NOVEL TRANSCRIPTION FACTOR, BP1

Abstract: An isolated DNA of SEQ ID NO: 1 is provided that encodes the transcription factor BP1, which is believed to be a repressor of the beta -globin gene. A host cell that is transformed with a vector that contains the DNA may be used to produce BP1. Vectors having a controllable promoter operably connected to the BP1 open reading frame may be used to transform beta -globin producing cells of patients with sickle cell anemia, thereby providing a treatment. Because BP1 is overexpressed in leukemia and breast cancer cells,acute myeloid leukemia, acute lymphocytic leukemia, and breast cancer can be screened for and diagnosed by determining whether PB1is overexpressed in cell samples of patients who may have these conditions. An antisense DNA or RNA to the DNA encoding BP1 may be used as a treatment for acute myeloid leukemia, acute lymphocytic leukemia, and breast cancer.

Description: NOVEL TRANSCRIPTION FACTOR, BPl The present application claims the benefit of the filing date of United States Provisional Application No. 60/148,940, filed August 13, 1999. The provisional application is incorporated by reference herein. Statement As To Federally Sponsored Research Work described herein was supported by NIH grant R01DK53533. The U.S. Government has certain rights in the invention. Technxcal Field The present invention relates to a DNA that encodes the transcription factor BPl, a vector containing the DNA and a host cell containing the DNA. The invention also relates to an antisense DNA or RNA to the DNA encoding BPl, methods for treating sickle cell anemia by administering an effective amount of BPl, and methods for screening for acute myeloid leukemia, acute lymphocytic leukemia, and breast cancer. (In the provisional application, the transcription factor BPl was sometimes called "BPl/Dlx9" or "BPl/D1x9". Herein, for greater clarity, the transcription factor is simply referred to as "BPl".) Expression of globm genes m the β-globin cluster is restricted to erythropoietic cells, with five different genes expressed during embryonic (ε), fetal (Gy and Ay) and adult (δ and β) development. Transcriptional activation of globm genes occurs not only by binding of transcriptional activator proteins to the promoter of the gene being activated, but also by a regulatory element located 6 - 18 kb upstream of the β-globm cluster, the Locus Control Region (LCR) (See, for example, Berg, P. E. and A. N. Schechter. 1992. Molecular αenetics of disorαers of he oαlobm. In T. Friedmann (ed), Molecular Genetic Medicine. Academic Press, San Diego.; Forrester, W. C. , C. Thompson, J. T. Elder, and Groudine, M. 1986. A developmentally stable chromatin structure in the human β-globin gene cluster. Proc. Natl. Acad. Sci. USA 83: 1359-1363.; and Tuan, D., . Soloman, Q. Li, and I. M. London. 1985. The "β-like-globin" gene domain in human erythroid cells. Proc. Natl. Acad. Sci. USA 82: 6384-6388.). Sequential activation of the β-globin cluster genes during ontogeny must be countered by repression of the globin genes inactive during a given developmental stage. Repression is caused by binding of repressor proteins to promoter/upstream DNA and, in the case of the adult β-globin gene, is probably also due to lack of activation by the LCR (see, for example, Crossley, M. and S. H. Orkin. 1993. Regulation of the β-globin locus. Curr. Opinion Gen. Dev. 3: 232-237.) . While much is known about transcriptional activators that bind to DNA sequences near the β-globin gene, little is known about the proteins that repress its transcription. As discussed below, BPl is shown to bind to two silencer DNA sequences upstream of the β-globin gene and therefore, there is strong evidence suggesting that BPl protein is a repressor of the β-globin gene. The present invention provides for a DNA sequence that encodes BPl, and methods of using information derived from knowledge of the DNA sequence to screen for conditions such as breast cancer, acute myeloid leukemia and acute lymphocytic leukemia. The DNA sequence was found to be closely related to two other human genes, DLX4 and DLX7, described in Quinn, L. M. , B. V. Johnson, J. Nicholl, G. R. Sutherland, and B . Kalionis . 1997. Isolation and identification of homeobox genes from human placenta including a novel member of the Distal-less family, DLX4. Gene 187: 55-61 and Nakamura S, Stock DW, Wydner KL, Bollekens JA, Takeshita K, Nagai BM, Chiba , Kitamura T, Freeland TM, Zhao Z, Minowada J, Lawrence JB , Weiss KB, and Ruddle FH . Genomic analysis of a new mammalian Distal-less gene: Dlx-7. Genomics 1996; 38: 314-324. Survival rates for many types of cancers correlate with early detection and treatment. Further, it is helpful to monitor ongoing cancer treatments to determine effectiveness. Accordingly, there is a continuing need for reliable cancer cell screening methods. One method of screening is to detect and monitor the expression of genes that are overexpressed or underexpressed m particular types of cancer cells, in comparison to normal cells. Detection techniques and particular markers have been disclosed, for example, in the following U.S. patents, incorporated herein by reference: U.S. Patent No. 5,776,683 to Smith et al; U.S. Patent No. 5,965,409 to Pardee et al; U.S. Patent No. 6,037,129 to Cole, et al; U.S. Patent No. 5,677,125 to Holt, et al; U.S. Patent No. 6,004,756 to Watson, et al; U.S. Patent No. 5,994,062 to Mulshine, et al; U.S. Patent no. 5,700,927 to Zon, et al; and U.S. Patent No. 5,981,218 to Rio et al. Because no marker or method of screening is completely reliable, there is a continuing need in the art for additional genetic markers. In particular, there is a continuing need for genetic markers for identifying breast cancer, acute myeloid leukemia and acute lymphocytic leukemia. The present invention overcomes this problem in the art by providing DNA BPl compositions and methods for screening for breast cancer, acute myeloid leukemia and acute lymphocytic leukemia. The problem also exists in the art of lack of information regarding repression of the β-globm gene. As discussed below, there are medical conditions such as sickle cell anemia that can be treated by repressing the expression of β-globin and the present invention, provides for the production of BPl, a putative β-globin repressor. Disclosure of Invention It is an object of the present invention to provide a DNA sequence encoding BPl . It a further object of the present invention to provide a marker and a method for detecting acute myeloid leukemia and acute lymphocytic leukemia. It is a further object of the present invention to provide a marker and a method for detecting breast cancer. It is a further object of the present invention to provide a treatment for conditions that can be alleviated by repressing the expression of the β-globin gene. It is a further object of the present invention to provide a treatment for sickle cell anemia. It is a further object of the present invention to provide a treatment for conditions that can be alleviated by blocking the expression of BPl. These and other objects are achieved in present invention by providing an isolated DNA having SEQ ID NO: 1, encoding BPl. BPl exhibits a repressor function: it binds to Silencers I and II upstream of the β-globin gene and binds to a sequence of -530 bp upstream of the δ-globin gene. The invention also includes a vector comprising the DNA encoding BPl and a host cell transformed with the vector. In an alternative embodiment, the invention is directed to an antisense DNA or an antisense RNA of a DNA having SEQ ID NO: 1, encoding BPl. The invention further provides a method of treating sickle cell anemia comprising administering an effective amount of BPl. The invention provides a method of screening for and/or diagnosing acute myeloid leukemia or acute lymphocytic leukemia, the method comprising the steps of (a) obtaining a cell sample from a patient, and (b) determining whether BPl is overexpressed by said cell sample as compared to normal cells. The invention also provides a method of screening for and/or diagnosing breast cancer, the method comprising the steps of (a) obtaining a cell sample from a patient, and (b) determining whether BPl is overexpressed by said cell sample as compared to normal cells. The invention further provides for a polyclonal antibody to BPl. The invention further provides a set of PCR primers for amplifying the DNA of SEQ ID NO. 1. The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description and figures, wherein only the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention. As is readily recognized the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention. Brief Description of the Drawings Figure 1 is a schematic representation of the region -100 to -600 bp upstream of the β-globm, showing that BPl binds within Silencer I at -530 bp relative to the cap site (+1) and within Silencer II at -300 bp, and that BP2, another DNA-bmdmg protein, binds within Silencer II at -270 bp. Figure 2 is an autoradiogram showing the results of an EMSA competition assay to verify that the cDNA encoded a protein with the expected binding specificities of BPl, namely, the ability to bind to Silencers I and II upstream of the β-globin gene and to a sequence -530 bp upstream of the δ-globm gene. The putative BPl protein, fused to β-galactosidase, was expressed from a λ lysogen. This fusion protein was partially purified and incubated with the Silencer II probe used for screening the library, resulting m a single shifted band, indicated by the arrow, lane 1. Figure 3A is an autoradiogram showing the results of a Northern blot of K562 RNA using part of the 630 bp BPl cDNA (obtained as described herein) as a probe. An RNA band of 2.1 kb was recognized. Fiqure 3B is an autoradiogram showing the results of Northern blot of fetal tissue, using the same probe as described m Figure 3A Fiqure 4 is the sequence of BPl . This sequence is also set forth in the attached sequence listing as SEQ ID NO:l. The open reading frame of the sequence is indicated as well as the predicted protein sequence, which is also set forth in the sequence listing as SEQ ID NO: 2 and SEQ ID NO: 12. Figure 5A and Figure 5B are graphs showing the relative CAT activity of K562 cells in transient transfection assays in relation to increasing amounts of pRSV/BPl-ORF (sub-cloned as described herein) or to an empty vector. In Figure 5A, pεCAT/SI was the target DNA; in Figure 5B, pεCAT/SII was the target DNA. K562 cells were transiently co-transfected with increasing amounts of either an empty vector or pRSV/BPl-ORF, along with a target plasmid. Figure 6 is an autoradiogram showing the expression of BPl in erythroid and myeloid lineage cell lines, as determined by RT- PCR. Figures 7A and 7B are autoradiograms showing the expression of BPl, DLX7 and DLX4 in T-cell ALL cell lines and erythromyloid cell lines, as determined by RT-PCR. Figure 8 is an autoradiogram showing the expression of BPl, DLX7 and DLX4 in normal bone marrow, B-cells and T cells. Semi- quantitative RT-PCR was used to measure expression. Figure 9 is an autoradiogram showing the expression of BPl, DLX7 and DLX4 in acute myeloid leukemia. Samples from six patients are shown. (-) indicates expression equal to or less than normal controls; (+) indicates expression greater than controls; (+) indicates an expression ratio at least three times greater than the ratio for ( + ) . Figure 10 is a graph comparing the expression of BPl, DLX7 and DLX4 in AML and T-cell ALL. Black bars represent adult AML, and white bars represent pediatric T-cell ALL. Figure 11 is an autoradiogram showing BPl expression in CD34 + and CD34 cells. Figure 12 is a graph showing the percent viability over the course of four days of cell lines transformed with an BPl antisense-producing plasmid (10B and 10D) in comparison with control cells receiving an empty vector (9A and 9B) . Figure 13 is an autoradiogram showing BPl expression of the cell lines 9A - 9B receiving an empty vector (lanes 1 - 2) and cell lines transformed with an BPl antisense-producing plasmid 10B-10D(lanes 3 - 4) . Figure 14 is an autoradiogram showing the expression of BPl by several breast cancer cell lines, including breast cancer lines MCF7 ADR, MDA468 and T47D, and no expression in novel breast tissue. Figure 15 is an autoradiogram showing the expression of BPl in normal and malignant breast tissues. Best Mode For Carrying Out The Invention The present invention relates to an isolated DNA, having or consisting essentially of SEQ ID NO:l and encoding the transcription factor BPl. The transcription factor BPl exhibits β-globin repressor function and binds to Silencers I and II upstream of the β-globm gene, to a sequence of -530 bp upstream of the δ-globin gene and to an Indian haplotype D sequence. BPl is typically expressed in placenta and kidney. The predicted sequence of BPl, deduced from the open reading frame of the isolated DNA of SEQ ID NO: 1, is given as SEQ ID NOs : 2 and 12. Both the DNA sequence and the protein sequence are shown together m Fig. 4. As used herein, the term "isolated DNA" refers to DNA that is not in its native environment. In a preferred embodiment of the invention, the isolated DNA encoding BPl is a cDNA of 1251 bp . The isolated DNA also comprises an open reading frame (ORF) . The present invention further relates to a vector containing the DNA and to a host cell transformed with the vector containing the DNA. In a preferred embodiment, rhe transformed host cell is a eukaryotic host cell. The invention further includes an antisense DNA or an antisense RNA of a DNA having SEQ ID NO:l. The invention further includes a method of treating sickle cell anemia, the method comprising the step of administering an effective amount of BPl to a patient in need thereof. In the method of the invention, BPl decreases β-globin expression in the patient and decreases mtracellular concentrations of HbS . The invention further includes a method of screening for and diagnosing acute myeloid leukemia or acute lymphocytic leukemia, the method comprising the steps of (a) obtaining a cell sample from a patient, and (b) determining if BPl is overexpressed by said cell sample as compared to normal cells. The invention also includes a method of screening for and diagnosing breast cancer, the method comprising the steps of (a) obtaining a cell sample from a patient, and (b) determining if BPl is overexpressed by said cell sample as compared to normal cells. In the methods for detecting leukemia and breast cancer, the overpression of BPl may determined by detecting BPl-encodmg mRNA by reverse transcπptase polymerase chain reaction (RT-PCR) or by detecting BPl itself by lmmunohistochemical detection. The invention further includes PCR primers that amplify a unique DNA molecule from a substrate of cDNA of mRNA that encodes for BPl. Such PCR primers can be readily derived from the DNA sequence of SEQ ID NO.: 1. As used herein, the term "unique DNA molecules" means that the DNA is of sufficient specifity to determine that the substrate being amplified originated from the sequence of SEQ ID NO:l. The invention further includes polyclonal and monoclonal antibodies to BPl. A polyclonal antibody can be obtained by immunizing a mammal with BPl or an antigenic fragment thereof m order to induce the production of sera containing polyclonal antibodies, as described below. A monoclonal antibody to BPl may be obtained by hybridoma technology, which typically includes the further steps of isolating splenocytes from immunized mammals, fusing them with myeloma cells, then identifying fused cells that secrete antibodies that bind to BPl . The invention further includes a methoα of repressing the β-globin gene comprising administering an effective amount of BPl . These aspects of the invention are further characterized below. First, it is shown that the cloning of BPl cDNA and that its overexpression in a transient co-transfection assay causes repression of a reporter gene linked to either Silencer I or Silencer II DNA of the β-globm gene. This provides evidence that BPl is a repressor of the β-globm gene. Sequencing reveals that the DNA encoding BPl contains a homeobox, placing the DNA in a family of conserved genes that regulate each other and that regulate unrelated genes as well (see, for example, Jaynes, J. B. P. H. and O' Farrell. 1988. Activation and repression of transcription by homeodomain-containmg proteins that bind a common site. Nature 336: 744-749. and references therein; Lev ne, M. and T. Hoey. 1988. Homeobox proteins as sequence-specific transcription factors. Cell 55: 537-540.). Sequence comparison indicates that the DNA encoding BPl belongs to the Distal-less family of homeobox genes, which are expressed during early development (see, for example, Cohen, S. M. and G. Jurgens . 1989. Proximal-distal pattern formation m Drosophila : cell autonomous requirement for Distal-less gene activity m limb development. EMBO J 8: 2045-2055; Cohen, S. M. , G. Bronner, F. Kuttner, G. Jurgens, and H. Jackie. 1989. Distal -l ess encodes a homeodomam protein required for limb development in Drosophila . Nature 338: 432-434; Dolle, P., M. Price and D. Duboule . 1992. Expression of the murine Dlx-1 homeobox gene during facial, ocular, and limb development. Differentiation 49: 93-99; Robinson, G. W. and K. Mahon. 1994. Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes incraniofacial development. Mech. Dev. 48: 199-215; Simeone, A., D. Acampora, M. Pannese, M. D'Esposito, A. Stornaiuolo, M. Gulisano, A. Malla aci, K. Kastury, T. Druck, and K. Huebner . 1994. Cloning and characterization of two members of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci. USA 91: 2250-54; and Stock, D. W. , D. L. Ellies , Z. Zhao, M. Ekker, F. H. Ruddle, and K. M. Weiss. 1996. The evolution of the vertebrate Dlx family. Proc. Natl. Acad. Sci. USA 93: 10858- 10863.). The expression of the DNA encoding BPl is highly restricted, which is a characteristic of developmentally important genes. Cloninσ and Characterizing BPl Materials and Methods: Cloning BPl: A λgtll K562 cell cDNA expression library (Clontech, Inc.) was screened with an oligonucleotide probe containing the BPl binding site located m Silencer II DNA, which included sequences between -336 and -278 bp upstream of the β- globin gene. Screening was as described (see, for example, Vinson, C. R. , K. L. LaMarco, P. F. Johnson, W. H. Landschulz, and S. L. McKnight. 1988. In situ detection of sequence-specific DNA binding activity specified by a recombmant bacteriophage . Genes & Devel . 2: 801-806), with several modifications. Filters were prehybπdized m binding buffer (see, for example, Berg, P. E., D. M. Williams, R.-L. Qian, R. B. Cohen, S. X. Cao, M. Mittelman, and A. N. Schechter . 1989. A common protein binds to two silencers 5' to the human β-globm gene. Nucl. Acids Res. 17: 8833-8852) containing 5% Carnation instant dry milk at 4°C for 30 minutes, then hybridized m the same buffer for 2 to 12 hours at 4°C with shaking. Positive plaques were purified and subjected to three additional rounds of screening. Electrophoretic mobility shift assay (EMSA) : The binding reaction included 500 pg of fusion protein or 250 ng of nuclear extract added tc binding buffer (10 M Tris, pH 7.5, 50 mM NaCl, 5% glyceroi, ImM DTT, lμl of lmg/ml BSA, 1 μg poly(dl-dC)) and 5,000-10,000 cpm of -"P-dCTP labeled probe. Incubation was for 30 minutes at room temperature. Nuclear extracts were prepared according to Digna et al (Dignam, J. D., R. M. Lebovitz , and R. G. Roeder . 1983. Accurate transcription initiation by RNA polymerase II m a soluble extract from isolated mammalian nuclei. Nucl . Acids Res. 11: 1475-1489). Protein extracts from lysogens were prepared as described (see, for example, Cowell, I. G. and H. C. Hurst. 199. Cloning transcription factors from a cDNA expression library, p.120-122. In D. S. Latchman (ed.), Transcription factors: a practical approach. IRL Press, New York) . Competitors were pre-incubated with nuclear extract for 20 minutes prior to addition of the probe. Oligonucleotides sequences of probes and competitors for EMSA analyses are shown below. Name Sequence -530 Reference TGTATATATACACATATATATATATATTTTTTTT CTTTTCTTACCAGAAGGTTT (SEQ ID NO: 3) -530 Indian TGTACATATACACATATATATATATATATA TTTTTTCTTTTCTTACCAGAAGGTTT (SEQ ID NO: 4) -300 beta TTCTTATTTGTGTAATAAGAAAATTGGGAAAACG ATCTTCAATATGCTTACCAAGCTG (SEQ ID NO: 5) -530 delta TTCTTTTAA TGGATATTTATTTCAATATAAT AAAAAATTAGAGTTTTA (SEQ ID NO: 6) non-specific TGCATATATATGTATATGTATGTGTGTATA :SEQ ID NO: 7) Plasmid construction: To construct a plasmid expressing the BPl cDNA open reading frame, BPl cDNA was amplified by RT-PCR from K562 cell mRNA. The resulting 1000 bp product was cloned into pGEM7 vector (Promega, Inc.) and its identity confirmed by sequencing. This plasmid was cleaved with Hind III and Xba I to release the BPl ORF, which was directionally cloned into pRc/RSV (Invitrogen, Inc.). Transfections and CAT assays: Transient transfection assays were performed m K562 cells using DMRIE-C reagent (Gibco-BRL, Inc.). Cells were grown to a density of 5 X 10- cells/ml and a total of 2 X 10 6 cells were used per transfection reaction. Transfections were performed in six well plates using OPTI-MEM serum-free medium (Gibco-BRL, Inc.). A total of 8 μg of plasmid DNA and salmon sperm DNA were added to complex with 16 ul of DMRIE-C reagent in 1 ml of OPTI-MEM I Reduced Serum Medium (Gibco-BRL, Inc.) for 15-45 mm. at room temperature. 2 X 10 6 cells in 0.2 ml serum-free medium were added to each well and plates were incubated for 4-5 hrs at 37 °C . Two ml of growth medium were added and cells were harvested after 48 hr . CAT assays were performed as described (see, for example, Berg, P. E., D. M. Williams, R.- L. Qian, R. B. Cohen, S. X. Cao, M. Mittelman, and A. N. Schechter. 1989. A common protein binds to two silencers 5' to the human β-globm gene. Nucl. Acids Res. 17: 8833-8852). Northern blot analysis: RNA was isolated using RNeasy and Oligotex kits (Qiagen, Inc.) . Two micrograms of mRNA was electrophoresed per lane, and then transferred to Hybond N membrane (Amersham) by capillary action. RNA was crosslmked to the membrane using a Stratolmker (Stratagene) , and the filter was hybridized using either standard methods or a sandwich method, which eliminates the background (see, for example, Wu, S., Q . Lu, and A. L. Kriz . 1995. Multiple-sandwich, one-step hybridization of Nortnern and Southern blots. BioTechniques 18. 585-586. . An RNA dot blot was purchased (Clontech, Inc., ; the amounts of RNA loaded on the blot had been normalized by the manufacturer to eight different housekeeping genes, obviating the need to normalize the samples. The probe was a 630 bp DNA fragment obtained by PCR amplification of BPl cDNA cloned m pBluescπpt with subsequent Eco Rl digestion to release a partial 3' BPl cDNA lacking homeobox sequences. It was labeled by random primmq and hybridized overnight at 65°C. Blots were washed and exposed to λ-ray film. RT-PCR: Total RNA was isolated using TPIzcl Reagent (GIBCO BRL accordmq to the manufacturer's specifications. One microgram of RNA was reverse transcribed using Superscript II RT (GIBCO BRL) in a total reaction volume of 20 μl . PCR was performed with lμl of reverse-transcription product in 25 μl total reaction volume consisting of 16.8 μl of distilled H 2 0, 2.5 μl of 10 X PCR buffer (200 mM Tris-HCl [pH 8.4], 500 mM KC1) , 1.5 μl 25 mM MgCl 2 , 0.5 μl 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) , 0.2 μl (1 unit) Ampli-Taq DNA polymerase (Perk Elmer) and 1 μl each of 10 μM β-actm or BPl forward and reverse primers, respectively. Based on linearity assay results, the following PCR conditions for BPl were used. Each PCR cycle consisted of a denaturation step (94°C, 1 mm), an annealing step (58°, 1 minute) and an elongation step (72°C, 1.5 minute) for 27 cycles, followed by an additional extension (72°C, 5 minutes). Primers, designed to amplify a product of 581 bp, were: forward: 5'- CACCTCCTGTCTTACCCCTACACC-3' SEQ ID NO: 8; reverse: 5'- GCCCTTCCCCAGATTCACATCATC-3' SEQ ID NO: 9. PCR products were electrophoresed on a 2% agarose gel and visualized with ethidium bromide. The product was verified by cleavage with restriction enzymes and hybridization with an internal probe. Alternatively, a product of 225 bp may be obtained with the following primers: forward 5 ' -GTATGGCCACCTCCTGTCTT-3 ' (SEQ ID NO: 10) and reverse: 5 ' -GAGTAGATGGTCCTCGGCTT-3 ' (SEQ ID NO: ll)under the following conditions: 94° C for 2 minutes; then 94° C for 1 minute, 62° C for 1 minute and 72° C for 1.5 minutes, 30 cycles; 72° C for 10 minutes. Cloning of BPl cDNA: A λgtll cDNA expression library made from human K562 cells was probed using a multimenzed oligonucleotide containing the -300 bp BPl binding site. Two million plaques were screened and one positive plaque was isolated that expressed a protein that recognized the -300 bp BPl binding site but not a negative control sequence. To verify that the cDNA encoded a protein with the expected binding specificities of BPl, namely, the ability to bind to Silencers I and II upstream of the β-globm gene and to a sequence -530 bp upstream of the δ-globm qene (see, for example, Berg, P.E., S . Abhyankar, and M. Chase. 1994. The high mobility group protein HMG-I (Y) binds to a silencer DNA sequence upstream of the human β-globin gene. Blood 84 Suppl 1: 262a ) , an EMSA competition assay was performed. The putative BPl protein, fused to β-galactosidase, was expressed from a λ lysogen, as described above. This fusion protein was partially purified and incubated with the Silencer II probe used for screening the library, resulting in a single shifted band, indicated by the arrow ( Fig. 2, lane 1 ) - The band at the top marks the position of the wells, and the band at the bottom contains the unshifted probe. Cold competitors were added at a 100 X and 200 X molar excess to determine specificity. The shifted band was competed by self DNA ( unlabeled -300 binding site, lanes 2 -3), the BPl binding site at -530 bp in Silencer I containing either the reference ( wild type ) or Indian haplotype sequences (lanes 6-9 ) , and the BPl binding site found upstream of the δ-globin gene ( lanes 10-11 ) , but not to a non-specific DNA (lanes 4-5). Note that the Indian sequence is a better competitor than the reference sequence, indicating tighter binding of BPl to the Indian sequence, as was previously observed using K562 nuclear extracts ( see, for example, Elion, J . , P. B. Berg, C. Lapoumeroulie , G. Trabuchet, M. Mittelman, R. Krishnamoorthy, A. N. Schechter, andD. Labie. 1992. DNA sequence variation in a negative control region 5' to the β -globin gene correlates with the phenotypic expression of the β s mutation. Blood 79: 787-792.) . A negative control, pure β - galactosidase protein, did not exhibit binding activity towards the probe ( data not shown). The EMSA analysis thus demonstrated that the binding properties of the fusion protein conform to those expected for BPl. The BPl cDNA obtained was 630 bp in length. When part of this sequence was used as a probe in a Northern blot containing K562 RNA, an RN A band of 2.1 kb was recognized ( Fig. 3A ) . This result i ndicated an incomplete cDNA had been cloned. To extend the cDNA sequence, rapid amplification of cDNA ends ( RACE ) was used Addit i onal sequences in both the 5' and 3' directions were obtained, giving a cDNA of 1366 bp. Subsequently fine-tuning led to the 1251 bp sequence of SEQ ID:1 (and Figure 4) . Further, the predicted open reading frame (ORF) was obtained. Computer analysis using BLAST indicated that BPl contains a homeobox (HB) , placing it a family of genes known to be important development. Several sub-families of homeobox genes have been defined. BPl is a member of the Distal-less (Dlx) family, based on sequence homology to the homeobox of these genes. In Figure 4, the homeobox is underlined and the ammo acids comprising the three predicted α helices found in homeodoma s (see, for example, Pabo, C. O. and R. T. Sauer. 1992. Transcription factors: structural families and principles of DNA recognition. Annu . Rev. Biochem. 61: 1053-1095.) are also indicated. The second and third helices comprise the helix-turn- helix (HTH) motif which is characteristic of homeodomams. A putative translational start site was determined by homology with the Kozak consensus sequence (see, for example, Kozak, M. 1987. An analysis of 5 ' -noncod g sequences from 699 vertebrate messenger RNAs. Nucl. Acids Res. 15: 8125-8148) . The open reading frame (ORF) and its predicted ammo sequence are shown. Transient transfection assays: To examine the function of the cloned BPl ORF, transient transfection assays were performed. The open reading frame (ORF) described above was sub-cloned to create pRSV/BPl-ORF. K562 cells were transiently co-transfected with increasing amounts of either an empty vector or pRSV/BPl-ORF, along with a target plasmid (Figs. 5A and 5B) . The target DNA consisted of Silencer I (pεCAT/SI) or Silencer II (pεCAT/SII '1 cloned into an expression vector containing the epsilon globm promoter fused to the CAT reporter gene. (This plasmid was oriq nally used in experiments which defined Silencers I and II (see, for example, Berg, P. E., D. M. Williams, R.-L. Qian, R. B. Cohen, S. X. Cao , M. Mittelman, and A. N. Schechter. 1989. A common protein binds to two silencers 5' to the human β-globin αene Nucl. Acids Res. 17: 8833-8852) . CAT activity was normalized against a co-transfected internal standard, pCMV/βgal. Relative CAT activity was calculated as the ratio of CAT activity in cells receiving pRSV/BPl-ORF to the CAT activity of cells receiving only the empty vector. When increasing amounts of pRSV/BPl-ORF DNA were added to pεCAT/SI (Fig. 5A) , CAT activity was repressed in a dose dependent manner, with a plateu beginning at 3 μg of pRSV/BPl- ORF. Likewise, increasing addition of pRSV/BPl-ORF DNA to pεCAT/SII caused increasing repression of the CAT reporter gene (Fig. 5B) , but the lowest amount added, 1 μg, caused a sharp decrease in CAT expression. A control, an εCAT plasmid lacking silencer DNA, was unresponsive to the addition of pRSV/BPl-ORF (data not shown) . Thus, it was shown that BPl exhibits repressor activity towards both silencers of the β-globm gene. Expression Analysis of BPl: Northern analysis was performed to determine the pattern of BPl expression human tissues and cell lines. In placenta, the BPl probe recognized three bands, of 2.1, 2.6 and 3.2 kb (Fig. 3A) . A Northern blot of fetal tissue was also probed, with the results shown m Fig. 3B. There was expression m both fetal kidney and lung (lanes 1 and 3), with bands of 2.1 and 6.3 kb, while no expression was detected m fetal liver or brain (lanes 2 and 4,. The 2.1 kb band likely corresponds to BPl, since it is the sole band in K562 cells from which BPl was cloned. The other bands may represent additional isoforms, commonly associated with homeobox genes (see, for example, Cohen, S. M. and G. Jurgens. 1989. Proximal-distal pattern formation in Drosophila : cell autonomous requirement for Distal-less gene activity in limb development. EMBO J 8: 2045-2055; Lowney, P., J. Corral, M. M. LeBean, L. Deaven, H. J. Lawrence, and C. Largman. 1991. A human Hoxl homeobox gene exhibits myeloid-specific expression of alternative transcripts in human hematopoietic cells. Nucl. Acids Res. 19: 3443-3449. and O Connor, M. B., R. Binaπ , L. A. Perkins, and W. Bender. 1988. Alternative RNA products from the Ultrabithorax domain of the bithorax complex. EMBO J 7: 435-445.) To analyze expression in a broad spectrum of tissues, a dot blot containing normal human RNA from 50 tissues was probed (Table 1, below) . Strong expression was seen in placenta and kidney, and weak expression was observed in cerebral cortex, spleen, mammary gland, small intestine, lung, fetal lung and fetal kidney (the latter two being present on both the Northern blot of Fig. 3B and the RNA dot blot) . RNA from 41 additional tissues showed no expression. TABLE 1 Tissue specific expression of BPl Strong Weak Negative Negative placenta cerebral cortex whole brain thyroid gland kidney spleen amygdala salivary gland mammary gland caudate nucleus pituitary small intestine gland trachea lung cerebellum peripheral fetal lung hippocampus leukocyte fetal kidney frontal lobe lymph node medulla oblongata bone marrow appendix occipital lobe thalamus putamen liver substantia nigra spinal cord fetal spleen temporal lobe fetal thymus subthalamic fetal brain nucleus fetal heart thymus fetal liver heart aorta skeletal muscle colon bladder uterus prostate stomach testis ovary pancreas adrenal gland Thus far, twenty-five human homeobox genes belonging to the OX family have been described that are expressed during normal hematopoietic development and m leukemic cell lines, often in a lineage-specific manner (see, for example, Lawrence, H. J. and C. Largman. 1992. Homeobox genes in normal hematopoiesis and leukemia. Blood 80: 2445-2453) . Therefore, the expression of BPl in erythroid and myeloid lineage cell lines were examined by RT- PCR (Fig. 6) . The strongest expression was seen in erythroid K562 and HEL cells and monocytic THP-1 and U937 cells, with less expression in megakaryocyt ic MEG-01 cells and monocytic/granulocytic HL60 cells. Although the cell lines used in this experiment are often examined for expression of genes in hematopoietic lineages, it should be noted that they are derived from leukemias. As described above, BPl exhioits repressor function. Using increasing amounts of a plasmid containing the ORF causes increasing repression of a target εCAT reporter gene containing Silencer I or II DNA. Expression of the reporter genes was not completely extinguished, which is believed to be due to a possible requirement for binding of additional, low abundance repressor proteins. One such protein may be BP2, which also binds to Silencer II DNA and appears to have repressor function (Berg, P. E., D. M. Williams, R.-L. Qia , R. B. Cohen, S. X. Cao, M. Mittelman, and A. N. Schechter . 1989. A common protein binds to two silencers 5' to the human β-globin gene. Nucl. Acids Res. 17: 8833-8852 and Ebb, D., D. C. Tang, L. Drew, K. Chin, P. E. Berg, and G. P. Rodgers. 1998. Identification of regulatory elements that repress adult beta-like globm genes. Blood Cells, Mol., Dis. 24: 356-369.). The data and the fact that an mtact BPl binding site is required for Silencer II function (see, for example, Ebb, D . , D . C . Tang , L . Dre , K . Chin , P . E . Berg , and G . . Rodgers . 1998. Identification of regulator^ elements that repress adult beta-like globm genes. Blood Cells, Mol., Dis. 24: 356-369.) shows that BPl is most likely a repressor of the β-globm gene. As such, it is the first β-globin repressor to be cloned. BPl is a homeobox gene and a memoer of the Distal-less family (see, for example, Cohen, S. M. , G. Bronner , F. Kuttner, G. Jurgens, and H. Jackie. 1989. Distal -less encodes a homeodomam protein required for limb development n Drosophila . Nature 338: 432-434.). Homeobox genes are known as master regulator genes during development because, upon mutation, vast morphologic abnormalities occur. Distal-less genes are found in organisms as diverse as mice, Xenopus, zebrafish and humans (see, for example, Stock, D. W. , D. L. Ellies, Z. Zhao, M. Ekker, F. H. Ruddle, and K. M. Weiss. 1996. The evolution of the vertebrate Dlx family. Proc. Natl. Acad. Sci. USA 93: 10858-10863) . In Drosophila , Distal-less (called Dll) is required for the normal development of larval sensory organs of the head and thorax. In larval and adult flies, Dll plays an important role in the development of the distal regions of the limbs (see, for example, Cohen, S. M. and G. Jurgens. 1989. Proximal-distal pattern formation in Drosophila : cell autonomous requirement for Distal-less gene activity in limb development. EMBO J 8: 2045-2055 and Cohen, S. M. , G. Bronner, F. Kuttner, G. Jurgens, and H. Jackie. 1989. Distal-less encodes a homeodomam protein required for limb development in Drosophila . Nature 338: 432-434.). There is a single Dll gene Drosophila , but multiple Distal-less genes m other organisms. In mammals, seven Distal-less (called Dlx in mammals) genes have been identified so far. During muπne development, Dlx genes are expressed m branchial arches, forebram, limbs, eyes, teeth, bones and facial mesenchyme (see, for example, Dolle, P., M. Price and D. Duboule . 1992. Expression of the murine Dlx-1 homeobox gene during facial, ocular, and limb development. Differentiation 49: 93-99, Robinson, G. W. and K. Mahon. 1994. Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes mcraniofacial development. Mech. Dev. 48: 199-215 and Simeone, A., D. Acampora, M. Pannese, M. D'Esposito, A. Stornaiuolo, M. Gulisano, A. Mallamaci , K. Kastury, T. Druck , and K. Huebner . 1994. Cloning and characterization of two members of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci. USA 91: 2250-54). BPl and two recently discovered human qenes, DLX7 (see, for example, Naka ura, S., D. W. Stock, K. L. Wydner, J. A. Bollekens, K. Takeshita, B. M. Nagai, S. Chiba, T. Kitamura, T. M. Freeland, Z. Zhao, J. Minowada, J. B. Lawrence, K. B. Weiss, and F. H. Ruddle. 1996. Genomic analysis of a new mammalian Distal-less gene: Dlx-7. Genomics 38: 314-324) and DLX4 (formerly called Dlx8; see, for example, Quinn, L. M. , B. V. Johnson, J. Nicholl, G. R. Sutherland, and B. Kalionis . 1997. Isolation and identification of homeobox genes from human placenta including a novel member of the Distal-less family, Dlx4. Gene 187: 55-61) appear to be isoforms, since their homeoboxes are identical and they exhibit extensive areas of homology. Using fluorescence in situ hybridization, BPl was mapped to chromosome 17q21-23, also the locus of DLX7 and DLX4. Further, BPl and DLX7 (see, for example, Nakamura, S., D. W. Stock, K. L. Wydner, J. A. Bollekens, K. Takeshita, B. M. Nagai, S. Chiba, T. Kitamura, T. M. Freeland, Z. Zhao, J. Minowada, J. B. Lawrence, K. B. Weiss, and F. H. Ruddle. 1996. Genomic analysis of a new mammalian Distal-less gene: Dlx-7. Genomics 38: 314-324 and Price, J. A., D. W. Bowden, J. T. Wright, M. J. Pettenati, and T. C. Hart. 1998. Identification of a mutation in Dlx3 associated with tricho-dento- osseous (TDO) syndrome. Hum. Mol. Gen. 7: 563-569) share complete sequence identity from nucleotides 565 to 1250 of BPl while, further upstream of nt 565, there is no homology. Sequence homology between BPl and DLX4 also begins at nt 565 of BPl and includes the homeobox. The functions of DLX7 and DLX4 are not known. However, abrogation of DLX7 expression in K562 cells causes apoptosis (see, for example, Shimamoto, T., S. Nakamura, J. Bollekens, F. H. Ruddle and K. Takeshita. 1997. Inhibition of Dlx-7 homeobox gene causes decreased expression of GATA-1 and c- myc genes and apoptosis. Proc. Natl. Acad. Sci. USA 94: 3245- 3249). Preliminary data suggests lack of BPl causes apoptosis in K562 cells. A striking feature of BPl is its restricted expression. BPl mRNA is most strongly expressed in placenta and kidney (Table 1 and Fiα. 3) . Although no homeobox sequences were present in the probe, there was partial sequence homology to DLX7. Thus, expression of BPl in placenta and bone marrow by RT-PCT was assessed using BPl-specific primers; a band of the correct size was observed. The seven tissues exhibiting weak hybridization may be expressing BPl and/or DLX7. Forty one tissues lacked BPl expression. It seemed surprising that BPl mRNA was absent in fetal liver and fetal spleen, if BPl represses adult globin gene expression. However, both of these tissues were obtained from 24 week fetuses, by which time β-globin expression has already begun (see, for example, Ley, T. J. , K. A. Maloney, J. I. Gordon and A. L. Schwartz. 1989. Globin gene expression in erythroid human fetal liver cells. J. Clin. Invest. 83: 1032-1038, Mavilio, F. , A. Giampaolo, A. Care, G. Migliaccio, M. Calandrini , G. Russo, G. L. Pagliardi, G. Mastroberardino, M. Marinucci, and C. Peschle. 1983. Molecular mechanisms of human hemoglobin switching: selective undermethylation and expression of globin genes in embryonic, fetal, and adult erythroblasts . Proc. Nat. Acad. Sci. USA 80: 6907-6911, and Papayannopoulou, T. , T.H. Shepard, and G. Stamatoyannoupoulos .1983. Studies of hemoglobin expression in erythroid cells of early human fetuses using anti-γ- and anti-β- globin chain fluorescent antibodies, p.421-430. In G . Stamatoyannopoulos and A.W. Nienhuis (ed.), Globin Gene Expression and Hematopoietic Differentiation. Alan R. Liss, New York). To examine this point further, RT-PCR was performed on four 10 week fetal liver samples. Even by 10 weeks, β-globin transcription has begun (see, for example, Ley, T. J. , K. A. Maloney, J. I. Gordon and A. L. Schwartz. 1989. Globin gene expression in erythroid human fetal liver cells. J. Clin. Invest. 83: 1032-1038) and BPl expression using 30 cycles of amplification samples were not detected; at 35 cycles a detectable band of the correct size in each of the four was observed. Thus, there are very low amounts of BPl in fetal liver; yolk sac tissue is not available to better trace its pattern of expression. At least 25 HOX genes, the largest family of human homeobox genes, are expressed during hematopoietic development, often in a lineage-specific manner (reviewed in Lawrence, H. J. and , C. Largman. 1992. Homeobox genes in normal hematopoiesis and leukemia. Blood 80: 2445-2453. ). However, the functions of these genes are unknown. The identification of BPl as a regulator of the β-globin gene makes this the first human homeobox gene with a known function in hematopoiesis. In addition to the Silencer I and Silencer II binding sites discussed above, additional potential gene targets that may be regulated by BPl have been identified. Sequence analysis was performed to identify DNA sequences with at least 75% homology to known BPl binding sites. Three sites were identified upstream of the gamma globin genes. These sites are located at -1427 bp and -1091 bp upstream of the Gγ-globin gene and at -1091 bp upstream of the Aγ-globin gene. These sites were shown to be binding sites for BPl protein using the electrophoretic mobility shift assay. Creation of a Polyclonal Antibody, Immunoprecipitation and Western Blotting A polyclonal antibody was raised in rabbits against the unique N-terminal BPl peptide SYPYTEPANPGDSYLSCQQ (SEQ ID NO: 13) (Research Genetics) . Freshly prepared K562 cell protein lysate was immunoprecipitated using Protein A Sepharose beads (Bioradj . Equal Amounts of K562 protein lysate, immunoprecipitated lysate and cold in vi trc transcription/translation protein product were used for analysis by 12.5% SDS-PAGE gel. The proteins were electrophoretcally tranferred to nitrocellulose membranes. BPl antibody was diluted in PBS (1:2500) and added for 2 hours at room temperature, then the anti-rabbit secondary antibody was added. Detection of the signal was accomplished using an ECL kit according to the recommended protocol (Amersham Pharmacia Biotech) Creation of a Plasmid containing Antisense BPl DNA A BPl DNA fragment of 239 bp (nt 219 tc 558) was cloned into the vector pMT in an antisense orientation, under control of the sheep metallothionein promoter, as follows. RT-PCR was performed using primers to amplify DNA between nt 219 and nt 1231 of the BPl cDNA. This fragment was blunt ended and cloned into pGEM7. A 5' BamHI site in the polylmker of pGEM7 and an Xho I site at nt 558 (internal to BPl), were used to release BPl DNA from the vector. This BPl fragment of 239 bp was directionally cloned in an antisense orientation into pMT . (see, for example, Canelles, M., Delgado, M.D., Hyland, K.M. Lerga, A., Richard, C, Dang, C.V.and Leon, J. (1997) Max and inhibitory cMyc mutants induce erythroid differentiation and resistance to apoptosis human myeloid cells. Oncogene 14, 1315 - 1327.) Treatment of Sickle Cell Anemia A mutation in the sixth codon of the beta globin gene causes sickle cell anemia. Current treatments of sickle cell anemia are focused on reactivation of the fetal hemoglobin genes. Such treatments may include gene therapy, which would be used either to replace the beta globm gene or to add a fetal globin gene engineered to overexpress fetal globm protein. However, these treatments are hampered by the presence of the defective sickle cell beta protein, which interferes with the formation of normal hemoglobin. As discussed above, there are two silencer regions upstream of the β-globm gene and two proteins which bind to them, BPl and BP2 (see, for example, Berg, P. E., D. M. Williams, R.-L. Qian, R. B. Cohen, S. X. Cao, M. Mittelman, and A. N. Schechter. 1989. A common protein binds to two silencers 5' to the human β-globm gene. Nucl. Acids Res. 17: 8833-8852). BPl binds within Silencer I at -530 bp relative to the cap site (+1) and within Silencer II at -300 bp, while BP2 binds within Silencer II at -270 bp, shown in Fig. 1. Interestingly, it was found that High Mobility Group Protein I (HMG-I (Y) ) , an architectural protein, binds to and benos the DNA at or near the BPl binding site Silencer I, but not in Silencer II (see, for example, Chase, M. B., S. Haga, W. D. Hankins, D. M. Williams, Z. Bi , J. W. Strovel, C. Obriecht, and P. E. Berg. 1999. Binding of HMG-I (Y) elicits structural changes in a silencer of the human β-globm gene. Am. J. Hem. 60: 27-35). HMG-I (Y) may facilitate binding of BPl and possibly other repressor proteins m this region. Mutation of the BPl site in Silencer II partially relieves repression (see, for example, Ebb, D . , D . C . Tang, L . Drew, K. Chi , P . E . Berg, and G . P . Rodgers . 1998. Identification of regulatory elements that repress adult beta-like globm genes. Blood Cells, Mol., Dis. 24: 356-369), suggesting that BPl may act as a repressor of the β-globin gene. Furthermore, BPl binds m a negative regulatory region upstream of the other adult globin gene, delta (see, for example, Berg, P.E., S. Abhyankar, and M. Chase. 1994. The high mobility group protein HMG-I (Y) binds to a silencer DNA sequence upstream of the human β- glob gene. Blood 84 Suppl 1: 262a) . The inventor believes that BPl coordmately regulates adult globm gene expression. BPl protein may be directly involved in the switch from fetal to adult hemoglobin during development since it binds to DNA near the fetal gamma globin genes, as well as near the adult delta and beta globm genes. Experiments have demonstrated that BPl binding activity is increased upon pharmacologic reactivation of fetal globm genes. Since BPl is believed to activate fetal genes while repressing adult genes, it can potentially be used to treat sickle cell anemia, as an alternative to treatments involving adding normal beta gene or gamma gene. An inverse correlation between the binding affinity of BPl and the severity of sickle cell anemia (SCA) has been observed (see, for example, Elion, J. , P. E. Berg, C. Lapoumeroulie , G. Trabuchet, M. Mittelman, R. Kπshnamoorthy, A. N. Schechter, and D. Labie. 1992. DNA sequence variation m a negative control region 5' to the β-globm gene correlates with the phenotypic expression of the β° mutation. Blood 79: 787-792.). In sickle cell anemia, the mutated β-globm gene (β s ) is m strict linkage disequilibrium with five restriction haplotypes (see, for example, Kulozik, A. E., J. S. Wainscoat, G. R. Serjeant, B. D. Kar, B. Al- Awamy, G. J. F. Essan, A. G. Falusi, S. K. Hague, A. M. Hilali, S. Kate, W. A. E. P. Ranasmghe, and D. J. Weatherall . 1986. Geograpmcal survey of β s -globm gene haplotypes: Evidence for an independent Asian origin of the sickle-cell mutation. Am. J. Hum. Genet. 39: 239-244; Lapoumeroulie, C, O. Dunda, G. Trabuchet, M. Mony-Lobe, D. Labie, J. Elion, and R. Krishnamoor h . 1989. A novel sickle gene of yet another origin m Africa: The Cameroon type. Blood 74: 225a and Pagnier, J. , J. G. Mears, O. Dunda- Belkhod^a, K. E. Schaefer-Rego , C. Beldjord, R. L. Nagel, andD. Labie. 1984. Evidence of the multicentπc origin of the hemoglobin S gene m Africa. Proc. Natl. Acad. Sci. USA 81: 1771-1773.). There are differences m the clinical severity of sickle cell anemia among the haplotypes, with the Indian-Arabo (hereafter referred to as the Indian haplotype) being the mildest and the Bantu the most severely affected (see, for example, Ali, S. A. 1970. Milder variant of sickle cell disease in Arabs in Kuwait associated with unusually high level of foetal haemoglobin. Br. J. Haematol. 19: 613-619 and Perrine, R. P., M. E. Pembrey, S. Perπne, and F. Shoup. 1978. Natural history of sickle cell anemia in Saudi Arabs. A study of 270 subjects. Ann. Internal. Med. 88: 1-6) . A sequence polymorphism within the -530 bp binding site for BPl is also m linkage disequilibrium with these five SCA haplotypes (see, for example, Chebloune, Y., J. Pagnier, G. Trabuchet, C. Faure, G. Verdier , D. Labie, and V.M. Nigo . 1988. Structural analysis of the 5 flanking region of the β-g obm gene m African sickle cell anemia patients: further evidence for three origins of the sickle cell mutation m Africa. Proc. Natl. Acad. Sci. USA 85: 4431-4435) . Using electrophoretic mobility shift competition assays, it was found that BPl binds five to six times more tightly to the Indian naplotype sequence than to the normal or reference sequence (see, ror example, Berg, P. E., M. Mittelman, J. Elion, D. Labie, and A. N. Schechter. 1991. Increased protein binding to a -530 mutation of the human β-globm gene associated with decreased β- globin synthesis. Am. J. Hematol . 36: 42-47; Elion, J. , P. E. Berg, C. Lapoumeroulie, G. Trabuchet, M. Mittelman, R. Krishnamoo thy, A. N. Schechter, and D. Labie. 1992. DNA sequence variation in a negative control region 5' to the β-globin gene correlates with the phenotypic expression of the β s mutation. Blood 79: 787-792 and Zeng, F.-y., G. P. Rodgers, S.-z. Huang, A. N. Schechter, M. Salamah, S. Perrine, and P. E. Berg. 1994. Sequence of the -530 region of the beta globin gene of sickle cell anemia patients with the Arabian haplotype. Human Mutation 3: 163-165) . On the other hand, BPl binds two to three times more weakly to the Bantu haplotype sequence than the reference sequence (see, for example, Elion, J. , P. E. Berg, C. Lapoumeroulie, G. Trabuchet, M. Mittelman, R. Krishnamoorthy, A. N. Schechter, and D. Labie. 1992. DNA sequence variation in a negative control region 5' to the β-globin gene correlates with the phenotypic expression of the β s mutation. Blood 79: 787-792) , which may allow more transcription of β s resulting in an increased concentration of Hemoglobin S. This correlates with the increased severity of sickle cell disease observed in Bantus. As stated above, BPl is a strong candidate for use in therapy of sickle cell anemia (SCA) . Decreasing β s -globin expression is expected to decrease the intracellular concentration of HbS, in turn decreasing the intracellular HbS concentration and polymer formation, believed to be the primary cause of the clinical effects of SCA (see, for example, Schechter, A. N. , C. T. Noguchi and G. P. Rodgers. 1987. Sickle cell disease, p. 179-218. In G. Stamatoyannopoulos, A. W. Nienhuis, P. Leder, Majerus, P. W. (ed.), The Molecular Basis of Blood Diseases. Saunders, Philadelphia) . In addition, treatment with a repressor such as BPl may also indirectly reactivate fetal globin genes. This idea is based on well established observations, both in vivo and in vitro, which demonstrate reciprocal regulation of fetal and adult globin genes. Dover and Boyer (see, for example, Dover, G. J. and S. H. Boyer. 1987. Fetal hemoglobm-contammg cells have the same mean corpuscular hemoglobin as cells without fetal hemoglobin: a reciprocal relationship between gamma- and beta-globin gene expression normal subjects ano in those with high fetal hemoglobin production. Blood 69: 1109-1113) demonstrated that reciprocal regulation exists in individuals who express high levels of HbF and low levels of HbA due to SCA, heterocellular hereditary persistence of fetal hemoglobin (HPFH) , and β- thalasse ia. Perrine et al (see, for example, Perrine, S. P., B. A. Miller, M. F. Greene, R. A. Cohen, N. Cook, C. Shackleton, and D. V. Faller. 1987. Butyric acid analogues augment γ globin gene expression in neonatal erythroid progenitors. Biochem. Biophys . Res. Comm. 148: 694-700.) showed that two factors, α-amino-n- butyric acid and insulin, present in high levels in the plasma of infants born to diabetic mothers, increase γ-globin expression accompanied by decreased β-globm expression. Furthermore, cultured erythroid progenitors from β-thalassemia and SCA patients, when treated with sodium butyrate, 4-phenyl butyrate or phenylacetate exhibited an increase in γ-globm expression and a reciprocal decrease in β-globin expression (see, for example, Fibach, E., P. Prasanna, G.P. Rodgers, and D. Samid. 1993. Enhanced fetal hemoglobin production by phenylacetate and 4- phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and β-thalassemia . Blood 82: 2203-2209 and Perrine, S.P., B. A. Miller, D. V. Faller, R. A. Cohen, E. P. Vichinsky, D. Hurst, B. H. Lubm, and T. Papayannopoulou . 1989. Sodium butyrate enhances fetal globm gene expression m erythroid progenitors of patients with HbSS and β thalassemia. Blood 74: 454-459). Since current treatments of SCA focus on chemical reactivation of fetal globm genes, repression of HbS may reduce or eliminate the need for such chemicals. As a treatment of sickle cell anemia, the preferred method is to incorporate DNA encoding BPl into a vector that can be administered to a patient by gene therapy, preferably targeted to tissues that express globin genes. To avoid potential problems caused by overexpression of BPl, the DNA could be linked to a controllable promoter. Screening of Leukemia Acute myeloid leukemia (AML) is the second most frequent pediatric leukemia and the most frequent adult leukemia (see, for example, Pui C-H. Childhood leukemias . New Eng J Med 1995; 332: 1618-1630 and Karp JE. Acute leukemia: mechanisms of cell survival as targets for therapy. Int J Oncol 1997; 11: 657-674.). Survival is poor m AML, with only 30-40% survival m children and 10-35% survival m adults. On the other hand, acute lymphocytic leukemia (ALL) is the-most frequent leukemia m children, but is rare in adults. Although several molecular markers have been associated with AML and ALL (see, for example, Look AT. Oncogenic transcription factors m the human acute leukemias. Science 1997; 278: 1059-1064; Tenen DG, Hromas R, Licht JD, Zhang D-E. Transcription factors, normal myeloid development and leukemia. Blood 1997; 90: 489-491; and Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role of HOX homeobox genes m normal and leukemic hematopoiesis. Stem Cells 1996; 14: 281-291), there is clearly a need to identify additional markers which could aid in diagnosis and in determining appropriate therapy, and/or potentially serve as therapeutic targets. Altered expression of transcription factors, including homeobox genes (HB) , has been implicated in acute leukemias, and aberrant expression of homeobox genes has been described m leukemia cell lines and m primary leukemia blasts. Homeobox genes are characterized by a conserved 180 bp DNA sequence encoding the DNA binding domain (see, for example, Levme M, Hoey T. Homeobox proteins as sequence-specific transcription factors. Cell 1988; 55: 537-540). They are "master regulator genes" which are extremely important to the physical development of a wide range of organisms. Recent studies have demonstrated that HB genes are also expressed during hematopoiesis (see, for example, Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role of HOX homeobox genes in normal and leukemic hematopoiesis. Stem Cells 1996; 14: 281-291' 1 . There are several families cf HB genes, defined by the extraordinary sequence conservation among their homeoboxes. The largest family, consisting of 39 members m humans, is called HOX. Some leukemias aberrantly express homeobox genes as fusion proteins (reviewed in Look AT. Oncogenic transcription factors in the human acute leukemias. Science 1997; 278: 1059-1064). In these cases, DNA encoding the activation domain from one gene is fused to DNA encoding the DNA binding region of another, bringing a different function to the new protein product. The downstream targets of fusion genes associated with AML or ALL have yet to be determined. In pre-B-cell lymphoblastic leukemia, for example, the HB gene Pbxl is fused to E2A (see, for example, Lu Q, Wright DD, Kamps MP. Fusion with E2A converts the Pbxl homeodomam protein into a constitutive transcriptional activator in human leukemias carrying the t(l;19) translocation. Mol Cell Biol 1994; 14: 3938- 3948) and, m acute myeloid leukemia (AML), HOXA9 is fused to NUP98-HOXA9 (see, for example, Borrow J, Shearman A.M., Stanton Jr. V.P., Becher R, Collins T, Williams AJ, Dube I, Katz F, Kwong YL, Morris C, Ohyashiki K, Toyama K, Rowley J, Housman DE. The t (7 ; 11) (pl5;pl5) translocation acute myeloid leukaemia fuses the genes for nucleoponn NUP98 and class I homeoprotein HOXA9. Na ture Genetics 1996; 12: 159-167 and Nakamura T, Yamazaki Y, Hatano Y, Miura I. NUP98 is fused to PMXl homeobox gene in human acute myelogenous leukemia with chromosome translocation t (1;11) (q23;pl5) . Blood 1999; 94: 741-747). In other cases of leukemia not involving obvious cytogenetic changes, a gene which is normally silent is either activated or pernaps not downregulated m an early hematopoietic progenitor. This was observed for four of nine HOXB genes which were expressed in T- or B-ALL, but not m normal T or B lymphocytes (see, for example, Petrini M, Quaranta MT, Testa U, Samoggia P, Tπtarelli E, Care A, Cianetti L, Valtieπ M, Barletta C, Peschle C. Expression of selected human HOX genes in B/T acute lymphoid leukemia and ιnterleukιn-2/ιnterleukm-l β-stimulated natural killer lymphocytes. Blood 1992; 80: 185-193). BPl expression was investigated in erythromyeloid and lymphoid leukemia cell lines and in bone marrow samples from adult and pediatric acute myeloid leukemia (AML) , pediatric acute T-cell lymphocytic leukemia (ALL) and pediatric pre-B-cell ALL. Because there are two apparent isoforms of BPl called DLX7 and Dlx4, their expression m the same samples was measured. All three are co- expressed in erythroid and monocytic cell lines and in at least half of the lymphoid cell lines. They are barely if at all detectable in normal bone marrow, PHA-stimulated T cells or B cells. BPl RNA was found m 63% of AML cases, including 81% of the pediatric and 47% of the adult cases, and m 32% of T-ALL cases, but was not expressed m any of the pre-B ALL cases. Similar results were obtained with DLX7 and Dlx4, with co-expression of all three in a significant number of leukemias. The data shows that BPl expression occurs in early progenitors. The presence of BPl RNA m leukemic blasts may therefore be a molecular marker for primitive cells and/or may indicate BPl is an important upstream factor m an oncogenic pathway. The hypothesis that BPl is leukemogenic is supported by the present studies showing that overexpression of BPl causes increases clonogenicity in the cell line K562, as well as causing decreased ability of these cells to undergo erythroid differentiation. Overexpression of BPl in the erythroeleukemia cell line K562 results in increased growth m soft agar, which is an indicator of leukemogenic potential. BPl expression is believed to cause increased proliferation and survival and decreased differentiation in K562 cells. Similar results were found m K562 cells when either of the two well known oncogenes were overexpressed, c-myc and c-myb. Therefore, the data suggests that BPl functions as an oncogene in AML and T-cell ALL conditions . The studies carried out tc determine BPl expression are described in greater detail below: Materials and Methods: Culture of leukemic cell lines: T and B-ALL cell lines were grown RPMI 1640 (Gibco-BRL ' ) supplemented with 2 mM glutamme, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential 3C amino acids, 1 mM sodium pyruvate and 10% fetal bovine serum. Erythromyeloid cell lines were cultured in RPMI 1640 supplemented with 2 mM glutamme, 100 U/ml penicillin and 100 μg/ml streptomycin, 10 mM HEPES and 10% fetal bovine serum. Clinical samples: Normal bone marrows (BM) were obtained from the Brain Tissue Bank for Developmental Disorders at the University of Maryland. Adult AML bone marrow samples were obtained from the University of Maryland Greenebaum Cancer Center and The Johns Hopkins Cancer Center Cytogenetics Core Laboratory. Bone marrow samples from pediatric T and B- ALL patients were obtained from the Pediatric Oncology Group (POG) . Informed consent was obtained from each patient or guardian at their local institutions. Leukemia samples contained at least 85% blasts. T and B lymphocytes from peripheral blood of normal donors were obtained by countercurrent elutriation followed by lineage specific separation using magnetic beads (Miltenyi Biotec, Auburn, CA) . RT-PCR: Mononuclear cells were isolated for RNA analysis by Ficoll-Paque . Total RNA was extracted from all samples using TRIzol Reagent (Gibco-BRL, Gaithersburg, MD) . For the reverse transcription (RT) reaction, one microgram of DNase I-treated RNA was added to 2.5 μM olιgo-d(T) primers ( PE Biosvstems), 10 U RNase inhibitor (Gibco-BRL), 0.5 mM dNTPs, and 100 U MMLV-reverse transcriptase (Promega). The RT reaction was performed in a thermal cycler at 25° C for 10 minutes, 4 minutes ramp, 42° C for 50 minutes, and 99° C for 5 minutes. One to two microliters of this reaction was used in the PCR reaction. Primers specific for BPl, DLX4 and DLX7 were designed, and the PCR products were verified by restriction enzyme analysis. Linearity assays for each primer set have been performed and cycling conditions adjusted accordingly (data not shown) . Primer sequences and PCR cycling conditions are in the following Table 2. TABLE 2: PCR Primers Primers Sequence PCR conditions Product Reference BPl Upper: 94° C/l min.; 62° C/l 581 bp 5 ' CACCTCCTGTCTTACC min. ; CCTACACC3' (SEQ ID 72° C/1.5 min. for 30 NO: 8) cycles Lower: 5 ' GCCCTTCCCCAGATTC ACATCATC3' (SEQ ID NO: 9) DLX7 Upper: 94° C/l min. ; 60° C/l 406 bp 5 ' CCTACACCGTGTTGTG min . ; CTGC3 ' (SEQIDNO 14) 72° C/1.5 mm. for 30 Lower: cycles 5 ' CTGTTGCCATAGCCAC TG3 ' (SEQ ID NO: 15) DLX4 Upper: 94° C/l min.; 60° C/l 350 bp 5 ' CACGGTGTGGCGGGGG min . ; AGACAT3' (SEQ ID 72° C/1.5 min. for 30 NO: 16) cycles Lower: 5 ' CTGCGGTGGGAGGTCG GAGTTC3' (SEQ ID NO: 17) β-actin Upper: 94° C/l min. ; 60° C/l 626 bp Raff et 5 ' GGATCTTCATGAGGTA min. ; al, 1996 GTCAGTC3' (SEQ ID 72° C/1.5 min for 20 NO: 18 j cycles Lower: 5 ' CCTCGCCTTTGCCGAT CC3 ' (SEQ ID NO: 19) c-myb Upper : 94° C/30 sec; 55° C/30 228 bp Majello et 5 ' ATTAGGTAATGAATTG sec . ; al, 1986 TAGCCAG3' (SEQ ID 72° C/l mm. for 28 NO: 20) cycles Lower : Shimamoto 5 'ACTTAGAGTAATGCTT et al, TTACTGA3' (SEQ ID 1997 NO: 21) GATA-1 Upper: 94° C/30 sec; 58° C/30 249 bp Tsai et 5 ' CCATTGCTCAACTGTA sec; al, 1989 TGGAGGG3' (SEQ ID 72° C/l mm. for 28 NO: 22) cycles Lower : Shimamoto 5 ΑCTATTGGGGACAGGG et al, AGTGATG3' (SEQ ID 1997 NO: 23) SCL Upper: 94° C/45 sec; 55° C/45 144 bp Chen et 5 ' CAATCGAGTGA sec . ; al, 1990 AGAGGAGACCTCC3 ' 72 ° C/ 1 . 5 mm for 30 (SEQ ID NO: 24) cycles Lower : 5 ' TTGCGGAGCTCGGCAA AGGC3' (SEQ ID NO: 25) Semi-quantitative RT-PCR: An oligonucleotide specific for each product was end-labelled with γ- 32 P-dATP and added to 10 percent of the PCR product (2.5 μl ) . Hybridization was in the thermal cycler (94° C for 15 seconds; 42° C for 1 minute) . The hybridized product was electrophoresed on a 5% polyacrylamide gel, the gel was dried, and then exposed to film. The autoradiograph was aligned with the gel and bands were excised and quantitated by scintillation counting. Expression was scored as negative (-) , positive (+) or ambiguous (+/-) by normalizing against β-actin: a ratio was calculated by dividing the cpm in the band representing BPl, DLX7 or DLX4 by the cpm in the β-actin band from the same sample. One normal BM and one remission BM were included in each AML experiment; their ratios (6 repeats of each) averaged 0.01 for BPl, DLX7 and DLX4 , and were the guide against which a sample was scored as -(0.0- 0.10), +/-(0.11-0.15) , +00.15) or ++(>0.45). Two phytohemagglutinin (PHA) -stimulated normal T cell cultures were the control for T-cell ALL; their average ratios (10 repeats) were 0.05; the same criteria were used for scoring BPl, DLX7 and DLX4 as above, c-myb and GATA-1 are expressed in normal BM (our observations and also noted in Gewirtz AM, Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science 1988; 242: 1303-1306. Guerrasio A, Saglio G, Rosso C, Alfarano A, Camaschella C, Lo Coco F, Biondi A, Ranbaldi A, Nicolis S, Ottolenghi S. Expression of GATA-1 mRNA in human myeloid leukemic cells. Leukemia 1994; 6: 1034-1038). The average ratios in BM for c-myb and GATA-1 were 0.57 and 2.1, respectively. Samples with a ratio between 0.10 and 1.14 { c-myb) or 0.10 and 4.2 (GATA-1) were scored as (+), i.e., within the range of normal BM, and ratios greater than 1.14 { c-myb) or 4.2 (GATA-1) were scored as (++). Each RT-PCR and hybridization was performed twice, independently. Isolation of CD34 cells: Bone marrow cells were aspirated from the posterior iliac crest of consenting healthy adult donors following guidelines approved by the Institutional Review Board for NIH. Cells were first prepared with Histopaque-1077 density gradient centrifugation. Mononuclear cells were then incubated with CD34 (QBEndlO) -conjugated magnetic microbeads ( AmCellCorp, Sunnyvale, CA) and processed through a MACS magnetic separation column ( Miltenyi Biotec, Bergisch Gladbach, Germany ) to obtain purified CD34+ cells. For higher purity of CD34+ cells, a second column run was used. Purity of isolated CD34+ cells was generally greater than 90%, and cell viability as evaluated by propidium iodide exclusion was always higher than 95%. Purity of the remaining CD34- cells was greater than 95%. Construction of cell lines overexpressing BPl: A BPl cDNA fragment of 1013 bp containing the complete open reading frame was amplified by RT-PCR from K562 cell RNA using primers designed from the original cDNA. This fragment was cloned into pGEM7, sequenced, then sub-cloned into pRC/RSV (Invitrogen) using Hind III and Xba I. Statistical Analysis: The significance of correlation between expression of BPl, DLX4, and DLX7 was assessed by Fisher's exact test. All p-values reported were two-sided. Detailed Results: Expression of BPl in normal cells: RNA levels for BPl were determined in four normal bone marrow samples and one bone marrow from an AML patient in remission, and in five PHA-stimulated T- cell and five B-cell preparations from normal bone marrow. Results obtained after semi-quantitative RT-PCR analysis of six representative samples are shown in Fig. 8. BPl RNA was barely visible in any of these control samples; results were similar for DLX4 and DLX7. After a longer exposure of the autoradiograph, a faint band was seen in all six samples for several of the isoforms (data not shown) . Expression of BPl in cell lines: Expression of BPl was examined by RT-PCR in a number of leukemia cell lines. Fourteen cell lines of lymphoid origin were analyzed for BPl expression. BPl RNA was present in four of eight T-cell ALL cell lines and in three of four lymphoma cell lines (Table 3) . TABLE 3. Expression of BPl in Hemopoietic Cell Lines BPl BPl Lvmphoid Expression Mveloid Expression B Cell Lineage Erythroid Normal +/- K562 + ALL HEL + REH + Monocytic RS4;1 1 + THP-1 + Burkett's lymphoma U937 + Raji - Megakarycocytic Daudi + MEG-01 +/- Monocytic/granulocytic T Cell Lineage HL60 +/- Normal +/- ALL Jurkat + MOLT-3 + MOLT-4 + MOLT- 13 + CCRF-CEM - HSB2 - MOLT- 16 - RPMI 8402 - Lymphoma HUT78 + Sup-Tl + DLX7 and DLX4 RNA levels were also determined m the T-cell ALL cell lines (Fig. 7A) , with co-expression of the three isoforms in the same cell lines. BPl mRNA was also readily detectable in four of six erythromyeloid cell lines (K562, HEL, THP-1 and U937), with less expression in MEG-01 and HL60 cells. 1 Analysis of DLX7 and DLX4 in those cell lines showed that BPl, DLX7 and DLX4 were frequently co-expressed, with greatest expression of all three in K562 and U937 cells (Fig 7B) . The observation that there was little or no expression of any of the isoforms normal bone marrow (described above) , compared with the expression found in diverse myeloid and lymphoid leukemia cell lines, led us to examine the relative expression of BPl, DLX7 and DLX4 in the bone marrow of acute leukemia patients using semi-quantitative RT-PCR. Expression of BPl acute myeloid leukemias: Expression of BPl was examined m both pediatric and adult AML patients. A total of 39 AML patients were studied, of whom 18 were under the age of 18 (pediatric; cases 8-27) and 21 were 18 years of age or older (adult; cases 29-49). Table 4, below, summarizes the analysis of the AML samples and the clinical data available for each patient, including cytogenetics, expression of surface markers and initial response to treatment; outcome data was not available for all patients and thus was excluded. Data are grouped according to the French-American-British (FAB) criteria, (see, for example, Bennett M, Catovsky D, Daniel M , Flandrm G, Galton DAG, Gralnick HR, Sultan C. Proposed revised criteria for the classification of the French-American-British Cooperative Group. Ann In tern Med 1985; 103: 620-625. Assignment of expression levels was made as described in Materials and Methods. For this analysis, normal bone marrow was used as a negative baseline against which the clinical samples were compared. Samples classified as +/- were excluded from statistical analysis. BPl was overexpressed in a definitive manner m 81% (13/16) of the pediatric bone marrow samples and m 47% (9/19) of the adult cases. DLX7 was overexpressed 59% (10/17) of pediatric and 38% (6/16) of adult cases, while DLX4 was overexpressed in 79% (11/14) of pediatric and 79% (15/19) of adult cases. An example of RT-PCR analysis showing expression in selected samples is seen in Fig. 9. Here, patient 15 showed the greatest BPl and DLX7 levels, with less DLX4 expression. The correlation between BPl and DLX7 (p=0.0002), BPl and DLX4 (p=0.0016), and DLX7 and DLX4 (p=0.023) were statistically significant by Fisher's exact test. Table 4. Expression of BP1, DLX& and DLX4 and clinico-biological features in patients with AML FAB Patient BP1 DLX7 DLX4 c-mvb GATA-1 CD34 CD33 CD13 Karyotype Response * Normal B.M. + + MO 1 2 * ++ ND PR 37 +/- 46XYt{115)1rιsomylq NR 47 - ND ND 4 7,XY,+13 NR M1 15" ++ 4 6XYt(119)(q23,p131) CR 17* +/- 6,Y.t(X,7)(q13,p15),dup(1 2 )(p11p13) CR 19- - 46 XX CR 22* ++ ++ 4 7,XX,+21 ND 26* + +/- 46 XX CR 27* - 46.XY CR 29 - 46,XYdel(5)(q31q34) NR 0 ++ +/- 46,XY,t(1,12) CTC 46 + ND ND 6 XY del(1)(q32l,del(13)(q12-22) CR M2 18* - 4 7,XX,+8 CR 20- + 4 6XY CR ΛΛ - ND ND ND 46.XY CR 45 - ND ND 45 X,-Y CR ( 6 mo ) -18 + ND ND ND 4 7,XY,+8 NR M3 46XXK1517)(q21 q12-21) CR M4 23* 46,XX,ιnv(16)(p13q22) CR 31 45 XY -7 NR 32 45.XX.-16 NR 35 46 XX NR 49 ND ND 46,XY,ιnv(16)(p13q22) CR M5 8" 46 XX t( 11 17),q23 25) CR 9 * ND CR ii- 46 XY NR is * 4 6,XY,der(1)t(1,6)(q32,p211), CR add(11)(q23) der(22)t(1,22)(q23,q112) 14- 6XX CR 16 * 4 6,XX NA 30 47 XY +3 NA 33 46.XY NR 34 48 XX add(4),p151) del(5)(q21q33) NR t(712)(p10q10)add(11>(q23) +ι(12)(p 10) add(16)(q112) -17 +18 -20 +der(21)t{1721)(q10 q10) +mar 36 4 6,XY del(7)(q22),+(8)(q2 4 3) NR 38 46 XX CR 39 48,XY.t(1,16)+7,+19 CTC 42 47 X t(69)fq23 34)+8 RD 43 ND ND 46 XY, t(3,5)(q25,q34) NR M7 2V 46 X CR 24 * 45XYt(33)(q21,q26)-7 NR • AGE LESS THAN 18 "COMPLETE RESPONSE(CR) NO RESPONSE (NR) PARTIAL RESPONSE (PR) COMPLETE TUMOR CLEARANCE (CTC) RESIDUAL DISEASEIRDl. NOT DETERMINED (ND). DATA NOT AVAILABLE(NA) Analysis of GATA-1 and c-myb, markers of primitive cells, was performed on the AML samples to indicate the differentiation state of cells expressing BPl. GATA-1 is believed to be involved in positive regulation of myeloid development and thought to be expressed in early progenitors, then downregulated in myeloid (but not erythroid) differentiation, making its expression in the myeloid pathway a sign of immaturity. (see, for example, Crotta S, Nicolis S, Ronchi A, Ottolenghi S, Ruzzi , Shi ada Y, Migliaccio AR. Progressive mactivation of the expression of an erythroid transcriptional factor m GM- and G-CSF-dependent myeloid cell lines. Nucl Acids Res 1990; 18: 6863-6869) It is detected m acute leukemias characterized by expansion of early progenitors. (see, for example, Guerrasio A, Saglio G, Rosso C, Alfarano A, Camaschella C, Lo Coco F, Biondi A, Ranbaldi A, Nicolis S, Ottolenghi S. Expression of GATA-1 mRNA in human myeloid leukemic cells. Leukemia 1994; 6: 1034-1038) c-myb is expressed in immature hematopoietic cells, and its expression decreases as cells differentiate. (see, for example, Gewirtz AM, Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis m vitro. Science 1988; 242: 1303- 1306; Gonda T, Metcalf D. Expression of myb, myc and fos proto- oncogenes during the differentiation of a muπne myeloid leukemia. Na ture 1984; 310: 249-251 and Luscher B, Eisenman RN. New light on Myc and Myb. Part II. Myb. Genes & Devel 1990; 4: 2235-2241.) Since c-myb and GATA-1 are expressed in normal bone marrow (data not shown), (see, for example, Gewirtz AM, Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis m vitro. Science 1988; 242: 1303- 1306 and Guerrasio A, Saglio G, Rosso C, Alfarano A, Camaschella C, Lo Coco F, Biondi A, Ranbaldi A, Nicolis S, Ottolenghi S. Expression of GATA-1 mRNA in human myeloid leukemic cells. Leukemia 1994; 6: 1034-1038.) A classification of positive was given to samples expressing c-myb at the level of normal cells (see above for details) . It was found that every AML sample exhibited c-myb expression at least equal to that in normal bone marrow, and 42% (13/31) exhibited greater expression. A higher proportion of pediatric than adult cases showed high level c-m b expression, 59' children compared with 21% in adults. GATA-1 was present in 77% (24/31) of AML cases, with 72% expression in children and 85% in adults. Co-expression of DLX7 and DLX4 with BPl was striking: 84% of BP1+ samples were DLX7+ and 100% were DLX4+. GATA-1 was co-expressed in 74% of the samples, and all were c-jnyr positive, with 45% exhibiting high levels of c-myb. Another parameter in this study was analysis of surface markers. Interestingly, 64% of the BPl positive samples were CD34 negative, while 73% were CD13 positive and 100% were CD33 positive. Among the 39 AML patients, two had an abnormal chromosome 17q arm, the locus of BPl. (see, for example Fu S, Strovel JW, Haga SB, Stamberg J, Berg PE. Mapping of a new homeobox gene, BPl, near its isoform DLX7 and characterization of their roles in repression of the beta globin gene. Am . J. Hum . Gen . 1998; 63: A181.) Patient 41, with acute promyelocytic leukemia, exhibited a t(15;17) translocation presumably involving the retinoic acid receptor on chromosome 17. (see, for example, Look AT. Oncogenic transcription factors in the human acute leukemias. Science 1997; 278: 1059-1064.) Patient 8 exhibited a t(ll;17) (q23;q25) translocation. This translocation has been identified as fusing the MLL gene on chromosome llq23 to either the AF17q25 gene or to MSF, both located on 17q25 and differing by a single base. (see, for example, Baer MR, Stewart CC, Lawrence D, Arthur DC, Mrozek K, Strout MP, Davey FR, Schiffer CA, Bloomfield CD. Acute myeloid leukemia with llq23 translocat ions : myelomonocytic immunophenotype by multiparameter flow cytometry. Leukemia 1998; 12:317-325; Taki T, Ohnishi H, Shinohara K, Sako M, Bessho F, Yanagisawa M, Hayashi Y. AF17q25, a putative septin family gene, fuses the MLL gene in acute myeloid leukemia with t(ll;17) (q23;q25). Cancer Res 1999; 59: 4261-4265 and Osaka M, Rowley JD, Zeleznik-Le NJ. MSF (MLL septm-like fusion) , a fusion partner gene of MLL, in a therapy-related acute myeloid leukemia with a t (11;17) (q23;q25) . Proc Na tl Acad Sci USA 1999; 96: 6428- 6433.) Since the MLL gene is known to activate several HOX genes, (see, for example, Yu BD, Hess JL, Horning SE, Brown GAJ, Korsmeyer S. Altered Hox expression and segmental identity in Mil-mutant mice. Na ture 1995; 378: 505-508.) it is tempting to speculate that the fusion protein may activate BPl in this case. Expression of BPl in acute lymphoid leukemias: For comparison with the myeloid lineage leukemias, nineteen cases of pediatric T-cell ALL were examined (Table 5) . TABLE 5. Expression of BPl, DLX7 and DLX4 in Children with T-Cell ALL Patent BPl DLX7 DLX4 1 2 - + - 3 - + - 4 + - - 5 + + + 6 - - - 7 - + + 8 - - - 9 - - - 10 + + + 11 - nd - 12 + - - 13 - - + 14 + + + 15 - nd nd 16 + + + 17 - nd nd 18 - nd nd 19 - nd nd Here, 32% (6/19) were BPl positive. Outcome data were not available for these patients. Analysis of DLX7 and DLX4 showed that 40% (6/15) of the cases were DLX7 positive, and 40% (6/15; were DLX4 positive. A comparison of expression of the three isoforms in AML and T-cell ALL is seen in Fig. 10. Next, 19 pediatric patients with pre B-ALL were analyzed. No detectable BPl was observed in any of these cases, although the β-actin controls were normally expressed (data not shown) . BPl expression in CD34+ and CD34- cells: To examine more precisely the reason for very low BPl expression in normal bone marrow and to determine whether BPl is expressed in early progenitors, BPl expression was measured in CD34+ and CD34- cells. There was clear expression in two independent isolates of CD34- cells (Fig. 11, lanes 2 and 3) . In contrast, there was barely detectable BPl mRNA in three independent samples of CD34+ bone marrow (lanes 4, 5 and 6) . Expression in K562 cells is shown for comparison in lane 1, and b-actin was measured as a loading control. Clonogenicity of K562 cells overexpressing BPl: It has been found that enforced expression of BPl in K562 cells leads to decreased differentiation and increased clonogenicity. The human erythroleukemia cell line K562 was used to examine the effects of ectopic BPl expression on erythroid differentiation. Four independent K562 clones overexpressing BPl were isolated and compared with two clones containing an empty vector. All of the overexpressing cell lines were found to exhibit a diminished ability to undergo erythroid differentiation (data not shown) . Clonogenicity, considered to be a measure of oncogenicity, can be measured by the ability of leukemia cells to grow in 0.5% soft agar. Three of the cell lines overexpressing BPl exhibited significantly increased numbers of colonies able to grow in soft agar per 15,000 cells plated compared with controls (Table 6, below) . These results suggest BPl overexpression may be associated with increased oncogenicity in K562 cells and may increase proliferative capacity or cell survival. The average number of cells per colony (an indicator of proliferation) was determined by pooling 30 colonies to obtain an average number of cells per colony (Table 6) . This number did not increase in the overexpressing cell lines, and the proliferation curves were the same for the overexpressing cell lines and the controls (data not shown) , making it likely that the overexpressors show increased survival relative to 7a and 7b rather than increased proliferation. Taken together, these data imply that BPl expression may direct K562 cells towards survival at the expense of differentiation, a characteristic exhibited by some oncogenes. TABLE 6. Clonogenicity of K562 cells overexpressing BPl Cell Line Relative BPl Col. /15.000 Avσ. no. plated Exp:ression cells/colony 7 7aa ((ccoonnttrrooll)) 1 1 23±15 4400 7b (control) 1 21±7 1300 8a 21 5171108 2900 8c 12 9241199 5300 8d 5 223134 5800 8 8ee 7 7 57118 2300 K562 cells are BCL-2 negative, p53 negative and contain the BCR-ABL translocation; the effects of overexpressing BPl in K562 cells occur against this transformed background. For this reason, NIH 3T3 cells, which are immortalized but not transformed (they do not cause malignancy if injected into mice), were used. Enforced expression of BPl in those cells led to increased growth in soft agar, but at a reduced level of about 4-fold. NIH 3T3 cells overexpressing BPl also exhibited about a 4-fold increase in focus formation. The difference in the effect of high BPl expression on K562 cells and NIH 3T3 cells may mean that additional genetic changes are required for the approximately 10-fold larger increase in clonogenicity seen in K562 cells. Discussion : To summarize, significant RNA expression of BPl was detected in the bone marrow of 81% of pediatric AML patients, compared with 47% of adult patients. In contrast, expression of BPl was not reproducibly found in normal bone marrow. The highest percentage of BPl positives occurred in the FAB classification M5 (monocytic) , in which 77% of AML cases were BPl positive; bone marrow cells from 100% of the children in this category were BPl positive. Two splice variants, DLX7 and DLX4, were co-expressed in 48% and 79% of AML patients, respectively. BPl, DLX7 and DLX4 levels were also assessed in 19 cases of pediatric T-cell ALL. Although the frequency of expression was less in comparison with AML, BPl was overexpressed in 32%, DLX7 in 50% and DLX4 in 40% of T-cell ALLs, compared with weak or no expression in normal PHA- stimulated T lymphocytes. In sharp contrast, no BPl expression was detected in pre-B ALL. Although the reason for expression of BPl in T-cell ALL but not in pre B-cell ALL is not obvious, this difference may provide a useful diagnostic distinction. c-myb was expressed in all AML samples, either at a level comparable to normal bone marrow or at a higher level. Expression of c-myb is associated with immaturity, (see, for example, Gewirtz AM, Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science 1988; 242: 1303-1306; Gonda T, Metcalf D. Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukemia. Na ture 1984; 310: 249-251 and Luscher B, Eisen an RN. New light on Myc and Myb. Part II. Myb. Genes & Devel 1990; 4: 2235-2241.) so those cases within the normal range may be arrested at an early progenitor stage. Higher c-myb expression may be part of the leukemogenic process since activation of c-myb causes leukemia in mice. (see, for example, Wolff L, Roller R, Bies J, Nazarov V, Hoffman B, Amanullah A, Rrall M, Mock B. Retroviral insertional mutagenesis murine promonocytic leukemias: c-myb and Atoll. Curr Topi cs Mi cro Immuno 1996; 211: 191-199.) In this regard, of 11 samples which highly expressed c-myb and could be evaluated for BPl expression, 9 were BPl positive. There is a substantial body of data on expression of HOX genes in malignant hematopoietic cell lines. The HOX genes are clustered on four chromosomes, and the DLX genes are located in pairs on the same chromosomes. (see, for example Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role of HOX homeobox genes in normal and leukemic hematopoiesis. Stem Cells 1996; 14: 281- 291 and van Oostveen JW, Biji JJ, Raaphorst FM, Walbooners JJM, Meijer CJLM. The role of homeobox genes in normal hematopoiesis and hematological malignancies. Leukemia 1999; 13: 1675-1690; Nakamura S, Stock DW, Wydner KL, Bollekens JA, Takeshita K, Nagai BM, Chiba , Kitamura T, Freeland TM, Zhao Z, Minowada J, Lawrence JB, Weiss KB, and Ruddle FH. Genomic analysis of a new mammalian Distal-less gene: Dlx-7. Genomics 1996; 38: 314- 324.; Simeone A, Acampora D, Pannese M, D'Esposito M, Stornaiuolo A, Gulisano M, Mallamaci A, Kastury K, Druck T, Huebner K. Cloning and characterization of two members of the vertebrate Dlx gene family. Proc Na tl Acad Sci USA 1994; 91: 2250-2254.) BPl is situated at the 3' end of the HOXB cluster on chromosome 17. (see, for example Fu S, Strovel JW, Haga SB, Stamberg J, Berg PE . Mapping of a new homeobox gene, BPl, near its isoform DLX7 and characterization of their roles in repression of the beta globin gene. Am . J. Hum . Gen . 1998; 63: A181.) HOXB genes are preferentially expressed in erythroid cells, including K562 and HEL cell lines, (see, for example, Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role of HOX homeobox genes in normal and leukemic hematopoiesis. Stem Cells 1996; 14: 281-291; Shen W-F, Largman C, Lowney P, Corral JC, Detmer K, Hauser CA, Simonitch TA, Hack FM, Lawrence HJ. Lineage-restricted expression of homeobox-containing genes in human hematopoetic cell lines. Proc Na tl Acad Sci USA 1989; 86: 8536-8540; Magli CM, Barba , P, Celetti A, De Vita G, Cillo, C, Boncinelli E. Coordinate regulation of HOX genes in human hematopoietic cells. Proc Na tl Acad Sci USA 1991; 88: 6348- 6352; and Mathews CHE, Detmer K, Boncinelli E, Lawrence HJ, Largman C. Erythroid-restricted expression of homeobox genes of the human HOX2 locus. Blood 1991; 78: 2248-2252) It is believed that genes in the HOX clusters are switched off or on in blocks m myeloid cells, (see, for example, 38,40 The data presented herein suggest that BPl may be part of this coordinate regulation since its pattern of expression in erythromyeloid cell lines is similar to that of the adjacent HOXB genes . Transcripts of HOX genes have also been found in AML, T-ALL and pre B-ALL but, unlike BPl, they are readily detectable in normal bone marrow. (see, for example, Petrini M, Quaranta MT, Testa U, Samoggia P, Tritarelli E, Care A, Cianetti L, Valtieri M, Barletta C, Peschle C. Expression of selected human HOX genes m B/T acute ly pnoid leukemia and interleukm-2/interleukm-l β-stimulated natural killer lymphocytes. Blood 1992; 80: 185-193; Lawrence HJ, Sauvageau G, Ahmadi N, Lopez AR, LeBeau MM, Link M, Humphries K, Largman C. Stage- and lmeage-specific expression of the HOXA10 homeobox gene in normal and leukemic hematopoietic cells. Exp Hem 1995; 23: 1160-1166; Biji JJ, van Oostveen JW, Walboomers JMM, Brink ATP, Vos W, Ossenkoppele GJ, Meijer CJLM. Differentiation and cell-type-restricted expression of H0XC4, H0XC5 and H0XC6 m myeloid leukemias and normal myeloid cells. Leukemia 1998; 12: 1724-1732 /Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE . Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells. Leukemia 1999; 13: 687-698; Salvati PD, Ranford PR, Ford J, Kees UR. H0X11 expression in pediatric acute lymphoblastic leukemia is associated with T-cell phenotype. Oncogene 1995; 11:1333-1338) Notably, the expression of HOX genes is downregulated during normal hematopoietic differentiation but not AML. (see, for example, Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE . Expression of HOX genes, HOX cofactors, and MLL m phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells. LeuKemia 1999; 13: 687-698) . BPl RNA expression in acute leukemias may represent a marker for the differentiation stage of the leukemic blasts and/or may be directly involved in leukemogenesis . The data presented herein point to the possibility that BPl expression in AML occurs in early progenitors: (i) As described above, all of the BPl positive cells are also c-myb positive and 74% are GATA-1 positive, two indicators of early progenitors . (see, for example, 20,22-24 (ii) The barely detectable expression of BPl seen in normal bone marrow is compatible with expression in primitive cells, which comprise a very small sub-population of normal bone marrow. (lii) Overexpression of BPl in both myeloid and lymphoid leukemia argues that leukemogenesis may occur in a stem cell or multipotent hematopoietic progenitor. (iv) Further supporting this idea is the observation that 59% of BPl positive blasts are found m FAB classes considered to be primitive and associated with stem cell leukemias, i.e., MO (minimally differentiated) , M5 (monocytic) or M7 (megakaryocytic) . (see, for example, Cuneo A, Mecucci C, Keri S, Vandenberghe E, Dal Cin P, Van Orshoven A, Rodhain J, Bosly A, Michaux JL, Martiat P., Boogaerts M, Carli MG, Castoldi G, Van Den Berghe H. Multipotent stem cell involvement in megakaryoblastic leukemia: cytologic and cytogenetic evidence in 15 patients. Blood 1989; 74: 1781-1790; Bonnet D, Dick JE. Human acute leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Na ture Medi cine 1997; 3: 730-737; Venditti A, Del Poeta G, Buccisano F, Tamburini A, Cox MC, Stasi R, Bruno A, Aronica G, Maffei L, Suppo G, Simone MD , Forte L, Cordero V, Postorino M, Tufilli V, Isacchi G, Masi M, Papa G, Amadori S. Minimally differentiated acute myeloid leukemia (AML-M0): Comparison of 25 cases with other French-American-British subtypes. Blood 1997; 89: 621-629.) (v) 64% of BPl positive cases are CD34 negative. CD34+ stem cells express several HOX genes, and this expression is down-regulated in CD34- cells. (see, for example, van Oostveen JW, Biji JJ, Raaphorst FM, Walbooners JJM, Meijer CJLM. The role of homeobox genes in normal hematopoiesis ano hematological malignancies. Leukemia 1999; 13: 1675-1690 and Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH, Reid DS, Largman C, Lawreence J, Humphries RK. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci USA 1994; 91: 12223-12227.) In contrast, BPl is expressed in CD34- cells and down-regulated in CD34+ cells. These results are m agreement with the data AML samples (point v) , in which BPl was primarily found m CD34- cells. The very low expression in CD34+ cells could represent either expression in a few CD34+ cells or contamination of the CD34+ cells with CD34- cells. Recent papers support the existence of a sub- population of primitive CD34- lin- stem cells with repopulatmg ability m both mice and humans, (see, for example, 49-51 In mice, CD34- lm- stem cells can convert to CD34+ stem cells upon activation; this has not been investigated in humans. (see, for example, Goodell MA. CD34+ or CD34-: does it really matter? Blood 1999; 94: 2545-2547 and Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hematopoietic stem cells. Blood 1999; 94: 2548-2554) Since our CD34- cells contain both lm+ and lm- subpopulations, it is not known whether BPl is expressed in stem cells. However, it is clear that BPl is activated early in hematopoesis . It is therefore hypothesized that that BPl expression is then repressed during differentiation. This idea is strengthened by the observation that BPl is down-regulated during erythroid differentiation of the cell line MB-02. In support of a possible oncogenic role for BPl, it was observed that stable cell lines overexpressing BPl exhibited up to a 45-fold increase m clonogenicity. Moreover, its high frequency of expression in AML may indicate BPl is an upstream factor m an oncogenic pathway. Further experiments are needed to delineate the roles of BPl in normal hematopoiesis, to directly determine if it plays a role in neoplastic transformation, and to examine the clinical significance of its expression m acute leukemias. In view of the above findings cf increased expression of BPl leukemias, a methoα of screening for acute myeloid leukemia or acute lymphocytic leukemia includes the steps of obtaining a cell sample from a patient and determining whether BPl is overexpressed by cells in the cell sample, as compared to normal cells. Typically, to test for leukemias, the cell sample is taken from appropriate sources such as bone marrow or peripheral blood. The determination of whether BPl is overexpressed is preferably carried out by measuring BPl RNA levels or BPl protein levels in a cell sample. RNA levels can be determined by RNA assays known m the art such as Northern blot analysis, slot and dot blot analysis, RT-PCR and in situ hybridization. For example, the technique of reverse transcription polymerase chain reaction (RT-PCR) , such as in the typical examples described above, can be used to determine whether cells are producing BPl RNA. Specifically, RNA is isolated from the sample cells and transcribed to obtain a reverse-transcription product (cDNA) . A polymerase chain reaction is then carried out using forward and reverse PCR primers derived from SEQ ID NO: 1. Suitable PCR primer pairs for BPl include, for example, SEQ ID NOs 8 and 9, which amplify a product of 581 bp and SEQ ID NOs 10 and 11, which amplify a product of 225 bp. The polymerase chain reaction is carried out under any suitable reaction conditions for amplifying the BPl product. For example, for the primers of SEQ ID NOs 8 and 9, typical reaction conditions comprise a denaturation step (94°C, 1 mm), an annealing step (58°, 1 mm) and an elongation step (72°C, 1.5 mm) for 27 cycles, followed by an additional extension (72°C, 5 mm) . For the primers of SEQ ID NOs 10 and 11, typical reaction conditions comprise holding the sample at 94° C for 2 minutes; then carrying out 30 cycles of 94° C for 1 minute, 62° C for 1 minute and 72° C for 1.5 minutes, 30 cycles; and then holding the sample at 72° C for 10 minutes. The PCR products are then separated from the sample and visualized or quantified by typical methods such as electrophoresis. BPl protein levels may be measured directly by protein assays known m the art such as immunohistochemical assays. To provide polyclonal or monoclonal antibodies for immunoassays, the BPl DNA disclosed herein can be useα, by techniques known in the art, to produce sufficient quantities of substantially purified BPl to use to innoculate a mammal to produce polyclonal or monoclonal antibodies that bind specifically to BPl. Treatment of Leukemia Decreasing the BPl expression in K562 leukemia cells causes apoptosis. To demonstrate this, a vector containing an inducible metallothionem promoter regulating antisense BPl expression was constructed and stably introduced it into K562 cells. Four cell lines were studied, two controls containing an empty vector ( 9A and 9B) and two containing the antisense plasmid (10B and 10D) . CdS0 4 , an inducer of the metallothionem promoter, was added at 50 M for up to four days to induce antisense BPl expression. Induction of antisense BPl led to loss of viability, measured by trypan blue (Fig. 12) . There was some loss of viability of the controls, but significantly greater cell death for 10B and 10D. This correlated with a great reduction of BPl mRNA at this time. Fig. 13 shows the expression of BPl mRNA assessed by RT-PCR for the controls, 9A and 9B (lanes 1 and 2, respectively), compared with 10B and 10D (antisense, lanes 3 and 4, respectively) in the presence of 50 M CdSO, . BPl RNA expression is almost extinguished in 10B and 10D. To determine whether loss of viability was due to increased apoptosis, cells were assessed using Annexm V and scored for Annexm V positive, propidium iodide negative cells; apoptosis of the antisense-conta mg cells significantly increased (data not shown) . These experiments indicate that decreasing BPl expression is associated with apoptosis and, in conduction with the fact that cells overexpressing BPl appear to exhibit increased survival, confirm that modulation of BPl expression has strong consequences for cell survival m K562 cells. TABLE 7. Percent apoptosis of K562 cells after induction of BPl antisense RNA 9A 9±1 7 +3 1313 9B 5+3 911 1311 10B 6±1 1512 5314 10D 9+1 2011 6811 These results suggest that a possible treatment for leukemia may be to introduce antisense oligonucleotides of the DNA encoding BPl into a patient whereby the antisense oligonucleotides block the expression of BPl in leukemia cells, thereby causing apoptosis of these cells. It has also been found that enforced expression of BPl causes increased sensitivity of leukemia cells to cytosme arabinoside (Ara C) , a drug used to treat AML patients. K562 cell lines overexpressing BPl were challenged with different concentrations of ara C. After challenge with 50 μm ara C for 4 days, the viability of control cells was 82-86% while the viability of cells overexpressing BPl ranged 27-29% to 66%, i.e., there was up to a three-fold decrease in viability (data not shown) . No significant differences m the data were seen at 100 μm Ara C. In an Annexm V assay at 30 minutes and 60 minutes, the highest frequency of apoptotic cells was observed for the cells with the lowest viability. Thus, it is clear that the cell lines with enforced BPl expression exhibit increased sensitivity to Ara C, evidenced by an increase in apoptosis . The above findings can be used therapeutically to optimize the dosage of Ara C given to a patient m accordance with the BPl expression level of that patient. Screening and Treatment of Breast Cancer In order to determine whether BPl is overexpressed breast cancer cell lines, the inventor conducted studies to detect overexpression of BPl, as compared to normal breast tissue. Figure 14 shows that expression of BPl by several breast cancer cell lines, including breast cancer lines MCF7 ADR, MDA468 and T47D. In further studies, BPl expression was examined by RT-PCR in breast cancer cell lines (Table 8) . TABLE 8. BPl expression in breast cancer cell lines Cell line ER PR Malignancy Tumorigenic + BPl Hs578T - - ductal ca . no +/- MCF7 + + ductal ca. ** + MCF7ADR * ADR resistant yes +++ MDA-MB-231 - - ductal ca . yes ++ MMDDAA--MMBB--443355Ss -- -- ductal ca . no + MDA-MB-468 - - ductal ca . yes ++ T47D + + ductal ca . NA +++ MCF10A + + normal no +/- * non-responsive to estradiol + Data from the American Type Culture Collection. NA, data not available. ** Not tumorigenic without the addition of estradiol The most striking correlation was between BPl expression and the ability of a cell line to cause mammary tumors in mice. Interestingly, high BPl expression was observed in the adriamycin (ADR) resistant MCF7 cell line, MCF7ADR. Whether there is any relationship between BPl expression and ADR resistance is unknown. Cell line MCF10A, which is derived from normal breast epithelium, showed barely detectable BPl mRNA. To further demonstrate that BPl is expressed in breast cancer, BPl expression was examined in frozen breast tumor tissues and in surrounding normal tissue. A total of 15 tumor tissues were analyzed (Table 9) . BPl was expressed in all 12 of the ER negative cases, but in only one of three ER positive cases. These data indicate a trend of BPl expression in high grade, ER-, PR- tumor tissues. TABLE 9. Comparison of BPl expression with ER and PR status in breast cancer BP1+ BP1- ER+PR+ 1 2 ER-PR- 12 0 RT-PCR analysis of representative samples from normal and malignant breast tissues are shown in Fig. 15. Normal tissue is indicated by N and tumor by T. All of the tumor tissues shown are ER- PR-. In lanes 1-4, tumor and corresponding normal tissues are shown. No expression was seen for the normal samples, while BPl was expressed in all of the tumors. A total of six normal breast tissues have been analyzed. Five of the six were BPl negative and one showed low BPl expression (data not shown) . Three additional tumor tissues are shown in lanes 5-7. β-actin expression demonstrates the integrity of the RNA and serves as a loading control for each sample. In view of the above, the detection of overexpression of BPl is a screening tool for breast cancer. 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(1993) SCL, the gene implicated in human T-cell leukemia, is oncogenic in a murine T-lymphocyte cell line. Oncogene 8: 3093 -3101. The purpose of the above description and examples is to illustrate some embodiments of the present invention without implying any limitation. It will be apparent to those of skill in the art that various modifications and variations may be made to the composition and method of the present invention without departing from the spirit or scope of the invention. All patents and publications cited herein are incorporated by reference in their entireties.

Claims: I Claim : 1. An isolated DNA having SEQ ID NO:l, encoding BPl. 2. The isolated DNA according to claim 1, wherein said BPl exhibits repressor function. 3. The isolated DNA according to claim 1, wherein said BPl binds to Silencer I at -530 bp upstream of the β-globin gene and binds to Silencer II at -300 bp upstream of the β-globin gene . 4. The isolated DNA according to claim 1, wherein said BPl binds to a DNA sequence at -530 bp upstream of the δ-globin gene . 5. The isolated DNA according to claim 1, wherein said BPl binds to sequences at -1400 bp and -1100 upstream of the G γ- globin gene and to a sequence at -1100 of the ^γ-globin gene. 6. The isolated DNA according to claim 1, wherein said DNA is a cDNA of 1251 bp. 7. The isolated DNA according to claim 1, wherein said DNA contains an open reading frame (ORF) . 8. A vector comprising the isolated DNA of claim 1. 9. A host cell transformed with the vector of claim 8. 10. A host cell according to claim 9, wherein said host cell is a eukaryotic host cell. 11. An antisense DNA or an antisense RNA complementary to the DNA having SEQ ID NO:l. 12. A method of treating sickle cell anemia, the method comprising the step of administering an effective amount of BPl to a patient. 13. The method of claim 12 wherein the BPl is administered by transforming β-globm-producmg cells of a patient with a vector containing an open reading frame of SEQ ID NO. 1 and a controllable expression promoter, so that BPl is controllably produced the β-globm-producing cells of the patient. 14. The method of claim 12, wherein said BPl decreases β- globm expression and decreases intracellular concentrations of HbS. 15. A method of screening or diagnosing acute myeloid leukemia or acute lymphocytic leukemia, the method comprising the steps of (a) obtaining a cell sample from a patient, and (b) determining whether BPl is overexpressed by said cell sample as compared to normal cells, wherein overexpression, if any, of BPl indicates a positive diagnosis of acute myeloid leukemia or acute lymphocytic leukemia. 16. The method of claim 15 wherein the cell sample is obtained from bone marrow or peripheral blood. 17. The method according to claim 15, wherein step (b) of determining whether BPl is overexpressed is accomplished by carrying out RT-PCR (reverse transcπptase polymerase chain reaction) using PCR primers derived from SEQ ID NO:l. 18. The method of claim 17 wherein the PCR primers are the oligonucleotides of SEQ ID NOs 8 and 9 and wherein the expression of BPl is indicated by a PCR product of 581 base pairs (bp) . 19. The method of claim 17 wherein the PCR primers are the oligonucleotides of SEQ ID NOs 10 ano 11 and wherein the expression of BPl is indicated by a PCR product of 225 base pairs (bp) . 20. The method of claim 15 wherein the method further includes the step of providing an antibody to BPl and wherein step (b) of determining whether BPl is overexpressed by said cell sample as compared to normal cells is accomplished by immunohistochemical detection of BPl using the antibody to BPl. 21. A method of screening/diagnosis of breast cancer, the method comprising the steps of (a) obtaining a cell sample from breast tissue of a patient, and (b) determining whether BPl is overexpressed by said cell sample as compared to normal cells, wherein the overexpression, if any, of BPl indicates a positive diagnosis of breast cancer. 22. The method according to claim 21, wherein step (b) of determining whether BPl is overexpressed is accomplished by carrying out RT-PCR (reverse transcriptase polymerase chain reaction) using PCR primers derived from SEQ ID NO:l. 23. The method of claim 22 wherein the PCR primers are the oligonucleotides of SEQ ID NOs 8 and 9 and wherein the expression of BPl is indicated by a PCR product of 581 base pairs (bp) . 24. The method of claim 22 wherein the RT-PCR primers are the oligonucleotides of SEQ ID NOs 10 and 11 and wherein the expression of BPl is indicated by a PCR product of 225 base pairs (bp) . 25. The method of claim 21 wherein the method further includes the step of providing an antibody to BPl and wherein step (b) of determining whether BPl is overexpressed by said cell sample as compared to normal cells is accomplished by immunohistochemical detection of BPl using the antibody to BPl. 26. An mRNA fragment encoding BPl protein. 27. The isolated DNA according to claim 1, wherein said BPl, wherein said BPl is a homeobox gene and a member of the distal-less family. 28. The isolated DNA according to claim 1, wherein said BPl, wherein said BPl maps to chromosome 17q21-22. 29. A polyclonal antibody to BPl. 30. A monoclonal antibody to BPl 31. A composition comprising a set of PCR primers that amplifies a unique DNA molecule from a substrate of cDNA of mRNA that encodes for BPl . 32. The composition of claim 31 wherein the set of PCR primers comprises the oligonucleotides of SEQ ID NO: 8 and SEQ ID NO: 9. 33. The composition of claim 31 wherein the set of PCR primers comprises the oligonucleotides of SEQ ID NOs: 10 and SEQ ID NO: 11.

Inventors: BERG PATRICIA E, BERG, PATRICIA, E.
Applicants: UNIV GEORGE WASHINGTON, THE GEORGE WASHINGTON UNIVERSITY
Application year: 2000
Main classification: C07H21
IPC codes: A61K38, A61K48, A61P7, A61P35, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, G01N33