DIAGNOSTIC FOR METASTATIC PROSTATE CANCER

Abstract: The present disclosure provides methods for the diagnosis of metastatic prostate cancer and/or the prediction of the metastatic ability of prostate cancer in prostate biopsy tissue. Metastatic ability of prostate cancer is positively correlated with the level of transcriptional and translational expression of the MUC18 coding sequence in the neoplastic tissue. Methods for the determination of MUC18 protein synthesis include Western blots, ELISA, radioimmunoassay, immunofluorescence, and other immunoassays using MUC18-specific antibody and suitable detection means. Methods for measurement of transcriptional expression of the MUC18 coding sequence include Northern hybridizations and quantitative reverse transcriptase-polymerase chain reaction analyses. Absence of or very low MUC18 expression in the prostate tumor tissue is associated with nonmetastatic cancer, while relatively high levels of MUC18 expression are predictive of prostate cancer which is likely to metastasize or which has already metastasized. The present disclosure provides an improved diagnostic tool to aid the medical community in the choice of appropriate treatment regimens for prostate cancer patients.

Description: DIAGNOSTIC FOR METASTATIC PROSTATE CANCER BACKGROUND OF THE INVENTION Prostate cancer accounts for about 1 in 10 cancer cases in the United States, and it is the most often diagnosed cancer in males [Henderson et al. ( 1991 ) Science 254, 1131-1138]. While in many affected patients, the tumors are slow-growing and nonmetastatic, in others the malignant prostate tumors are aggressive and metastasize. When prostate cancer metastasizes, the prognosis for the patient is poor, especially without treatment. To date, the most frequently used test for prostate cancer is the serum level of prostate specific antigen (PSA) and the radionuclide bone scan for detecting prostate cancer metastases before definitive therapy is initiated. However, the elevated level of PSA in serum is not predictive of the pathologic stage of the prostate cancer or the presence of metastatic disease. PSA, a serine protease, is not exclusively expressed in the epithelial cells of metastatic prostate cancer, but it is also expressed in normal epithelial cells, primary tumors and benign prostate hyperplasia. The altered expression of cell-adhesion molecules has been correlated with metastasis of many cancers. Low or no expression of E-cadherin, a cell-adhesion molecule, has been found in high-grade prostate carcinoma, and this indicates a poor prognosis for those prostate cancer patients. However, the absence of an antigen is not very useful as a diagnostic marker for cancer metastasis. MUC18 is a glycoprotein of about 113 kDa which serves as a cell adhesion molecule on the surface of melanoma cells, and it has been correlated with the ability of melanomas to metastasize [See, e.g., Lehmann et al. (1989) Proc. Nat. Acad. Sci. USA 86, 9891-9895; Luca et al. (1993) Melanoma Res. 3, 35-41; Johnson et al. (1996) Curr. Top. Microbiol. Immunol. 213, 95-105; Xie et al. (1997) Cancer Res. 57, 2295-2303; Tang and Honn (1994-1995) 2 Invasion Metas. 14, 109-122; Rummel et al. (1996) Cancer Res. 56, 2218-2223]. MUC18 is also known as MCAM and CD 146. MUCl 8 carries a carbohydrate modification known as HNK-1 or CD57 [Shih et al. (1994) Cancer Res. 54, 2514-2520]. Besides being associated with melanoma cells' ability to metastasize, MUCl 8 is also associated with normal vascular tissue, and on the smooth muscle of venules, and it expresses sporadically on capillary epithelium [Johnson, J. (1994-1995) Invasion Metas. 14, 123-130]. There is a longfelt need in the art for an improved diagnostic test for metastatic prostate cancer so that appropriate therapy can be initiated as soon as possible and so that the number of false positive results can be minimized. SUMMARY OF THE INVENTION The present invention provides an improved diagnostic test for prostate cancer which has a relatively high potential for metastasis or which has metastasized. This allows the physician to choose appropriate surgical, chemotherapeutic or radiation treatment regimens. This improved assay is based on the correlation of high levels of expression of the MUCl 8 coding sequence as measured by MUCl 8 mRNA or MUCl 8 protein. This expression can be detected at the transcriptional level, where mRNA levels are monitored, or detection of the MUCl 8 gene product at the translation level can be determined, for example, through the use of an immunoassay for the MUCl 8 protein. The source of the material for these tests is prostate biopsy tumor tissue (e.g., from a needle biopsy) from a patient needing a determination of the metastatic potential of a prostate tumor or from cells from a prostate tumor. Relative levels of transcriptional expression (mRNA) of the MUCl 8 coding sequence can be determined by Northern hybridization analysis or by quantitative reverse transcription polymerase chain reaction (RT-PCR) in normal and neoplastic prostate tissue samples and in biopsy material. Translational expression of MUC 18 can be determined by any of a number of adaptations of an immunoassay using antibody specific for the MUC 18 cell surface antigen. The relative level of MUC 18 can be determined by standard immunoassays using a MUC 18- specific antibody preparation and a detection system suitable for the assay. Immunoassays can include, but are not limited to, immunofluorescence assays, radioimmunoassays, enzyme- 3 linked immunosorbent assays, and Western (immuno) blot assays. In the context of the present invention, relative amounts of the MUC 18 protein are determined in tissue samples (e.g., biopsy material). It is a further object of the present invention to provide an antibody which inhibits prostate cancer metastasis. In particular, antibody specific to MUC 18 prevents metastasis of prostate cancer cells. Additional objects include vectors directing the expression of an immunogenic fragment of human MUC 18 and the corresponding recombinantly expressed protein. As specifically exemplified, an immunogenic fragment of human MUC 18 is encoded by the PvuII to Xhol fragment within the sequence given in Tables 1 A- IB. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the results for RT-PCR amplified human MUC 18 cDNA (left panel) and cloning of the whole cDNA and its fragments into a GST-fusion protein expression vector (right panel). RT-PCR amplification of human MUC 18 cDNA from the poly(A)+RNA isolated and purified from a human melanoma cell line, Sk-Mel-28. Left panel: Lane (a) shows the expected PCR product of 1957 bp (as indicated by an arrow head). Lane (ml) shows the 1 kb ladders and lane (m2) the 123-bp ladders as DNA molecular size markers. Right panel: The plasmid map of the cloned whole human MUCl 8 cDNA and three fragments in a GST-fusion expression system. Fig. 2 illustrates Northern blot analysis of expression of human MUC 18 in different prostate cancer cell lines. Poly(A)+RNA was isolated from human melanoma cells SK-MEL- 28 (SK), human melanocyte (M), and human prostate cancer cells PC-3 (PC-3), DU145 (DU), TSU-PR-1 (TSUPR1), and LNCAP (LNCAP). The size of the human MUC18 mRNA is 3.3 kb. The amount of poly(A)+RNA (2.5 to 10 μg) is indicated as a number on top of each lane. Fig. 3 depicts recombinant human MUC18-middle fragment. The plasmid map is shown in Fig. 1. The left panel shows the PAGE result and right panel the Superdex column purification. The GST-human MUCl 8 middle fragment fusion protein is shown after IPTG induction (a & b, indicated by a triangle on the left of the left panel). The fusion protein was first purified through a glutathione-Sepharose affinity column and then cleaved with the HRV-3C protease (left panel, lanes c-e). The affinity-purified recombinant human MUCl 8- 4 middle fragment was then further purified through a Superdex column (right panel) to remove high molecular weight contaminants (peaks I and II, fractions 3-13). The final recombinant human MUC18-middle fragment protein is about 22 kDa (peak III, fractions 14-20), as indicated by a triangle on the right in the left panel. Fig. 4 shows pGEX-6P, commercially available from Pharmacia Biotech, Piscataway, NJ. The specific multiple cloning site (MCS) sequences for pGEX-6P-l (SEQ ID NO:l 1, encoded amino acids, SEQ ID NO: 12), pGEX-6P-2 (SEQ ID NO: 13, encoded amino acids, SEQ ID NO:14) and pGEX-6P-3 (SEQ ID NO:15, encoded amino acids, SEQ ID NO:16) are provided. Fig. 5 provides diagrammatic illustrations of the whole cDNA sequence of human MUCl 8, its N-terminal fragment, middle fragment and C-terminal fragment, as cloned into the pGEX-6P-l vector are provided. These fragments as cloned result in the expression of the N-terminal, middle and C-terminal fragments of the MUC 18 protein. See also Tables 1A- 1B for the locations of the relevant restriction sites in the cDNA sequence for human MUC 18 and SEQ ID NO: 1 for sequence. Fig. 6 illustrates the results of Western blot analysis of huMUC18 protein expression in four prostatic cancer cell lines. Cellular extracts of four prostatic cancer cell lines were prepared, and the proteins were size-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The anti-huMUC18 antibodies generated by immunization of the recombinant huMUCl 8-middle portion protein in chicken were used for Western blot analysis. SK stands for cellular lysate prepared from human SK-Mel-28 cells (a), Tsu-PRl cells (b and c), DU145 for DU145 cells (d and e), PC-3 for PC-3 cells (f and g), and LNCaP for LNCaP.FGC cells (h and i). The number over each lane indicates the amount of protein (μg) loaded in each well. The huMUC18 protein band is indicated with an open triangle. The numbers on the right-most lane indicate the protein molecular weight (kDa) rainbow markers (RPN800, Amersham). Fig. 7 shows the results of Western blot analysis of human MUC 18 protein in normal prostatic gland, normal primary human prostatic epithelial cells, and tissues of a patient with malignant prostatic cancer. Cellular lysates were prepared from normal prostatic gland (b), cultured normal primary prostatic epithelial cells (c), and prostatic cancerous tissues from a patient with malignant prostatic cancers (e-g). Cellular extracts prepared from human SK- 5 Mel-28 cells (a) and from DU145 cells (d) were shown as the positive controls. 20μg protein of each lysate was loaded per well. The numbers on the right-most lane indicate the protein molecular weight (kDa) rainbow markers (RPN800, Amersham). DETAILED DESCRIPTION OF THE INVENTION Prostate cancer accounts for about 10% of all cancer cases in the United States. It is now the most frequently diagnosed cancer in American males [Rinker-Schaeffer et al. (1993) Cancer and Metas. Rev. 12, 3-10] . In some patients, prostate cancers metastasize rapidly, killing the patient within one year of the initial clinical presentation. In contrast, some other prostate cancer patients show a relatively slow growth of the malignant tumor without metastasis. The majority of the histologically localized prostate cancers remain subclinical and never require treatment. Prostate cancer, when truly localized, can be cured by radical prostatectomy. While in many affected patients, the tumors are slow-growing and nonmetastatic, in others prostate cancer takes a more aggressive course. Unfortunately, metastatic prostate cancer is a fatal disease without treatment. At present, the test for serum levels of prostate specific antigen (PSA) and the radionuclide bone scan are the only diagnostic tests available before therapy is initiated. However, an elevated level of PSA observed in serum is not predictive of the pathological state of prostate cancer, nor is it correlated with metastatic prostate cancer. This is, at least in part, because PSA, a serine protease, is not exclusively expressed in the epithelial cells of metastatic prostate cancer, but it is also expressed in normal epithelial cells, primary prostate cancerous tumors and benign prostatic hyperplasia [Wood et al. (1994) Cancer 74, 2533- 2540]. To date, it has not been possible to predict whether histologically detected localized tumors are likely to progress to clinical cancer or when these localized tumors are likely to metastasize to other sites within the body. Thus, there is an urgent need for biochemical markers which serve to identify prostate cancers which have progressed to a stage which requires immediate surgical removal and/or additional chemotherapeutic or radiation therapeutic treatments, i.e., those cancers which are likely to metastasize. To identify a diagnostic marker which is improved over PSA, it is crucial to understand the biochemical differences between the malignant state and the benign state and between tumor cells with high and low metastatic potential. Overexpression and 6 underexpression of certain cell adhesion molecules at the cell surface has been proposed to reflect metastasis of several cancers [Tang and Honn (1994-1995) Invasion Metas. 14, 109- 122]. For example, the low expression of E-cadherin has been correlated with poor prognosis of prostate cancer [Rinker-Schaeffer et al. (1993) supra]. The present invention provides a positive correlation between the level of MUC 18 expression and high metastatic potential of prostate cancer cells. The human MUC 18 (huMUC18) cDNA sequence (see Table 1A) obtained by the present inventors is different from the huMUClδ cDNA sequence given in GenBank Accession No. N28882 [Johnson et al. (1994)]. The deduced amino acid sequence of huMUC18 cDNA given in Table 1A was identical to the huMUC18 sequence deposited in GenBank by Johnson's group except seven amino acid residues. This discrepancy of amino acid sequence may be due to allelic differences. However, the amino acid sequence of the inventor's huMUC18 (646 amino acids) was very different from that published by Johnson's group in 1989 (603 amino acids), which 1989 sequence appears to contain sequence errors in the huMUC18 cDNA. The human MUC 18 cDNA sequence disclosed in Table 1A was amplified by RT- PCR from poly(A)+RNA, which was isolated from the human melanoma cell line SK-Mel- 28. The cDNA was cloned into the pGEM-T-easy vector (Promega, Madison, WI). The DNA sequence of the huMUClδ cDNA was determined by rapid DNA sequencing using ABI prism dye terminator cycle sequencing ready reaction kit (Perkin-Elmer) with various huMUC18 specific primers in an automated sequencer of ABI 373XL system. The Lasergen and GCG programs were used for comparison of nucleotide and amino acid sequences of the huMUC18 cDNA. A cDNA sequence of a human MUC 18 clone has been published in GenBank under Accession No. M 28882 (See Table IB). The translation initiation codon (ATG) is underlined, as is the translation termination codon (TAG). The PvuII (CAGCTG) and Xhol (CTCGAG) restriction sites are boxed, and cut sites are indicated by vertical arrows. Table IC (see also SEQ ID NO:5) shows the human MUC18 sequence as modified to introduce a BamHI site (GGATCC) just upstream of the translation start site to facilitate cloning. The translation initiaition (ATG) and termination (TAG) codons are boxed. The long arrows near the 5' and TABLE 1A HUMAN MUC 18 cDNA SEQUENCE WITH DEDUCED AMINO ACID SEQUENCE 1 ATG GGG CTT CCC AGG CTG GTC TGC GCC TTC TTG CTC GCC GCC TGC TGC TGC TGT CCT CGC GTC GCG GGT GTG CCC GGA GAG GCT GAG CAG 90 < Hat Gly Leu Pro λrg Lau Val Cys Ala Phβ Lou Leu Ala Ala Cys Cys Cys Cys Pro Arg Val Ala Gly Val Pro Gly Glu Ala Glu Gin SO 91 CCT GCG CCT GAG CTG GTG GAG GTG GAA GTG GGC AGC ACA GCC CTT CTG AAG TGC GGC CTC TCC CAG TCC CAA GGC AAC CTC AGC CAT GTC 180 so Pro Ala Pro Glu Leu Val Glu Val Glu Val Gly Sar Thr Ala Leu Leu Lys Cys Gly Lau Sβr G n Sβr G n Gly Λ**n Lau Sar His Val ^ JΛ lβl GAC TGG TTT TCT GTC CAC AAG GAG AAG CGG ACG CTC ATC TTC CGT GTG CGC CAG GGC CAG GGC CAG AGC GAA CCT GGG GAG TAC GAG CAG 270 tf Asp Trp Pha Sβr Val His Lys Glu Lys Arg Thr Leu lie Phβ Arg Val Arg Gin Gly Gin Gly Gin Sβr Glu Pro Gly Glu Tyr Glu Gin 271 CGG CTC AGC CTC CAG GAC AGA GGG GCT ACT CTG GCC CTG ACT CAA GTC ACC CCC CAA GAC GAG CGC ATC TTC TTG TGC CAG GGC AAG CGC 360 Arg Lau Sβr Leu G n λsp Arg Gly Ala Thr Leu Ala Leu Thr Glh Val Thr Pro Gin λsp Glu Arg l e Phe Leu Cys Gin Gly Lys Arg 361 CCT CGG TCC CAG GAG TAC CGC ATC CAG CTC CGC GTC TAC AAA GCT CCG GAG GAG CCA AAC ATC CAG GTC AAC CCC CTG GGC ATC CCT GTG 450 Pro Arg Ser Gin Glu Tyr Arg He Gin Leu Arg Val Tyr Lys Ala Pro Glu Glu Pro λsn lie Gin Val Asn Pro Leu Gly lie Pro Val 451 AAC AGT AAG GAG CCT GAG GAG GTC GCT ACC TGT GTA GGG AGG AAC GGG TAC CCC ATT CCT CAA GTC ATC TGG TAC AAG AAT GGC CGG CCT 540 λsn Sβr Lys Glu Pro Glu Glu Val Ala Thr Cys Val Gly Arg λsn Gly Tyr Pro lie Pro Gin Val lie Trp Tyr Lys λsn Gly Arg Pro 541 CTG AAG GAG GAG AAG AAC CGG GTC CAC ATT CAG TCG TCC CAG ACT GTG GAG TCG AGT GGT TTG TAC ACC TTG CAG AGT ATT CTG AAG GCA 630 Leu Lys Glu Glu Lys λsn Arg Val His l e G n Ser Sβr Gin Thr Val Glu Sβr Sβr Gly Leu Tyr Thr Leu G n Ser lie Leu Lys Ala 631 CAG CTG GTT AAA GAA GAC AAA GAT GCC CAG TTT TAC TGT GAG CTC AAC TAC CGG CTG CCC AGT GGG AAC CAC ATG AAG GAG TCC AGG GAA 720 G n Leu Val Lys Glu Asp Lys Asp Ala Gin Phe Tyr Cys Glu Leu λsn Tyr Arg Leu Pro Ser Gly Asn His Het Lys Glu Sβr Arg Glu 721 GTC ACC GTC CCT GTT TTC TAC CCG ACA GAA AAA GTG TGG CTG GAA GTG GAG CCC GTG GGA XTG CTG AAG GAA GGG GAC CGC GTG GAA ATC B10 Val Thr Val Pro Val Phβ Tyr Pro Thr Glu Lys Val Trp Leu Glu Val Glu Pro Val Gly Hβt Leu Lys Glu Gly Asp Arg Val Glu lie βll AGG TGT TTG GCT GAT GGC AAC CCT CCA CCA CAC TTC AGC ATC AGC AAG CAG AAC CCC AGC ACC AGG GAG GCA GAG GAA GAG ACA ACC AAC 900 λrg Cys Lau Ala Asp Gly λsn Pro Pro Pro His Phβ Ser lie Ser Lys Gin Asn Pro Ser Thr Arg Glu Ala Glu Glu Glu Thr Thr Asn ^ 901 GAC AAC GGG GTC CTG GTG CTG GAG CCT GCC CGG AAG GAA CAC AGT GGG CGC TAT GAA TGT CAG GGC CTG GAC TTG GAC ACC ATG ATA TCG 990 Asp Λsn Gly Val Leu Val Leu Glu Pro Ala Arg Lys Glu His Ser Gly Arg Tyr Glu Cys Gin Gly Leu Asp Leu Asp Thr Hβt He Ser 991 CTG CTG AGT GAA CCA CAG GAA CTA CTG GTG λλC TAT GTG TCT GAC GTC CGA GTG AGT CCC GCA GCC CCT GAG AGA CAG GAA GGC AGC λGC 1080 Leu Leu Sβr Glu Pro Gin Glu Leu Leu Val Asn Tyr Val Ser Asp Val Arg Val Ser Pro Ala Ala Pro Glu λrg Gin Glu Gly Ser Sβr 1081 CTC ACC CTG ACC TGT GAG GCλ GAG AGT AGC CAG GAC CTC GAG TTC CAG TGG CTG AGA GAA GAG ACA GGC CAG GTG CTG GAA AGG GGG CCT 1170 Leu Thr Lau Thr Cys Glu λla Glu Sβr Ser Gin λsp Lau Glu Phe Gin Trp Leu Arg Glu Glu Thr Gly Gin Val Leu Glu Arg Gly Pro 1171 GTG CTT CAG TTG CAT GAC CTG AAA CGG GAG GCA GGA GGC GGC TAT CGC TGC GTG GCG TCT GTG CCC λGC ATA CCC GGC CTG AAC CGC λCλ 1260 Val Lau Gin Leu His λsp Leu Lys λrg Glu Ala Gly Gly Gly Tyr λrg Cys Val λla Ser Val Pro Ser He Pro Gly Leu λsn λrg Thr 1261 CλG CTG GTC AAC GTG GCC ATT TTT GGC CCC CCT TGG ATG GCA TTC AAG GAG λGG AAG GTG TGG GTG Aλλ GAG AAT ATG GTG TTG AλT CTG 1350 G n Leu Val λsn Val λla lie Phe Gly Pro Pro Trp Het λla Phe Lys Glu λrg Lys Val Trp Val Lys Glu λsn Met Val Leu λsn Leu 1351 TCT TGT Gλλ GCG TCλ GGG CλC CCC CGG CCC λCC λTC TCC TGG λλC GTC λλC GGC λCG GCλ λGT GλA CAA GAC CAA GAT CCA CAG CGA GTC 14 0 Ser Cys Glu Ala Sβr Gly His Pro Arg Pro Thr He Ser Trp λsn Val λsn Gly Thr λla Ser Glu Gin λsp Gin λsp Pro Gin λrg Val 1441 CTG AGC ACC CTG AAT GTC CTC GTG λCC CCG GAG CTG TTG GλG λCλ GGT GTT Gλλ TGC ACG GCC TCC λλC GλC CTG GGC λλλ λλC λCC AGC 1530 Leu Ser Thr Leu Asn Val Leu Val Thr Pro Glu Leu Leu Glu Thr Gly Val Glu Cys Thr Ala Ser Asn Asp Leu Gly Lys Asn Thr Ser 1531 λTC CTC TTC CTG GAG CTG GTC AAT TTA λCC ACC CTC ACλ CCA GAC TCC AAC λCA ACC ACT GGC CTC AGC ACT TCC ACT GCC AGT CCT CAT He Leu Phβ Lau Glu Lau Val λsn Lau Thr Thr Lau Thr Pro λsp Ser Asn Thr Thr Thr Gly Leu Sβr Thr Sβr Thr Ala Ser Pro His 1621 ACC AGλ GCC λλC AGC λCC TCC λCλ GλG λGλ AAO CTG CCG GAG CCG GAG AGC CGG GGC GTG GTC ATC GTG GCT GTG ATT GTG TGC ATC CTG Thr Arg Ala λsn Sβr Thr Ser Thr Glu Arg Lys Leu Pro Glu Pro Glu Ser Arg Gly Val Val He Val Ala Val lie Val Cys He Leu CΛ O so 1711 GTC CTG GCG GTG CTG GGC GCT GTC CTC TAT TTC CTC TAT AAG AAG GGC AAG CTG CCG TGC AGG CGC TCA GGG AAG CAG GAG ATC ACG CTG 1800 O_ Val Leu Ala Val Leu Gly Ala Val Leu Tyr Phβ Lau Tyr Lys Lys Gly Lys Leu Pro Cys Arg Arg Ser Gly Lys Gin Glu He Thr Leu *-. 00 iaθl CCC CCG TCT CGT AAG AGC GAA CTT GTA GTT Gλλ GTT λλG TCλ GAT AλG CTC CCλ GλA GAG ATG GGC CTC CTG CAG GGC λGC AGC GGT GλC 1890 © Pro Pro Ser λrg Lys Ser Glu Leu Val Val Glu Val Lys Ser λsp Lys Leu Pro Glu Glu Met Gly Lau Leu Gin Gly Sβr Sβr Gly λsp 1891 AAG λGG GCT COG GGλ GλC CλG GGλ GAG λλλ TAC ATC GAT CTG AGG CλT TAG CCCCOAAAT Lys λrg λla Pro Gly λsp Gin Gly Glu Lys Tyr He λsp Leu λrg His End TABLE IB O SO HUMAN MUCl 8 cDNA (GENBANK ACCESSION NO. M28882). § (SEQ ID NO:3; deduced amino acid sequence, SEQ ID NO:4) M GGGAAGCATGGGGCTTCCCAGGCTGGTCTGCGCCTTCTTGCTCGCCGCCTGCTGCTGCTGTCCTCGCGTC 5 GCGGGTGTGCCCGGAGAGGCTGAGCAGCCTGCGCCTGAGCTGGTGGAGGTGGAAGTGGGCAGCACAGCCC TTCTGAAGTGCGGCCTCTCCCAGTCCCAAGGCAACCTCAGCCATGTCGACTGGTTTTCTGTCCACAAGGA GAAGCGGACGCTCATCTTCCGTGTGCGCCAGGGCCAGGGCCAGAGCGAACCTGGGGAGTACGAGCAGCGG CTCAGCCTCCAGGACAGAGGGGCTACTCTGGCCCTGACTCAAGTCACCCCCCAAGACGAGCGCATCTTCT TGTGCCAGGGCAAGCGCCCTCGGTCCCAGGAGTACCGCATCCAGCTCCGCGTCTACAAAGCTCCGGAGGA 10 GCCAAACATCCAGGTCAACCCCCTGGGCATCCCTGTGAACAGTAAGGAGCCTGAGGAGGTCGCTACCTGT GTAGGGAGGAACGGGTACCCCATTCCTCAAGTCATCTGGTACAAGAATGGCCGGCCTCTGAAGGAGGAGA AGAACCGGGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTTGTACACCTTGCAGAGTATTCT GAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTTTTACTGTGAGCTCAACTACCGGCTGCCCAGT C O GGGAACCACATGAAGGAGTCCAGGGAAGTCACCGTCCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGG 15 AAGTGGAGCCCGTGGGAATGCTGAAGGAAGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCAACCC TCCACCACACTTCAGCATCAGCAAGCAGAACCCCAGCACCAGGGAGGCAGAGGAAGAGACAACCAACGAC AACGGGGTCCTGGTGCTGGAGCCTGCCCGGAAGGAACACAGTGGGCGCTATGAATGTCAGGCCTGGAACT TGGACACCATGATATCGCTGCTGAGTGAACCACAGGAACTACTGGTGAACTATGTGTCTGACGTCCGAGT GAGTCCCGCAGCCCCTGAGAGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAGAGTAGCCAG 20 GACCTCGAGTTCCAGTGGCTGAGAGAAGAGACAGACCAGGTGCTGGAAAGGGGGCCTGTGCTTCAGTTGC ATGACCTGAAACGGGAGGCAGGAGGCGGCTATCGCTGCGTGGCGTCTGTGCCCAGCATACCCGGCCTGAA CCGCACACAGCTGGTCAAGCTGGCCATTTTTGGCCCCCCTTGGATGGCATTCAAGGAGAGGAAGGTGTGG GTGAAAGAGAATATGGTGTTGAATCTGTCTTGTGAAGCGTCAGGGCACCCCCGGCCCACCATCTCCTGGA ACGTCAACGGCACGGCAAGTGAACAAGACCAAGATCCACAGCGAGTCCTGAGCACCCTGAATGTCCTCGT 25 GACCCCGGAGCTGTTGGAGACAGGTGTTGAATGCACGGCCTCCAACGACCTGGGCAAAAACACCAGCATC CTCTTCCTGGAGCTGGTCAATTTAACCACCCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTT ^ CCACTGCCAGTCCTCATACCAGAGCCAACAGCACCTCCACAGAGAGAAAGCTGCCGGAGCCGGAGAGCCG ^ GGGCGTGGTCATCGTGGCTGTGATTGTGTGCATCCTGGTCCTGGCGGTGCTGGGCGCTGTCCTCTATTTC §j CTCTATAAGAAGGGCAAGCTGCCGTGCAGGCGCTCAGGGAAGCAGGAGATCACGCTGCCCCCGTCTCGTA so 30 AGACCGAACTTGTAGTTGAAGTTAAGTCAGATAAGCTCCCAGAAGAGATGGGCCTCCTGCAGGGCAGCAG CGGTGACAAGAGGGCTCCGGGAGACCAGGGAGAGAAATACATCGATCTGAGGCATTAGCCCCGAATCACT §oS TABLE IC HUMAN MUC18 cDNA WITH 5' MODIFICATION TO FACILITATE CLONING vθ • * ■ S UF ( GCGGGTGTGCCCGGAGAGGCTGAGCAGCCTGCGCCTGAGCTGGTGGAGGTGGAAGTGGGCAGCACAGCCC in-. W SO TTCTGAAGTGCGGCCTCTCCCAGTCCCAAGGCAACCTCAGCCATGTCGACTGGTTTTCTGTCCACAAGGA GAAGCGGACGCTCATCTTCCGTGTGCGCCAGGGCCAGGGCCAGAGCGAACCTGGGGAGTACGAGCAGCGG CTCAGCCTCCAGGACAGAGGGGCTACTCTGGCCCTGACTCAAGTCACCCCCCAAGACGAGCGCATCTTCT TGTGCCAGGGCAAGCGCCCTCGGTCCCAGGAGTACCGCATCCAGCTCCGCGTCTACAAAGCTCCGGAGGA GCCAAACATCCAGGTCAACCCCCTGGGCATCCCTGTGAACAGTAAGGAGCCTGAGGAGGTCGCTACCTGT GTAGGGAGGAACGGGTACCCCATTCCTCAAGTCATCTGGTACAAGAATGGCCGGCCTCTGAAGGAGGAGA AGAACCGGGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTTGTACACCTTGCAGAGTATTCT GAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTTTTACTGTGAGCTCAACTACCGGCTGCCCAGT GGGAACCACATGAAGGAGTCCAGGGAAGTCACCGTCCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGG AAGTGGAGCCCGTGGGAATGCTGAAGGAAGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCAACCC *> TCCACCACACTTCAGCATCAGCAAGCAGAACCCCAGCACCAGGGAGGCAGAGGAAGAGACAACCAACGAC AACGGGGTCCTGGTGCTGGAGCCTGCCCGGAAGGAACACAGTGGGCGCTATGAATGTCAGGCCTGGAACT TGGACACCATGATATCGCTGCTGAGTGAACCACAGGAACTACTGGTGAACTATGTGTCTGACGTCCGAGT GAGTCCCGCAGCCCCTGAGAGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAGAGTAGCCAG GACCTCGAGTTCCAGTGGCTGAGAGAAGAGACAGACCAGGTGCTGGAAAGGGGGCCTGTGCTTCAGTTGC ATGACCTGAAACGGGAGGCAGGAGGCGGCTATCGCTGCGTGGCGTCTGTGCCCAGCATACCCGGCCTGAA CCGCACACAGCTGGTCAAGCTGGCCATTTTTGGCCCCCCTTGGATGGCATTCAAGGAGAGGAAGGTGTGG GTGAAAGAGAATATGGTGTTGAATCTGTCTTGTGAAGCGTCAGGGCACCCCCGGCCCACCATCTCCTGGA ACGTCAACGGCACGGCAAGTGAACAAGACCAAGATCCACAGCGAGTCCTGAGCACCCTGAATGTCCTCGT GACCCCGGAGCTGTTGGAGACAGGTGTTGAATGCACGGCCTCCAACGACCTGGGCAAAAACACCAGCATC CTCTTCCTGGAGCTGGTCAATTTAACCACCCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTT o CCACTGCCAGTCCTCATACCAGAGCCAACAGCACCTCCACAGAGAGAAAGCTGCCGGAGCCGGAGAGCCG § GGGCGTGGTCATCGTGGCTGTGATTGTGTGCATCCTGGTCCTGGCGGTGCTGGGCGCTGTCCTCTATTTC | CTCTATAAGAAGGGCAAGCTGCCGTGCAGGCGCTCAGGGAAGCAGGAGATCACGCTGCCCCCGTGTCGTA I AGACCGAACTTGTAGTTGAAGTTAAGTCAGATAAGCTCCCAGAAGAGATGGGCCTCCTGCAGGGCAGCAG 1 CGGTGACAAGAGGGCTCCGGGAGACCAGGGAGAGAAATACATCGATCTGAGGCAT^ΪAGlCCCCGAATCACT 10 3' ends indicate primer positions (BF1 and ER6a, respectively). The cut site for BamHI within its recognition sequence (GGATCC) is indicated with a vertical arrow. The Genbank M 28882 sequence (given in SEQ ID NO:3) is identical to a human MUC 18 cDNA clone (huMUC18) from human SK-Mel-28 cells, a human malignant melanoma cell line which produces relatively high levels of the MUC 18 protein. This sequence is slightly different from the huMUClδ previously published [Johnson and Rummel (1996) in Immunology of Human Melanoma, ed, Maio, M., IOA Press, Washington, DC, pp. 31-38; Lehmann et al. (1989) Proc. Natl. Acad. Sci. USA 86, 9891-9895; Luca et al. (1993) Melanoma Res. 3, 35-41]. The two cDNAs have three stretches of amino acid (aa) residues that are different, such as 19 aa and 17 aa at the N-terminal portion and 17 aa near the C- terminal portion. Furthermore, the published human MUCl 8 cDNA sequence was missing 42 amino acids at the C-terminal end (see SEQ ID NO:4). The human and murine cDNAs have 74.5% identity in the deduced amino acid sequences. The 3'-end primer used previously, ER6, did not include the last few codons and the termination codon. To re-clone the intact correct human MUC 18 cDNA, a correct new 3 '-primer, ER6a, for amplifying the intact human MUC 18 cDNA was designed (see hereinbelow). Efforts to express the recombinant huMUCl 8 protein in the pCal-n expression system (Stratagene, La Jolla, CA) in E. coli failed. Finally, the expression was possible by using a GST-fusion protein expression system. The huMUC18 cDNA was cloned in the PGEX-6p-l vector (Pharmacia), a small amount of the nearly intact MUCl 8 protein in E. coli was expressed. Fortunately, the sequence of the middle portion of the huMUC18 cDNA was correct, and it was then used for making recombinant protein in E. coli. Only when the middle 166 amino acid portion encoded by the cDNA, but not the N-terminal or C-terminal portions, was used for expression, over-expression of the recombinant protein was possible. One pair of primers: BamHI-HMUC 18-pvuII (28-mer, GGATCCCAGCTGGTTAAAGAAGACAAAG ^ ) (SEQ ID NO:6) and HMUC18-xhoI (27- mer, CTGGAACTCGAGGTCCTGGCTACTCTC) (SEQ ID NO:7) were used for PCR- amplification of the region from PvuII to Xhol of the HuMUCl 8 cDNA. The amplified fragment was cloned into pGEM-T Easy vector. The DNA fragment that included the coding region from the PvuII site to the Xhol site was excised from the pGEM-T Easy recombinant plasmid by two restriction enzymes, BamHI and Sail, and cloned into the BamHI and Sail 11 cleaved pGEX-6P-l vector. The recombinant HuMUCl 8 -middle fragment after cleavage with PrScission protease and purification contained the following sequences (see also Table 1 A and SEQ ID NO:2). 211 376 (Gly-Pro-Leu-Gly-Ser-)(SEQ ID NO:8)Gln-Leu- Leu-Glu-Phe-Gln-(Asn-His). PGEX-6P- 1 vector (Amino acids 211 -376 of SEQ ID NO:2) pGEM T Easy vector A 22 kDa protein fragment was expressed from the PvuII to Xhol fragment of the cDNA (see Tables 1A and IC). A large-scale preparation of the recombinant human MUC 18 "middle portion" fragment was carried out. More than 6 mg of the purified recombinant protein was obtained after purification through a glutathione-affmity column, cleavage with the HRV-3C protease, being eluted and concentrated, and further purification through a Superdex column in a Pharmacia FPLC system. After final concentration of the eluant, 6 mg of the "middle portion" recombinant protein was sent to Lampire Biological Laboratories to make polyclonal antibodies in chickens. High antibody titers were reported. Eggs are collected and IgY (chicken antibody protein) is purified from these eggs. After the titers of these purified IgY preparations are determined, they are used for immunological testing. Using the MUC18-specifιc antibody preparation from chickens after immunization with the purified human recombinant huMUCl 8-middle fragment protein, the present inventor has shown that these antibodies can react with the human MUC 18 protein expressed in human prostate cancerous cell lines and prostatic cancerous tissues by Western blot analysis. The results showed that the human MUC 18 protein was only expressed in three metastatic prostate cancer cell lines (Tsu-PR-1, DU145 and PC-3), but not in one nonmetastatic cell line (LNCaP.FGC). These results are consistent with the Northern blot analysis of the expression of huMUCl 8 mRNA in these prostatic cancer cell lines, described herein. The human MUC 18 protein was weakly expressed in normal prostatic epithelial cells and in normal prostate gland, but highly expressed in human cancerous prostatic tissues, as shown by Western blot analysis of the extracts prepared from these tissues. See Fig. 6 for the results of Western blot analysis. Further immunohistochemical analysis revealed that huMUCl 8 is expressed in the membrane of the expected special cell types, such as metastatic melanoma tissues, endothelial 12 cells, and smooth muscle cells. This indicates that the antibodies are very specific for huMUCl 8 antigen and work well with the formaldehyde-fixed, paraffin-embedded tissue sections. As expected, the antibodies did not react with any antigens of the normal secretory epithelial cells in the acini of the prostate gland. Interestingly, they react with the secretory epithelial cells in the acini of the prostate cancer tissues. The expression of human MUC 18 protein (or antigen) only in cancerous epithelial cells, but not in normal epithelial cells, supports the use of human MUC 18 as a diagnostic marker for the metastatic potential of prostate cancers. Table 2 summarizes the results for MUC 18 expression in four prostate cancer cell lines in comparison with pertinent results published by other groups. Expression of MUC 18 in these cell lines is consistent with their low or no expression of E-cadherin and -catenin and their extent of invasiveness in vitro and metastasis in nude mice. See Fig. 6 for Western blot analysis. The present work has correlated relatively high levels of MUCl 8 with the ability of prostate cancer cells to metastasize. High levels of MUC 18 expression were observed in the three metastatic prostate cancer cell lines TSU-PR1, DU145 and PC-3. MUCl 8 expression was not detectable in the LNCAP prostate cell line, which is not metastatic. Nonmetastatic prostate cancer cells and normal prostate cells produce no or barely detectable expression of MUC 18 either as protein or mRNA. Experiments in which the LNCAP cell line is genetically engineered to express MUCl 8 at high levels demonstrate that when cells gain the capacity to express MUC 18 at high levels, those cells gain the ability to metastasize. Experiments in which the nonmetastatic prostate cancer cell line LNCaP.FGC is genetically engineered to express MUC 18 at high levels demonstrate that when the cells gain the capacity to express MUC 18 at high levels, the ability to metastasize is also gained. Thus, the relative level of MUC 18 expression in prostate tumor tissue is correlated with the ability to metastasize, and measurement of MUC 18 expression in prostate tumor biopsy tissue allows the medical practitioner to choose the most appropriate therapy for each prostate cancer patient, with high levels of MUC 18 expression mandating an aggressive treatment strategy, likely including surgery, chemotherapy and/or radiation. 13 TABLE 2 RELATIVE LEVELS OF MUC 18 EXPRESSION TSU-PR1 DU145 PC-3 LNCAP Reference E-cadherin 0 0.1 0.6 1.1 Morton et al. expression (1993) Cancer Res. 53,3585- 3590 α-catenin none none none yes Morton et al. expression (1993) Total RNA yes yes yes none this work (MUC 18) tumor growth yes (2000) yes (500) Yes (1400) yes (2000) Passaniti et al. in nude mice (1992) International J. Cancer 51,318-324 Metastasis in yes yes yes none Lalani et al. nude mice (1992) Cancer Metastasis Rev. 16,29-66 Invasiveness in yes (220) yes (25) yes (20) none Passaniti et al. vitro (1992) Monoclonal or polyclonal antibodies, preferably monoclonal, specifically reacting with MUCl 8, may be made by methods well known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York. Also, recombinant immunoglobulins may be produced by methods known in the art, including but not limited to, the methods described in U.S. Patent No. 4,816,567. Monoclonal antibodies with affinities of 10 8 M "1 , preferably 10° to 10 10 M "1 or more, are preferred. Antibodies (polyclonal or monoclonal) specific for MUC 18 are useful, for example, as probes for screening DNA expression libraries or for detecting the presence (and relative amounts) of MUCl 8 in a test sample, for example, prostate tumor biopsy tissue or a tissue slice of a metastatic prostate cancer, or cells in culture which were derived from a primary prostate cancerous tumor or a metastatic prostate cancer tumor. Desirably, the results are 14 normalized to cell number or to total cellular protein. Frequently, the polypeptides and antibodies are labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. Suitable labels include, but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include, but are not limited to, Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (ed.) (1993) Meth. Enzymol. 2\8, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein. All references cited in the present application are incorporated by reference herein to the extent that they are not inconsistent with the present disclosure. The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles and/or methods which occur to the skilled artisan are intended to fall within the scope of the present invention. 15 EXAMPLES Example 1. Recombinant MUC 18 Production and Antibody Production The human MUC 18 cDNA (1970 bp, RT-PCR amplified fragment) and three sub- fragments have been cloned in-phase in a GST-fusion protein expression system, pGEX-6p-l (Pharmacia), which contains the glutathione-S-transferase (GST) coding region as an affinity- tag for the inserted foreign protein at its C-terminus. Fig.l and Fig. 5 show the four possible fusions: the whole region, the N-terminal fragment, the middle fragment, and the C-terminal fragment. Only the middle fragment of the human MUC 18 protein can be induced by IPTG to express in a high amount in E. coli K- 12 strain BL-21. Thus, only this protein is further purified for immunization. When culture A 600 reaches 0.6 (2 to 3 hours after 1/100 inoculation of an overnight culture in L-broth with ampicillin), the expression of the recombinant middle fragment of MUC 18 protein fused to GST in recombinant E. coli is induced by addition of 0.1 mM of IPTG to 3-liter cultures (1.5 liters per 4-liter baffled flask). Two hours after addition of IPTG at 37 °C, cells are harvested by centrifugation at 3,000 rpm (2,323 x g) for 20 min in a horizontal HG-4L rotor in Sorvall RC-3 centrifuge. The cell pellet is suspended in 40 ml of ice-cold PBS (10 mM Na 2 HPO4, 1.8 mM KH 2 PO4, 2.7 mM KC1, and 140 mM NaCl, pH 7.3) and then lysed with a prechilled French pressure cell at 800 psi. The lysate is clarified by centrifugation for two to three times at 13,000 rpm (21,000 x g) for 30 min in SS-34 rotor in Sorvall RC-2 centrifuge. The protein concentration of the clear crude lysate adjusted to 10 mg/ml protein (about 60 ml) was used as the starting material for purification. The recombinant MUC 18 proteins are purified from the clear crude lysate by batchwise adsorption to the Glutathione-Sepharose 4B affinity resin (about 20 ml of 50% slurry) by inversion on an inversion shaker at room temperature for 30 min. The GST portion of the fusion protein mediates the binding of the protein to the resin via the glutathione. After twice washing with 10 volumes (50 ml per 5 ml packed resin) of 1 X PBS and followed by twice washing with 1 X PreCission protease cleavage buffer (50 mM TrisHCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) to remove unbound proteins, the fusion protein on the resin is cleaved with 100 units of HRV-3C protease (PreCission protease, 2 units/μl, from Pharmacia) by rocking on an inversion shaker for 16 hours at 4°C. The resin was spun down at 2,000 rpm for 10 min in a Sorvall RC-32 centrifuge. The supernatant and three washings 16 (10 ml 1 X PBS per 10 ml resin), which contain the recombinant MUC 18 protein, are then combined and concentrated by centrifuging through a Centricon-30 (Millipore/ Amicon). The purity of the protein is characterized by SDS-PAGE (8 to 10% polyacrylamide gel, slab gel). The 70kDa contaminated protein is removed by passing through a Superdex 200 HR 10/30 column in 1 X PBS (void volume about 7 ml for a 20 ml packed column), and the fractions containing the recombinant middle fragment MUC 18 protein (22 kDa) (eluted at about 15.5 ml) were pooled. Fig. 2 shows the SDS-PAGE results or recombinant huMUCl 8 protein in the GST-fusion system. Six mg of protein is sent to Lampire Biological Lab. (Pipersville, PA) for immunizing three chickens. The anti-MUC18 antibody titers are determined by ELISA assay using the purified recombinant MUCl 8 as the antigen. Eggs collected during the period of high serum antibody titers are used for purification of chicken immunoglobulin IgY. To confirm the association between MUC 18 expression and metastatic ability of prostate cancer cells, the human MUC 18 coding sequence is introduced into a non-metastatic, non-expressing human prostate cancer cell line (LNCAP), and clones with different levels of expression of MUC 18 are isolated. The human MUC 18 cDNA has been cloned into pCR3.1 Uni (Promega, Madison, WI), a mammalian expression vector in which high levels of gene expression are driven by the human CMV-IE promoter. The human MUCl 8 cDNA is also cloned into a murine amphitrophic retrovirus expression vector, e.g. pZipNeoSVX [Cepko et al. (1984) Cell 37, 1053-1062] or LXSN [Miller and Rosman (1989) BioTechniques 7, 980-990], in which LTR drives gene expression. These MUC 18 recombinant vectors are used to transfect a human prostate cancer cell line which does not express MUC 18, for example the LNCaP.FGC cell line [Umbas et al. (1992) Cancer Res. 52, 5104-5109; lizumi et al (1987) J. Urol. 137: 1304-1306]. The vectors are introduced into the cultured cells by lipofection [Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417] or by electropporation [Potter, H. (1988) Anal. Biochem. 174:361- 373]. G418-resistant clones are selected and purified [Yuo et al. (1992) Intervirol. 34, 94- 104] in view of the kanamycin resistance coding sequence expressed under the control of the SV40 promoter in each of the vectors. Relative expression levels of MUC18 expression in 17 different clones are determined by western blotting with polyclonal antibodies (described herein). Metastatic abilities are determined as described herein. Example 2. Determination of Metastatic Ability of Prostate Cancer Cells The degree of motility and the invasiveness of prostate cancer cells are quantitated using published methods [Tucker et al. (1994) Eur. J. Cell Biol. 58, 28-290; Repesh, L.A. (1989) Invasion and Metastasis 9, 192-208]. The Costar transwell chamber contains an inner well with a porous polycarbonate membrane, with 3 μm pore sizes in the bottom of the well. This is tightly fitted to the outer well. To determine the motility of human prostate cancer cells, 0.5 x 10 5 cells are seeded in the top well. The cells remain on the top well because the poly carbonate membrane only allows medium to pass through freely. After seeding and attachment, the cells in the top well gradually migrate through the pores in the polycarbonate membrane to the bottom side of the membrane. Eventually some cells establish growth at the bottom side of the membrane. When the pore size of the membrane is about 3 μm, it somewhat slows the movement of the cells from the top side of the membrane to the bottom side. Motility of the cells is measured over the next several consecutive days. The rate of motility of a given cell line can be determined quantitatively by counting the cell number at the bottom of the membrane after trypsinization. Using this in vitro method, the motility rates of PC-3 and PC-huMUC18 human prostate cancer cells, with and without an over-expression of the human MUC 18 protein, respectively, are determined and compared. For the invasiveness of the prostate cancer cells, a similar kind of chamber is used, except before seeding the cells to the top well, the polycarbonate membrane is pre-coated with matrigel that contains protein components of the basal membranes of blood vessels. When the concentration of matrigel is correct, the membrane thus formed is thick enough to form a barrier to stop the cells from penetrating immediately, but is thin enough to allow cells to gradually invade through the membrane and migrate to the bottom of the membrane. Matrigel, which contains protein components of the base membrane of blood cell membrane, such as laminin, collagen type IV, entactin (nedogen), and heparin sulfate proteoglycan, is available commercially through Collaborative Research (Bedford, MA). Each filter in each 6.5 mm well is coated with 100 μl of a 1 :20 dilution of commercial Matrigel in cold DMEM 18 (about 30 μg per filter). Using a similar method of counting the cells, which grow and attach to the bottom of the membrane, the rate of invasiveness of a given cell line can be quantitated [Repesh, L.A. (1989) supra]. To count the cells at the bottom of the membrane, the bottom of the membrane is treated with trypsin to detach the cells, and the cell number is counted directly using a Hemacytometer, or the cells on the membrane is stained with trypan blue and counted directly using a microscope. Alternatively, the cells are labeled with 0.6 μCi of 125 I- iododeoxyuridine (5 Ci/mg) for 18-24 h (about 95% of cells) and seeded to the top of the transwell chamber. After 72 h of invasion, the cells at the bottom and the cells on the top are trypsinized. The total input radioactivity is determined and compared to the radioactivities associated with cells from the top and bottom chambers. In this way the percentage of cells invading through the membrane can be accurately quantitated. Invasion rate can thus be determined [Repesh, L.A. (1989) Invasion & Metast. 9:192-208]. This type of in vitro test has been demonstrated to produce results in agreement with that of the in vivo animal tests [Repesh, L.A. (1989) supra]. Using this in vitro method, the invasion rates of LNCAP and LNCAP-huMUC 18 transformed or transfected to express MUC 18 human prostate cells, with and without over-expression of the human MUC 18 protein, respectively, are determined and compared. For comparison, the metastasis rates of these prostate cells are also tested in vivo in athymic nude mice [van Weerden et al. (1996) Am. J. Pathol. 149, 1055-1062]. The effect of the different expression levels of human MUC 18 in prostate cancer cells on metastatic ability of different clones is determined. Similar pairs of cells as used for in vitro assay are also used in animal tests. The cells are implanted subcutaneously into nude mice. The size of tumors after different times are measured. The time and extent of the tumor cell metastases to bone or to other organs are investigated [van Weerden et al. (1996) supra]. The experiments set forth above confirm that the relative level of MUCl 8 expression in prostate cancer cells correlates positively with metastatic ability. Example 3. Transcriptional Expression of MUC 18 Total cell RNA is prepared from cell lines or from prostate tumor tissue with the method of one step acid-guanidinium-thiocyanate-phenol-chloroform extraction [Chomczyuski and Sacchi (1987) Anal. Biochem. 162, 156-159]. We have shown that this 19 method consistently yields good quality RNA for RT-PCR. Poly(A)+RNA is prepared from total RNA by purifying tlirough an oligo(dT)-cellulose column [Aviv and Leder (1972) J. Molec. Biol. 134, 743; Wu et al. (1985) Int. J. Biochem. 17, 355-363]. Northern hybridization technology is used to determine MUC 18 mRNA levels expressed in the four prostate cancer cell lines [Ausubel et al. (1987) Current Protocols in Molecular Biology, Section 4]. Total RNA is extracted from cell preparations, electrophoretically separated in a formaldehyde-impregnated agarose gel, blotted and hybridized to a 32 P-labelled human MUCl 8 cDNA [Feinberg and Vogelstein (1983) Anal. Biochem. 132, 6-13]. Relative levels of MUC 18 mRNA are detected by radioautography. The amount of RNA loaded and transferred is estimated by the intensities of the 28S and 16S bands in each lane. Alternately, G3PDH mRNA can be estimated using a G3PDH-specific probe or 28S and 18S probes after stripping the MUC18-specific probe. Quantitative RT-PCR methodology [Innis et al. (1990) PCR Protocols, Academic Press; Quantitative RT-PCR (1993) Methods and Applications Book #3, Clontech, Palo Alto, CA]. Human Sk-Mel-28 cells are used as positive control for this method. Because the quantity of mRNA from a small amount of tissue is small, quantitative RT-PCR is used for quantifying MUC 18 mRNA expression in tissues [Innis et al. (1990) supra]. The isolation kit from Boehringer-Mannheim (Indianapolis, IN) using magnetic beads is suitable for obtaining a small amount of poly(A)+RNA from prostate tissue. The quality of mRNA isolated with this kit is also excellent for translation and RT-PCR. The quantitative RT-PCR method is first established from using mRNA of cultured cells, as described above, and then is used for quantifying the expression of MUC 18 mRNA in different prostate cancer tissues. The RT step is standard: a 20 μl RT reaction contains 1 μg of poly(A)+RNA from the human melanoma cell line, SK-Mel-28, as template, 0.5 μg of oligo(dT) ]6 as primer (Promega), 2 μl of 10X AMV-RT buffer (Promega, Madison, WI), 2 units of AMV -reverse transcriptase, 5 mM MgCl 2 , 1 mM of dNTP mix (Promega), 1 unit of RNase inhibitor (Promega), and 50 μg/ml of acetyl BSA. The reaction was carried out at 42 to 48 °C for one hour, and heated at 99 °C for 5 min. A 20 μl PCR reaction contained 2 μl of RT reaction mixture (containing the first- strain cDNA), 2 μl each of the two primers (20 pm μl), 0.01 mM dNTPs, 1 μg of acetyl BSA, 20 2 μl of 10X PCR buffer with 15 mM MgCl 2 (Promega), and 0.5 units of Taq DNA polymerase (Promega, 5 units/μl). PCR cycles are as follows: Hot start at 94°C 5 min, 80°C 30 sec 29 cycles of 94°C 30 sec, 64-66°C 30 sec, and 72°C 2 min 1 cycle of 94°C 30 sec, 64-66°C 30 sec, and 72°C 60 min The sequences of the primers for amplification of the human MUC 18 cDNA from poly(A)+RNA of human melanoma cell line SK-Mel-28 are as follows: BF1 27-mer 5'-CTCGGGATCCATGGGGCTTCCCAGGCT (SEQ ID NO:9) ER6A 25-mer 5'-TCGGGGCTAATGCCTCAGATCGATG (SEQ ID NO: 10) Example 4. Immunofluorescence Assay for MUC 18 Where the anti-human MUC 18 antibodies are made in chicken, fluorescence-tagged anti-chicken IgG are used for immunofluorescent staining. Tissue culture cells, or normal or cancer prostate tissue samples are fixed, and first reacted with the anti-human MUC 18 antibodies, washed, and then reacted with the fluorescence-tagged rabbit anti-chicken antibodies [Umbas et al. ( 1992) Cane. Res. 52, 5104-5109] . The presence of human MUC 18 on the surface of these cells or tissues readily detected using UV-fluorescence microscopy. Human melanoma cells, Sk-Mel-28 (ATCC HTB 72), which express MUC 18 a high level of MUC 18 are used as the positive control. Human melanoma cells, WM115 (ATCC CRL 1675) which express no MUC 18, are used as the negative control. Three human prostate cancer cell lines, LNCap.FGC, PC-3, and DU145 are available from American Type Culture Collection, Rockville, MD, as ATCC CRL 1740, ATCC CRL 1435 and ATCC HTB 81, respectively. One other cell line, TSU-PRl, isolated by Dr. lizumi in Japan [lizumi et al. (1987) J. Urol. 137, J Urol. 137:1304-1306] and provided by Dr. John T. Isaacs, John Hopkins University, Baltimore, MD] was tested. TSU-PRl cells are more metastatic than the other three cell lines [Graff et al. (1995) Cancer Res. 55, 5195-5199]. All four cell lines are grown as monolayer cultures in a 37 °C incubator with an atmosphere of 5% CO 2 . Tsu-PRl and LNCap.FGC cells are grown in RPMI 1640 supplemented with 10% fetal bovine serum. PC-cells are grown in F12K medium with 10% fetal bovine serum. DU145 cells are grown in EMEM medium supplemented with pyruvate, extra non-essential amino acids and vitamins, and 10% fetal bovine serum. 21 Biopsy samples are taken, fixed and subjected to immunoassay using polyclonal antibody specific for human MUC 18 described hereinabove, as described in Wood et al. (1994) Cancer 74:2533-2540.

Claims: 22 I CLAIM: 1. A method for identifying metastatic potential of a prostate cancer cell, said method comprising the step of: detecting expression of a MUC 18 coding sequence in a prostate cancer cell for which an identification of metastatic potential is sought relative to expression of a MUC 18 coding sequence in a normal prostate cell, wherein a higher level of expression of the MUC 18 coding sequence is positively correlated with metastatic potential of a prostate cancer cell, whereby metastatic potential of a prostate cancer cell is deemed high when the level of expression of a MUC 18 coding sequence is higher in said prostate cancer cell than in a normal prostate cell. 2. The method of claim 1, wherein said prostate cancer cell is from a biopsy tissue sample from a patient for whom a prediction of metastasis of prostate cancer is sought. 3. The method of claim 1 , wherein expression of MUC 18 coding sequence is determined by immunoassay. 4. The method of claim 3, wherein expression of the MUCl 8 coding sequence is determined by immunoassay using antibody made in an experimental laboratory animal in response to a MUC 18 antigen. 5. The method of claim 4, wherein the MUC 18 antigen is a middle portion of MUC 18. 6. The method of claim 5, wherein said middle portion of MUC 18 has an amino acid sequence as given in SEQ ID NO:2, amino acids 211-376. 23 7. The method of claim 1 , wherein expression of a MUC 18 coding sequence is determined by Northern hybridization. 8. The method of claim 7, wherein a probe used in Northern hybridization comprises at least 15 contiguous nucleotides of SEQ ID NO:l. 9. The method of claim 8, wherein a probe used in Northern hybridization comprises a nucleotide sequence as given in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10. 10. The method of claim 1 , wherein said expression of a MUC 18 coding sequence is determined by a reverse transcriptase-polymerase chain reaction. 11. The method of claim 10, wherein a primer used in the reverse-transcriptase polymerase chain reaction comprises a nucleotide sequence as given in SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 10. 12. The method of claim 1, wherein said prostate cancer cell is a cell line cell. 13. An immunoassay kit for diagnosing metastatic potential of a prostate cancer cell, said kit comprising an antibody made in response to immunization with an antigen consisting essentially of middle portion MUCl 8. 14. A nucleic acid vector comprising a nucleotide sequence encoding a middle portion MUC 18 protein, said middle portion MUC 18 protein consisting essentially of an amino acid sequence as given in SEQ ID NO:2, amino acids 211-376. 15. The nucleic acid vector of claim 14, wherein said vector comprises a nucleotide sequence encoding a middle portion MUC 18 protein as given in SEQ ID NO:l, nucleotides 631-1128. 24 16. A recombinant host cell comprising the nucleic acid vector of claim 14. 17. A nucleic acid vector comprising a nucleotide sequence encoding a MUC 18 protein, said MUC 18 protein being characterized by an amino acid sequence as given in SEQ ID NO:2. 18. The nucleic acid vector of claim 17, wherein said nucleotide sequence encoding a MUC18 protein is as given in SEQ ID NO:l, nucleotides 1-1938. 19. A recombinant host cell comprising the nucleic acid vector of claim 17.

Inventors: WU GUANG-JER, WU, GUANG-JER
Applicants: UNIV EMORY, WU GUANG JER, EMORY UNIVERSITY, WU, GUANG-JER
Application year: 1999
Main classification: G01N33
IPC codes: C07K14, C07K16, C12Q1, G01N33