexpression. The main conclusion drawn from our data is that, although ERBB2 remains a good candidate as one of genes under selection in the 17ql l-q21 amplicons, two novel candidate genes have been identified as driver genes of these amplicons. Thus, the elucidation of the physiological and pathological significance of MLN 62/CART1/TRAF4 and MLN 64 would confirm the involvement of these two genes in breast carcinogenesis. It will be appreciated to those skilled in the art that the invention can be performed within a wide range of equivalent parameters of composition, concentrations, modes of administration, and conditions without departing from the spirit or scope of the invention or any embodiment thereof. The disclosure of all references, patent applications and patents recited herein are hereby incorporated by reference. INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) Form PCr/RO/134 (July 1992) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13_>_s) A. The indications made below relate to the microorganism referred to in the description on page 3 . line 0 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet I I Name of depositary institution AMERICAN TYPE OJLTUKE COLLECTION Address of depositary institution (including postal code and country) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Date of deposit Accession Number 14 June 1996 ATCC 97608 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | | Plasmid pBS-MLN50(Lasp-1) D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the Internationa I Bureau later (specify the general nature of the indications eg., "Accession Number of Deposit") sr For receiving Office use only For International Bureau use only This sheet was received with the international application I I This sheet was received by the International Bureau on: Authorized officer Authorized officer Form PCT/RO/134 (July 1992) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) A. The indications made below relate to the microorganism referred to in the description on page 3 , line 21 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet I I Name of depositary institution AMERICAN TYPE CULTURE COLLECTION Address of depositary institution (including postal code and country) 12301 Parklawn Drive Rockville , Maryland 20852 United States of America Date of deposit Accession Number 14 June 1996 ATCC 97609 C. AD DITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | | Plasmid pBS-MLN64 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications eg., "Accession Number of Deposit") For receiving Office use only For International Bureau use only ~~ This sheet was received with the international application I I This sheet was received by the International Bureau on: Authorized officer c.wunaπ* tnal _ officer D_ ____w____ Authorized PO iπtemβlo fl_ Form PCT/RO/134 (July 1992) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13_ iLsr) Form PCT RO/134 (July 1992)

Claims: What Is Claimed Is: 1. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as shown in Figure 6 (SEQ ID NO:2), Figure 14 (SEQ ID NO:4), Figure 16 (SEQ ID NO:6), Figure 21 (A-D) (SEQ ID NO:8), or Figure 24(B) (SEQ ID NO: 10); (b) a polynucleotide encoding a polypeptide having an amino acid sequence as encoded by the cDNA contained in ATCC Deposit No. 97610, 97608, 97609, 97611 , or 97607; (c) a polynucleotide having a nucleotide sequence at least 90% identical to the nucleotide sequence of the polynucleotide of (a) or (b); (d) a polynucleotide that hybridizes under stringent conditions to any of the polynucleotides of (a)-(c) or the complement thereof; (e) a polynucleotide fragment of any of the polynucleotides of (a)-(d), wherein said fragment is at least 15 bp in length; and (f) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of any of the polynucleotides of (a)-(e). 2. The isolated nucleic acid molecule of claim 1, which is a DNA molecule. 3. The isolated nucleic acid molecule of claim 1, which is an in vitro RNA transcript. 4. The isolated nucleic acid molecule of claim 2, wherein said polynucleotide is cDNA. 5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding any one of the MLN 64 variants A-G disclosed in Table VI. 6. A method for making a recombinant vector comprising inserting the isolated nucleic acid molecule of claim 1 into a vector. 7. A recombinant vector produced by the method of claim 6. 8. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 7 into a host cell. 9. A recombinant host cell produced by the method of claim 8. 10. A recombinant method for producing a polypeptide comprising culturing the recombinant host cell of claim 9. 11. An isolated polypeptide selected from the group consisting of: (a) a polypeptide having the amino acid sequence as shown in Figure 6 (SEQ ID NO:2), Figure 14 (SEQ ID NO:4), Figure 16 (SEQ ID NO:6), Figure 21 (A-D) (SEQ ID NO:8), or Figure 24(B) (SEQ ID NO: 10); (b) a polypeptide having the amino acid sequence as encoded by the cDNA deposited ATCC Deposit No. 97610, 97608, 97609, 97611, or 97607; (c) a polypeptide having an amino acid sequence at least 90% identical to the polypeptide of (a) or (b); and (d) a polypeptide fragment of any one of (a)-(c), wherein said fragment is at least 15 amino acids in length. 12. An antibody specific for an isolated polypeptide of claim 11. 13. An isolated polypeptide which is any one of the MLN 64 variants A-G disclosed in Table VI. 14. A method useful during breast cancer prognosis, comprising: (a) assaying a first MLN 50, 51 , 62 or 64 gene expression level or gene copy number in breast cancer tissue; and (b) comparing said first gene expression level or gene copy number with a second MLN 50, 51, 62 or 64 gene expression level or gene copy number; whereby the comparison of said first gene expression level or gene copy number to said second gene expression level or gene copy number is a prognostic marker for breast cancer. 15. The method of claim 14, wherein said second gene expression level or gene copy number is assayed in non-tumorigenic breast tissue. 16. The method of claim 14, wherein said second gene expression level or gene copy number is assayed in tumorigenic breast tissue. 17. The method of claim 14, wherein said gene expression level is assayed by detecting MLN 50, 51, 62 or 64 protein with an antibody. 18. The method of claim 14, wherein said gene expression level is assayed by detecting MLN 50, 51, 62 or 64 mRNA. 19. The method of claim 14, wherein said gene copy number is assayed by performing or detecting extrachromosomal double minutes (dmin), integrated homogeneously staining regions (hsrs), comparative genomic hybridization (CGH), or fluorescence in situ hybridization. 20. A method for distinguishing between leukemia cells with myelocytic or erythroid characteristics, comprising: assaying leukemia cells for D52 or D53 gene expression, whereby the presence of D52 gene expression or the lack of D53 gene expression indicates that the leukemia cells have myelocytic characteristics and the presence of D53 gene expression or the lack of D52 gene expression indicates that the leukemia cells have erythroid characteristics.

Inventors: BASSET PAUL, BYRNE JENNIFER, RIO MARIE-CHRISTINE, TOMASETTO CATHERINE, BASSET, PAUL, BYRNE, JENNIFER, RIO, MARIE-CHRISTINE, TOMASETTO, CATHERINE
Applicants: CENTRE NAT RECH SCIENT, INST NAT SANTE RECH MED, SQUIBB BRISTOL MYERS CO, UNIV PASTEUR, BRISTOL-MYERS SQUIBB COMPANY, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE M, UNIVERSITE LOUIS PASTEUR
Application year: 1996
Main classification: C12N15
IPC codes: C07K14, C07K16, C12N5, C12N15, C12P21, C12Q1