WO2001019397A9 - Methods and compositions utilizing rad51 - Google Patents

Abstract

Herein methods are provided for determining the distribution of Rad51 foci in a first tissue type of a first individual, and then comparing the distribution to the distribution of Rad51 foci from a second normal tissue type from the first individual or a second unaffected individual. A difference in the distributions indicates that the first individual is at risk for a disease state which results in aberrant Rad51 foci. Preferred disease states include cancer and disease states associated with apoptosis. Also provided herein are methods for screening for modulators of Rad51 or homologues thereof and methods of using modulators of Rad51.

Description

METHODS AND COMPOSITIONS UTILIZING RAD51

The present application is a continuation-in-part application U.S. Serial No. 09/455,300, filed December 6, 1999, which is continuation-in-part application of 09/007,020, filed January 14, 1998, which claims priority to provisional applications 60/035,834, 60/045,668, and 60/119,578, filed January 30, 1997, May 6, 1997, and February 10, 1999 respectively. The present application is also a continuation-in-part application of and claims priority to provisional application 60/154,616, filed September 17, 1999. Each of these applications is expressly incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to methods of diagnosis, treatment and screening utilizing Rad51 molecules.

BACKGROUND OF THE INVENTION

Homologous recombination is a fundamental process which is important for creating genetic diversity and for maintaining genome integrity. In E.coli RecA protein plays a central role in homologous genetic recombination in vivo and promotes homologous pairing of double-stranded DNA with single-stranded DNA or partially single-stranded DNA molecules in vitro. Radding, C. M. (1988). Homologous pairing and strand exchange promoted by Escherichia coli RecA protein. Genetic Recombination. Washington, American Society for Microbiology. 193-230; Radding, C. M. (1991). J. Biol. Chem. 266: 5355-5358; Kowalczykowski, et al., (1994). Annu. Rev. Biochem. 63: 991-1043. In the yeast Saccharomyces cerevisiae there are several genes with homology to recA gene; Rad51 , Rad57 and Drnd . Rad51 is a member of the Rad52 epistasis group, which includes Rad50, Rad51, Rad52, Rad54, Rad55 and Rad57. These genes were initially identified as being defective in the repair of damaged DNA caused by ionizing radiation and were subsequently shown to be deficient in both genetic recombination and the recombinational repair of DNA lesions. Game, J. C. (1983). Yeast Genetics: Fundamental and applied aspects. J.F.T. Spencer, D.H.

Spencer and A.R.W. Smith, eds (New-York:springer-verlag) : 109-137; Haynes, et al., (1981). The molecular biology of the yeast Saccharomyces cerevisiae: Life cycle and inheritance. J.N. Strathern, E.W. Jones and J.M. Broach, eds (Cold Spring harbor, New York:Cold Spring Harbor laboratory press) : 371-414; Resnick, M. A. (1987). Meiosis, P.B. Moens, ed. (New York: Academic Press) : 157-210. During meiosis Rad51 mutants accumulate DNA double-strand breaks at recombination hot spots (Shinohara, et al., (1992). Cell 69: 457-470). Yeast rad51 gene was cloned and sequenced (Basile, et al., (1992). Mol. Cell. Biol. 12: 3235-3246; Aboussekhara, et al., (1992) Mol. Cell. Biol. 12: 3224-3234). Although yeast Rad51 gene shared homology with E.coli recA gene, the extent of homology was not very strong (27%). However, the extent of structural conservation between RecA protein and Rad51 protein became apparent when the yeast Rad51 protein was isolated and was shown to form nucleoprotein filaments that were almost identical to the nucleoprotein filaments formed by RecA protein (Ogawa, et al., (1993). CSH Symp. Quant. Biol. 58: 567-576; Ogawa, T., et al., (1993). Science 259: 1896-1899; Story, et al., (1993). Science 259: 1892-1896). Recently genes homologous to E.coli recA and yeast rad51 were isolated from all groups of eukaryotes, including mammals (Morita, et al., (1993). Proc. Natl. Acad. Sci. USA 90, 6577-6580; Shinohara, et al., (1993). Nature Genet. 4, 239-243; Heyer, W.D. (1994).

Experientia 50, 223-233; Maeshima, et al., (1995). Gene 160: 195-200). Phylogenetic analysis by Ogawa and co workers suggested the existence of two sub-families within eukaryotic RecA homologs: the Rad51-like (Rad51 of human, mouse, chicken, S. cerevisiae, S. pombe and Mei3 of Neurospora crassa) and the Dmc1-like genes (S. cerevisiae Dmc1 and Lilium longiflorum LIM15) (Ogawa, supra). All these Rad51 genes share significant homology with residues 33-240 of the E.coli RecA protein, which have been identified as a 'homologous core' region.

Yeast and human Rad51 proteins have been purified and characterized biochemically. Like E.coli RecA protein, yeast and human Rad51 protein polymerizes on single-stranded DNA to form a right-handed helical nucleoprotein filament which extends DNA by 1.5 times (Story, supra; Benson, et al., (1994) EMBO J. 13, 5764-5771). Moreover like RecA protein Rad51 protein promotes homologous pairing and strand exchange in an ATP dependent reaction (Sung, P. (1994). Science 265, 1241-1243; Sung, P. and D. L. Robberson (1995). Cell 82: 453-461; Baumann, et al., (1996) Cell 87, 57-766; Gupta, et al., (1997) Proc. Natl. Acad. Sci. USA 94, 463-468). Surprisingly, polarity of strand exchange performed by Rad51 protein is opposite to that of RecA protein (Sung and Robberson supra) and the relevance of this observation remains to be seen.

Studies with mouse models show that targeted disruption of the Rad51 gene leads to an embryonic lethal phenotype (Tsuzuki, et al., (1996). Proc. Natl. Acad. Sci. USA 93: 6236-6240). Moreover attempts to generate homozygous rad51 -/-embryonic stem cells have not been successful. These results show that Rad51 plays an essential role in cell proliferation, a surprise in view of the viability of S. cerevisiae carrying rad51 deletions. It is also interesting to note that Rad51 was found to be associated with RNA polymerase II transcription complex (Maldonado, et al., (1996). Nature 381, 86-89), the specificity and functional nature of these interactions remains to be seen but these observations point to a pleitropic role of hsRad51 in DNA metabolism.

While Rad51 transcripts and protein are present in all the cell types examined thus far, the highest transcript levels are found in tissues active in recombination, including spleen, thymus, ovary and testis (Morita, supra). Rad51 is specifically induced in murine B cells cultured with lipopolysaccharide, which stimulates switch recombination and Rad51 localizes to nuclei of switching B cells (Li, et al., (1996). Proc. Natl. Acad. Sci. USA 93: 10222-10227). These findings are consistent with the view that Rad51 plays an important role in lymphoid specific recombination events such as V(D)J recombination and immunoglobulin heavy chain class switching. In spermatocytes undergoing meiosis, Rad51 is enriched in the synaptonemal complexes, which join paired homologous chromosomes (Haaf, et al., (1995) Proc. Natl. Acad. Sci. USA 92, 2298-2302; Ashley, et al., (1995) Chromosoma 104: 19-28; Plug, et al., (1996). Proc. Natl. Acad. Sci. USA 93: 5920-5924). In cultured human cells, Rad51 protein is detected in multiple discrete foci in the nucleoplasm of a few cells by immunofluorescent antibodies. After DNA damage, the localization of Rad51 changes dramatically when multiple foci form in the nucleus and stain vividly with anti-Rad51 antibodies (Haaf, supra, 1995). After DNA damage the percentage of cells with focally concentrated Rad51 protein increases; the same cells show unscheduled DNA-repair synthesis.

Micronuclei (MN) originate from chromosomal material that is not incorporated into daughter nuclei during cell division. Different chemicals and treatment of cells induce qualitatively different types of micronuclei. MN caused by ionizing radiation or clastogens (i.e. 5-azacytidine) mostly contain acentric chromosome fragments (Verhaegen, F., and Vral, A. (1994). Radiation Res. 139, 208-213; Stopper, et al., (1995). Carcinogenesis 16, 1647- 1650). In contrast, MN induced by aneuploidogens (i.e. colcemid) result from lagging whole chromosomes and stain positively for the presence of kinetochores/ centromeres (Marrazini et al., 1994; Stopper, et al., (1994). Mutagenesis 9, 411-416). Determination of MN frequencies represents a good assay to measure genetic damage in cells, since it is much faster and simpler than karyotype analyses. In this light, the MN test has been widely used as a dosimeter of human exposure to radiation or clastogenic and aneugenic chemicals, and for the detection and risk assessment of environmental mutagens and carcinogens (Heddle, et al., (1991) Environmental Mol. Mutagenesis 18, 277-291; Norppa, et al., (1993). Environmental Health Perspect. 101, Supp. 3, 139-143; Hahnfeldt, et al., (1994) Radiation Res. 138, 239-245). However, although the MN assay is a convenient in situ method to monitor cytogenetic effects, the understanding of the connection between initial DNA damage and formation of MN is still poor. The tumor suppressor p53 prevents tumor formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinational events (Lane, D. P. (1992). Nature 358: 15-16; Lane, D. P. (1993). Nature 362: 786-787; Sturzbecher, et al., (1996). EMBO J. 15: 1992-2002) reported that wild-type tumor suppressor protein p53 interacts physically with human Rad51 protein and it inhibits the biochemical functions of Rad51 like ATPase and strand exchange. In vivo, temperature sensitive mutant p53 formed complexes with Rad51 only in wild type but not in mutant conformation. They suggested that gene amplifications and other types of chromosome rearrangements involved in tumour progression might occur not only as a result of inappropriate cell proliferation but as a direct consequence of a defect in p53 mediated control of homologous recombination processes due to mutations in the p53 gene. (Meyn, et al., (1994). Int. J. Radiat. Biol. 66: S141-S149) showed that normal cells transfected with a dominant-negative p53 mutant acquired interference with the G1-S cell cycle checkpoint and showed up to an 80-fold elevation in Rad51 mediated homologous DNA recombination rates compared with the normal parental control cells. Thus, loss of normal p53 function may cause a loss in control of normal DNA repair, recombination, and ultimately replication, resulting in inappropriate cell division and neoplastic growth. Breast tumour cells have mutated p53 genes and proteins and have various types of chromosomal aberrations like insertions, deletions, rearrangements, amplifications etc., indicative of abnormally controlled recombination.

Accordingly, the central role of Rad51 in cancer, DNA repair and recombination is recognized and characterized herein. Among other provisions, the invention provides methods of diagnosis and screening which focus on Rad51. Furthermore, the invention provides methods of using modulators of Rad51 , preferably inhibitors, in methods of treatment.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present invention provides methods of diagnosing individuals at risk for a disease state which results in aberrant Rad51 loci. The methods comprise determining the distribution of Rad51 foci in a first tissue type of a first individual, and then comparing the distribution to the distribution of Rad51 foci from a second normal tissue type from the first individual or a second unaffected individual. A difference in the distributions indicates that the first individual is at risk for a disease state which results in aberrant Rad51 foci. Preferred disease states include cancer and disease states associated with apoptosis.

In an additional aspect, the present invention provides methods for identifying apoptotic cells and cells under stress associated with nucleic acid modification. The methods comprise determining the distribution of Rad51 foci in a first cell, and comparing the distribution to the distribution of Rad51 foci from a second non-apoptotic cell. A difference in the distributions indicates that the first cell is apoptotic or under stress.

In a further aspect, the present invention provides methods for identifying a cell containing a mutant Rad51 gene comprising determining the sequence of all or part of at least one of the endogenous Rad51 genes.

In an additional aspect, the invention provides methods of identifying the Rad51 genotype of an individual comprising determining all or part of the sequence of at least one Rad51 gene of the individual. The method may include comparing the sequence of the Rad51 gene to a known Rad51 gene.

In a further aspect, the present invention provides methods for screening for a bioactive agent capable of binding to Rad51. The methods comprise adding a candidate bioactive agent to a sample of Rad51 , and determining the binding of the candidate agent to the Rad51.

In an additional aspect, the invention provides methods for screening for a bioactive agent capable of modulating the activity of Rad51. The method comprises the steps of adding a candidate bioactive agent to a sample of Rad51, and determining an alteration in the biological activity of Rad51. The method may also comprise adding a candidate bioactive agent to a cell, and determining the effect on the formation or distribution of Rad51 foci in the cell.

In another aspect, the present invention provides methods for inhibiting cell proliferation in an individual comprising administering to the individual a composition comprising a Rad51 inhibitor. Also provided herein is a method for inhibiting the growth of a cell comprising administering to said cell a composition comprising a Rad51 inhibitor. Such methods can further include the step of providing radiation or alkylating agents after administration of said Rad51 inhibitor. In preferred embodiments the methods are performed in vivo and/or on cancerous cells. In a further aspect, the invention provides methods of inducing apoptosis in a cell comprising increasing the activity of Rad51 in the cell. This can be done by overexpressing an endogenous Rad51 gene, or by administration of a gene encoding Rad51 or the protein itself.

In an additional aspect, the present invention provides composition comprising a nucleic acid encoding a Rad51 protein, and a nucleic acid encoding a tumor suppressor protein. The tumor suppressor protein may be p53 or a BRCA protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a digital image of photographs of cells that depict type I and type II Rad51 foci, respectively.

Figures 2A and 2B are digital images of photographs of two different breast cancer cells from a breast cancer cell line (BT20) that show Rad51 foci. The staining is localized to the nucleus, and does not occur in either the cytoplasm or the nucleolus.

Figures 3A, 3B, 3C and 3D show dynamic changes in the higher-order nuclear organization of Rad51 foci after DNA damage and cell-cycle arrest, (a-c) TGR-1 fibroblasts were irradiated with a lethal dose (900 rad) of ¹³⁷Cs and then allowed to recover for various times. Rad51 protein is stained (light), nuclei are counterstained with DAPI. Three hours after irradiation (a), Rad51 foci are distributed throughout the entire nuclear volume. Many foci have a double-dot appearance. After 16 hrs (b), clusters of Rad51 foci and linear higher-order structures are formed. Somatic pairing of linear strings of Rad51 foci is observed. After 30 hrs (c), Rad51 clusters move towards the nuclear periphery and are eliminated into micronuclei. (d) Simultaneous staining of Rad51 protein (light) and replicating DNA (dark) in an exponentially growing, XPA fibroblast culture. BrdU was incorporated into DNA for 30 hrs and detected with red anti-BrdU antibody. Note that the Rad5l-positive cell is devoid of BrdU label. Magnification 1000x.

Figure 4 depicts the exclusion of Rad51 -protein in micronuclei after DNA damage. TGR-1 fibroblasts, two days after ³⁷Cs irradiation with a dose of 900 rad. Rad51 protein is stained by (light), nuclei are counterstained with DAPI. Note the complete absence of Radδl-protein staining in nuclei. All Rad51 foci are excluded into micronuclei. Most micronuclei exhibit paired Radδl-protein structures. Magnification lOOOx. Figures 5A, 5B, 5C and 5D illustrates that apoptotic bodies (micronuclei) contain Rad51 protein and fragmented DNA. (a and b) TGR-I nuclei, 3 hrs (right), 16 hrs (middle), and 30 hrs (left) after ¹³⁷Cs irradiation. Rad51 -protein foci show light staining. The repair proteins Rad52 (a) and Gadd45 (b) are detected by antibody probes (darker staining). Nuclei are counterstained with DAPI. Note that neither Rad52 nor Gadd45 foci co-localize with Radδl. Only the Rad51 foci segregate into micronuclei. (c and d) Micronuclei induced by treatment of TGR-1 cultures with colcemid (c) and etoposide (d) contain Rad51 protein (light staining, left nucleus) and fragmented DNA (darker staining, right nucleus). Magnification 1000x.

Figures 6A, 6B and 6C show the association of Rad51 protein with linear DNA molecules, (a) Mechanically stretched chromatin prepared from a ¹³⁷Cs-irradiated cell culture and stained with light anti-HsRad51 antibodies. The Rad51 signals appear as beads-on-a-string on the linearly extended chromatin fibers, (b and c) DNA fibers excluded from TGR-1 nuclei, one day after ¹³⁷Cs irradiation. Preparations are not experimentally stretched. Chromatin is counterstained with DAPI. The DNA fibers are covered with Rad51 protein (c, light staining), whereas the remaining nuclei are devoid of detectable Rad51 foci. DNA-strand breaks in chromatin fibers are end labeled with fluorescent nucleotides (c, darker staining co-localizing with the Rad51 staining). Some fibers appear to form micronuclei. Magnification 1000x.

Figures 7A, 7B, 7C, 7D, 7E and 7F show the linear higher-order structures of Radδl protein in overexpressing nuclei and in colcemid-induced micronuclei. Rad5l protein is stained with anti-Rad5l antiserum, detected by green FITC fluorescence (light staining). Preparations are counterstained with DAPI, except the nucleus in b. (a and b) Human 7I0 kidney cells overexpressing Radδl fused to a T1-tag epitope. Nuclei are filled with a network of linear Radδl structures. Magnification lOOOx. (c) Subconfluent rat TGR 928.1-9 cells overexpressing HsRadδ Nuclear staining is most prominent in cells during G₀ and G., phase of the cell cycle. Magnification lOOOx. (d) TGR 928.1-9 nucleus filled with linear Radδl structures. Magnification 1000x. (e and f) Linear Radδl structures in colcernid-induced micronuclei. TGR-I fibroblasts were treated with colcemid for one day and then allowed to recover for two days. Note the absence of Radδl staining in the nuclei. Magnification 1000x.

Figure 8 is a schematic illustrating filter based assays to monitor strand exchange by Radδl . Single-stranded DNA used for making the nucleoprotein filament is unlabeled. Radδl is shown as ovals. The duplex DNA is labeled with fluorophore (R, rhodamine) on the complementary strand that will be displaced after the completion of DNA strand exchange. The labeled displaced strand binds to the filter and is detected in screening of the plate. Figure 9 shows a graph depicting DNA strand exchange reaction monitored by a filter binding assay. DNA joint molecules (intermediates in DNA strand exchange) are formed only when homologous DNA substrates are used. DNA joint molecules are not formed when the reaction is performed with either a heterologous DNA substrate or in the absence of RecA protein or with inactive RecA protein. Homologous DNA pairing and DNA strand exchange assays are highly dependent on the presence of DNA sequence homology, recombinase protein, nucleotide and magnesium cofactors. Hence, by employing appropriate controls, the specificity of the reaction and specific effects of inhibitors are established.

Figure 10 is a schematic illustrating homologous DNA pairing by a fluorescence resonance energy transfer (FRET) assay. Nucleoprotein filament is formed on a single-stranded DNA (black thick line) labeled with fluorescein (F). Recombinase is shown as ovals. Duplex DNA is labeled with rhodamine (R) on the complementary strand. Homologous pairing and subsequent DNA strand exchange results in quenching of emission from fluorescein.

Figure 11 is a schematic illustrating strand exchange assay measured by FRET assay. A nucleoprotein filament is formed on unlabeled single-stranded DNA (thick black line). Recombinase is shown as ovals. Duplex DNA (thin line) is labeled. The homologous DNA strand is labeled with rhodamine (R) and its complementary strand is labeled with fluorescein (F). DNA strand exchange results in enhanced emission from fluorescein.

Figure 12 is a schematic illustrating a D-loop assay. Nucleoprotein filament is formed on unlabeled single-stranded DNA. Duplex DNA (either supercoiled or linear) is end labeled with ³²P. The D-loop is formed after uptake of the single-stranded DNA into the duplex DNA. D-loops are trapped on nitrocellulose membranes because they contain single-strand DNA tails and/or single-strand regions and the unreacted duplex DNA is washed away in the filtrate.

Figure 13 depicts a photograph of a gel illustrating the down-regulation of Rad51 protein in MDA-MB-231 breast tumor cells by specific antisense oligodeoxynucleotides. The lanes are as follows: 1 and 8, untreated cells; 2, 200 mM AS3 (SEQ ID NO:1); 3, 200 nM AS4 (SEQ ID NO:2); 4, 200 nM AS3 (SEQ ID NO:1) 200 nM AS4 (SEQ ID NO:2); 5, 200 nM AS3 (SEQ ID NO:1); 6, 200 nM AS6 (SEQ ID NO:4); 7, 200 nM AS3 (SEQ ID NO:1) 200 nM AS6 (SEQ ID NO:4); 9, 200 nM AS4 (SEQ ID NO:2); 10, 200 nM AS6 (SEQ ID NO:4); 11, 200 nM AS4 (SEQ ID NO:2) 200 nM AS6 (SEQ ID NO:4). Figure 14 shows a graph which depicts the specific down-regulation of Radδl by antisense oligonucleotides in MDA-MB-231 cells. Cells were treated for 48 hours with a concentration of 200 nM of a single antisense oligonucleotide, combinations of two different antisense oligonucleotides, or control treatments. Total protein extracts were analyzed by SDS-PAGE δ and Radδl protein level was monitored by Western blotting using a polyclonal Rad51 antibody. The levels of Rad51 protein were reduced, depending on the oligonucleotide utilized, 30% to 96% compared to control treatment cells. The amount of Radδl present after each treatment was quantitated. The average from two independent experiments was obtained and plotted in the bar graph. This graph shows that in addition to single antisense 0 molecules, combinations of oligonucleotides work well, including the combination of AS3 and AS6 (SEQ ID NOS:1 and 4) which essentially completely inhibits the expression of Radδl .

Figures 15A and 1δB show the human Rad51 mRNA sequence wherein the regions complementary to the antisense molecules SEQ ID NOS: 1-9 are underlined.

Figures 16A-E show antisense oligonucleotides provided herein. Figure 16A shows 5 antisense in the coding region. Figure 16B shows antisense in the 5' untranslated region. Figure 16C shows antisense in the 3' untranslated region. Figure 16D shows sense oligonucleotides. Figure 16E shows scrambled oligonucleotides.

Figure 17 shows a recombinasome of an embodiment of the present invention.

Figure 18 shows a schematic for double-stranded break repair in a eukaryotic cell.

0 DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a series of discoveries relating to the pivotal role that Radδl plays in a number of cellular functions, including those involved in disease states. Thus, it appears that the levels, function, and distribution of the Radδl protein within cells may be monitored as a diagnostic tool of cellular health or fate. In addition, due to Radδl 's δ essential role in a number of cellular processes, Radδl is an important target molecule to screen candidate drug agents which can modulate its biological activity. Moreover, agents which modulate its biological activity are provided herein for use in methods of treatment.

Accordingly, in a preferred embodiment, the invention provides methods of diagnosing individuals at risk for a disease state. As will be appreciated by those in the art, "at risk for a 0 disease state" means either that an individual has the disease, or is at risk to develop the disease in the future. By "disease state" herein is meant a disease that is either caused by or results in aberrant Radδl distribution or biological activity. For example, as is more fully described below, aberrant distribution of Radδl foci in a cell can be indicative of cancer, apoptosis, cellular stress, etc., which can lead to the development of disease states. Similarly, disease states caused by or resulting in aberrant Radδl biological activity, δ including alterations caused by mutation, changes in the cellular amount or distribution of Radδl, and changes in the biological function of Rad51, for example altered nucleic acid binding, filament formation, DNA pairing (i.e. D-loop formation), strand-exchange, strand annealing, recombination or DNA repair, are also included within the definition of disease states which are related to or associated with Radδl .

0 Thus, disease states which may be evaluated using the methods of the present invention include, but are not limited to, cancer (including solid tumors such as skin, breast, brain, cervical carcinomas, testicular carcinomas, etc.), diseases associated with premature or incorrect apoptosis, including AIDS, cancers (e.g. melanoma, hepatoma, colon cancer, etc.), liver failure, Wilson disease, myelodysplastic syndromes, neurodegenerative diseases, 5 multiple sclerosis, aplastic anemia, chronic neutropenia, Tupe I diabetes mellitus, Hashimoto thyroiditis, ulcerative colitis, Canale-Smith syndrome, lymphoma, leukemia, solid tumors, and autoimmune diseases), diseases associated with cellular stress which is affiliated with nucleic acid modification, including diseases associated with oxidative stress such as cardiovascular disease, immune system function decline, aging, brain dysfunction and 0 cancer.

The present invention is directed to the use of Rad51 (and its analogs and homologs) in a variety of screening techniques. Thus, in one embodiment, Rad51 includes homologues of Radδl . In one aspect, Radδl homologues can be defined by the Radδl role in recombinational repair. In another aspect, Radδl genes encode proteins which share δ significant sequence identity with residues 33-240 of E.coli RecA protein, which has been identified as a homologous core region in the literature. Radδl homologues include RecA and Radδl homologues in yeast and in mammals. RecA and yeast Radδl have been cloned and are known in the art. Radding, Genetic Recom. 193-230 (1988); Radding, J. Biol. Chem. 266:δ3δδ-δ3δ8 (1991); Kowalczykoswski, et al., Annu. Rev. Biochem., 63:991- 0 1043 (1994); Basile, et al., Mol. Cell. Biol., 12:3236-3246 (1992); Aboussekhara, et al., Mol. Cell. Biol., 12:3224-3234 (1992). Genes homologous to E. Coli recA and yeast Radδl have been isolated from all groups of eukaryotes, including mammals. Morita, et al., PNAS USA, 90:6677-6680 (1993); Shinohara, et al., Nature Genet, 4:239-243 (1993); Heyer, Experentia, 60:223-233 (1994); Maeshima, et al. Gene, 160:196-200 (1996). Radδl has been δ identified in humans, mice, chicken, S. Cerevisiae, S. Pombe and Mei3 of Neurospora crassa. Human Radδl homologues include Radδl A, Radδl B, Rad51C, Radδl D, XRCC2 and XRCC3. Albala, et al., Genomics, 46:476-479 (1997); Dosanjh, et al. Nucleic Acids Res, 26:1179(1998); Pittman, et al, Genomics, 49:103-11 (1998); Cartwright, et al. Nucleic acids Res, 26:3084-3089 (1998); Liu, et al, Mol Cell, 1:783-793 (1998).

In another embodiment, Radδl is a dimer. The dimer may be a homodimer or a δ heterodimer. In a preferred embodiment, the heterodimer is formed from two different homologues. In one embodiment, the homologues are selected from the group consisting of RadδlA, RadδlB, RadδlC, and RadδlD. In a preferred embodiment, the dimer includes Radδl C or RadδlB in any combination.

Also included with the definition of Radδl are amino acid sequence variants. These variants 0 fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the Radδl protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant Radδl protein fragments δ having up to about 100-160 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the Radδl protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, 0 although variants can also be selected which have modified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the δ target codon or region and the expressed Radδl variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of Radδl protein activities.

0 Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger. Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the Radδl protein are desired, substitutions are generally made in accordance with the following chart:

Chart I Original Residue Exemplary Substitutions

Ala Ser

Arg Lys Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp Gly Pro

His Asn, Gin lie Leu, Val

Leu lie, Val

Lys Arg, Gin, Glu Met Leu, lie

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr Tyr Trp, Phe

Val lie, Leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the Rad51 proteins as needed. Alternatively, the variant may be designed such that the biological activity of the RAdδl protein is altered. In addition to Radδl, other proteins can be tested for their ability to effect either Radδl activity or other component activities. For example and referring to the schematic in Figure 17, it currently appears that in mammals, Radδl is associated with a variety of other proteins, including, but not limited to, RadδlB, Rad51C, RadδlD, Radδ2, Radδ4,BRCA1, BRCA2, pδ3, XRCC2, XRCC3, and RPA. Currently, it appears that a "recombinosome", comprising at least Rad51, Rad52, Rad54 and RPA, may function as the recA equivalent in mammals to exhibit double-stranded break repair. Accordingly, the recombinosome may be used in the assays outlined herein to either assay for alterations in Radδl activity, alterations in other components (e.g. Radδ2, Rad54, etc.), or for alterations in recombinosome activity. Again, analogs and homologs of these other recombinosome proteins are included as well.

In one embodiment, the method comprises first determining the distribution of Rad51 foci in a first tissue type of a first individual, i.e. the sample tissue for which a diagnosis is required. In some embodiments, the testing may be done on single cells. The first individual, or patient, is suspected of being at risk for the disease state, and is generally a human subject, although as will be appreciated by those in the art, the patient may be animal as well, for example in the development or evaluation of animal models of human disease. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc, and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of patient.

As will be appreciated by those in the art, the tissue type tested will depend on the disease state under consideration. Thus for example, potentially cancerous tissue may be tested, including breast tissue, skin cells, solid tumors, brain tissue, etc. Similarly, cells or tissues of the immune system, including blood, and lymphocytes; cells or tissues of the cardiovascular system (for example, for testing oxidative stress).

In a preferred embodiment, the disease state under consideration is cancer and the tissue sample is a potentially cancerous tissue type. Of particular interest is breast, skin, brain, colon, prostate, and other solid tumor cancers. As outlined in the Examples, cultured breast cancer cells and primary invasive breast cancer cells all demonstrate an increase in the presence of Rad51 foci.

Similarly, several diseases caused by defective nucleotide excision repair (NER) systems, including Xeroderma pigmentosium, show increased Radδl foci. In a preferred embodiment, primary cancerous tissue is used, and may show differential Radδl staining. While the number of cells exhibiting Radδl foci may be less than for cell lines, primary cancerous tissue shows an increase in Radδl foci. Thus for example, from O.Oδ to 10% of primary cancerous cells exhibit differential Radδl foci, with from about 1 to about δ% being common.

It should be noted that not all cancer cell lines exhibit aberrant Radδl protein foci. For example, the ovarian cancer cell line Hey does not show an increase in Radδl foci. Similarly, as outlined in the examples, transformed but non-malignant human cells can show an increased percentage of Radδl-positive cells (compared to non-transformed cells) , although it is generally not as great as in tumor cells.

In a preferred embodiment, the disease state under consideration involves apoptosis, and includes, but is not limited to, including AIDS, cancers (e.g. melanoma, hepatoma, colon cancer, etc.), liver failure, Wilson disease, myelodysplastic syndromes, neurodegenerative diseases, multiple sclerosis, aplasitic anemia, chronic neutropenia, Type I diabetes mellitus, Hashimoto thyroiditis, ulcerative colitis, Canale-Smith syndrome, lymphoma, leukemia, solid tumors, and autoimmune diseases. This list includes disease states that include too much as well as too little apoptosis. See Peter et al, PNAS USA 94:12736 (1997), hereby incorporated by reference.

In a preferred embodiment, the disease state under consideration involves cellular stress associated with nucleic acid modification, including aging, cardiovascular disease, declines in the function of the immune system, brain dysfunction, and cancer.

The distribution of Rad51 foci is determined in the target cells or tissue. To date, two main types of Radδl foci have been identified. As reported earlier (Haaf, 1996, supra) in situ immunostaining with Radδl antibodies reveals three kinds of nuclei: 1) nuclei that did not show any staining at all ( no foci); 2) nuclei that showed weak to medium staining and showed only a few foci (Type I nuclei); and 3) nuclei that showed strong staining and showed many foci (Type II nuclei). In general, the staining is excluded from the cytoplasm. Type I and Type II patterns of nuclei staining are shown in Figure 1; many of the foci have a double-dot appearance, typical of paired DNA segments. In normal cells, type I nuclei are found in 7-10% of cells and type II nuclei in less than 0.4 to 1 % of cells, with generally about 90% of the cells showing no foci. In contrast, some cells involved in disease states show a marked increase in Radδl foci. As outlined herein and shown in the examples, the numbers of cells showing Radδl foci in cells associated with disease states is significantly increased. Thus, in a preferred embodiment, the number of cells showing type I nuclei is generally from about 5% to about 60% of the nuclei, with from about 10% to about 40% generally being seen. Thus, in a preferred embodiment, there is at least a 6% increase in the type I foci, with at least about 10 % being preferred, and at least about 30% being particularly preferred. Generally, to see this effect, at least about 100 cells should be evaluated, with at least about δ 600 cells being preferred, and at least about 1000 being particularly preferred.

Similarly, the number of cells showing type II nuclei also increases, with from about 1% to about 10% of the nucleic exhibiting type II foci and from about 1% to about 5% being common. Thus, in a preferred embodiment, there is at least a 5% increase in type II foci, with at least about 10% being preferred, and at least about 30% being particularly preferred. 0 In a preferred embodiment, both types of foci increase simultaneously. In alternate embodiments, only one type of foci increases. Similarly, an increase in both types of foci (i.e. an increase in any foci, irrespective of type) can also be evaluated using the same numbers.

The distribution of Rad51 foci can be determined in a variety of ways. In a preferred 6 embodiment, a labeled binding agent that binds to Radδl is used to visualize the foci. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels 0 may be incorporated into the compound at any position. Preferred labels are fluorescent or radioactive labels. The binding agent can either be labeled directly, or indirectly, through the use of a labeled secondary agent which will bind to the first binding agent. The cells or tissue sample is prepared as is known for cellular or in situ staining, using techniques well known in the art, as outlined in the Examples.

6 In a preferred embodiment, the binding agent used to detect Radδl protein is an antibody. The antibodies may be either polyclonal or monoclonal, with monoclonal antibodies being preferred. In general, it is preferred, but not required, that antibodies to the particular Radδl under evaluation be used; that is, antibodies directed against human Rad51 are used in the evaluation of human patients. However, as the homology between different mammalian 0 Rad51 molecules is quite high (73% identity as between human and chicken, for example), it is possible to use antibodies against Radδl from one type of animal to evaluate a different animal (mouse antibodies to evaluate human tissue, etc.). Thus, in a preferred embodiment, antibodies raised against eukaryotic Rad51 are used, with antibodies raised against mammalian Rad51 being especially preferred. Thus, antibodies raised against yeast, δ human, rodent, primate, and avian Rad51 proteins are particularly preferred. In addition, as will be appreciated by those in the art, the protein used to generate the antibodies need not be the full-length protein; fragments and derivatives may be used, as long as there is sufficient immunoreactivity against the sample Radδl to allow detection. Alternatively, other binding agents which will bind to Radδl at sufficient affinity to allow visualization can be δ used.

Without being bound by theory, as outlined in the Examples, it does not appear that the quantitative amount of Radδl protein is necessarily altered in cells exhibiting the presence or altered distribution of foci. However, in some circumstances the quantitative amount of Radδl may be measured and correlated to the presence or absence of Radδl foci.

0 In addition, the appearance of the foci may be used in the determination of the presence of aberrant Rad51 foci. As noted in the Examples, in some cases linear "strings" of 5-10 Radδl foci are formed, with somatic association of "homologous" strings of similar length, tightly paired at one of the ends. These structures are generally associated with DNA fibers, as is shown in the Figures. Thus, the formation of these types of structures can be indicative δ of aberrant Radδl foci.

Furthermore, in a preferred embodiment, particularly in disease states involving apoptosis and DNA damage, aberrant Radδl foci includes the development of micronuclei containing Radδl . As shown in the Examples, evaluation of Radδl foci overtime, in particular after cellular stress, can lead to the concentration and exclusion of the Rad51 foci (which are 0 associated with DNA) into micronuclei, which frequently is accompanied by genome fragmentation. This effect is seen in a wide variety of apoptotic cells, as is shown in the Examples, even in the absence of induced DNA damage, such as through the use of colcemid, a spindle poison, thus indicating the role of Rad51 in normal apoptotic pathways. In addition to the evaluation of the presence or absence of Radδl foci, the cells may be 6 evaluated for cell cycle arrest, as is outlined in the Examples.

Once the distribution of Radδl foci has been determined for the target sample, the distribution of foci is compared to the distribution of Radδl foci from a second cell or tissue type. As will be appreciated by those in the art, the second tissue sample can be from a normal cell or tissue from the original patient or a tissue from another, unaffected individual, 0 which has been matched for correlation purposes. A difference in the distribution of Radδl foci as between the first tissue sample and the second matched sample indicates that the first individual is at risk for a disease state which results in aberrant Radδl foci. In a preferred embodiment, the difference in Radδl foci distribution is an increase in Radδl foci, of either type 1 or type 2 foci, as outlined above. In an alternate embodiment, the difference in Radδl foci distribution is a decrease in the number of Radδl foci.

In some embodiments, there need not be a direct comparison. For example, having once δ shown that a particular normal tissue only contains a small percentage of Radδl foci, the tissue or cells under evaluation may not need to be compared to a control sample; the presence of a higher percentage allows the diagnosis. Thus, for example, in breast cancer, the presence of at least 1% of the cells containing Radδl foci is indicative that the patient is at risk for breast cancer or in fact already has it.

0 In a preferred embodiment, a difference in the distribution of Radδl foci, in particular an increase in Radδl foci, indicates that the cell or tissue is cancerous.

In a preferred embodiment, a difference in the distribution of Radδl foci, in particular an increase in Radδl foci, indicates that the cell or tissue is apoptotic. These differences can include the association of Radδl with DNA fibers, the association of Radδl with damaged δ DNA in micronuclei, or the presence of Rad51 in micronuclei.

In addition, in a preferred embodiment, the extent of aberrant distribution indicates the severity of the disease state. Thus, for example, high percentages of cells containing Rad51 foci can be indicative of highly malignant cancer.

In addition to the evaluation of Rad51 foci, the presence or absence of variant (mutant) 0 Rad51 genes may also be used in diagnosis of disease states. Mutant forms of p53 have been found in roughly 50% of known cancers, and it is known that Rad51 and pδ3 can interact on a protein level. In addition, p53 and Rad51 have somewhat similar biochemical functions. Thus, the present discovery that Rad51 plays a pivotal role in some cancers and apoptosis thus suggests that variant Rad51, or incorrectly controlled Radδl levels or 6 functions may be important in some disease states.

Accordingly, in a preferred embodiment, the present invention provides methods for identifying a cell containing a mutant Radδl gene comprising determining the sequence of all or part of at least one of the endogenous Radδl genes. By "variant Rad51 gene" herein is meant any number of mutations which could result in aberrant Rad51 function or levels. 0 Thus, for example, mutations which alter the biochemical function of the Radδl protein, alter its half-life and thus its steady-state cellular level, or alter its regulatory sequences to cause an alteration in it's steady-state cellular level may all be detected. This is generally done using techniques well known in the art, including, but not limited to, standard sequencing techniques including sequencing by PCR, sequencing-by-hybridization, etc.

Similarly, in a preferred embodiment, the present invention provides methods of identifying the Radδl genotype of an individual or patient comprising determining all or part of the sequence of at least one Radδl gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. For example, putatively cancerous tissue of an individual is the preferred sample.

The sequence of all or part of the Radδl gene can then be compared to the sequence of a known Rad51 gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc.

In a preferred embodiment, the presence of a difference in the sequence between the Radδl gene of the patient and the known Rad51 gene is indicative of a disease state or a propensity for a disease state.

The present discovery relating to the role of Radδl in cancer and apoptosis thus provide methods for inducing apoptosis in cells. In a preferred embodiment, the methods comprise increasing the activity of Radδl in the cells. By "biological activity" of Radδl herein is meant one of the biological activities of Radδl, including, but not limited to, the known Radδl DNA dependent ATPase activity, the nucleic acid strand exchange activity, the formation of foci, single-stranded and double-stranded binding activities, filament formation (similar to the recA filament of yeast), pairing activity (D-loop formation), etc. See Gupta et al, supra, and Bauman et al, supra, both of which are expressly incorporated by reference herein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, the activity of Radδl is increased by increasing the amount of Radδl in the cell, for example by overexpressing the endogenous Rad51 or by administering a gene encoding Rad51, using known gene-therapy techniques, for example. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety.

In a preferred embodiment, the cells which are to have apoptosis induced are cancer cells, including, but not limited to, breast, skin, brain, colon, prostate, testicular, ovarian, etc. cancer cells, and other solid tumor cells. In a preferred embodiment, the methods may also comprise subjecting the cells to conditions which induce nucleic acid damage, as this appears to provide a synergistic effect, as outlined above.

In a preferred embodiment, the methods further comprise increasing the activity of p53 in the cell, for example by increasing the amount of p53, as outlined above for Radδl .

The present discoveries relating to the pivotal role of Radδl in a number of important cellular processes and disease states also makes Radδl , and its associated proteins, an important target in drug screening. There are a wide variety of screens that can be done, including screening for alterations and modulations in Radδl activity using Radδl (including homologs and analogs, and combinations of these), screening for alterations and modulations in Radδl activity using recombinasomes, screening for alterations and modulations in recombinasome activity (namely, double stranded break repair) using the recombinasome, and screening for alterations and modulations in the activities of other recombinasome components using the recombinasome.

Thus, in a preferred embodiment, the present invention provides methods for screening for a bioactive agent which may bind to Rad51 and modulate its activity.

In a preferred embodiment, the methods are used to screen candidate bioactive agents for the ability to bind to Radδl. In this embodiment, the methods comprise adding a candidate bioactive agent to a sample of Rad51 and determining the binding of the candidate agent to the Radδl. By "candidate bioactive agent" or "candidate drugs" or grammatical equivalents herein is meant any molecule, e.g. proteins (which herein includes proteins, polypeptides, and peptides), small organic or inorganic molecules, polysaccharides, polynucleotides, etc, which are to be tested for the capacity to bind and/or modulate the activity of Radδl . Candidate agents encompass numerous chemical classes. In a preferred embodiment, the candidate agents are organic molecules, particularly small organic molecules, comprising functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more chemical functional groups.

Candidate agents are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications to produce structural analogs.

In a preferred embodiment, candidate bioactive agents include proteins, nucleic acids, and organic moieties.

In a preferred embodiment, the candidate bioactive agents are proteins. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.

In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occuring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eukaryotic proteins may be made for screening against Radδl . Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

In a preferred embodiment, the candidate bioactive agents are peptides of from about δ to about 30 amino acids, with from about δ to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occuring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, pralines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc, or to purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleic acids. By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al, Eur. J. Biochem. 81:579 (1977); Letsinger et al, Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al, J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al, Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al. Nucleic Acids Res. 19:1437 (1991); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al, J. Am. Chem. Soc. 111:2321 (1989), O- methylphophoroamidite linkages (see Eckstein, oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al, Chem. Int Ed. Engl. 31:1008

(1992); Nielsen, Nature, 365:666 (1993); Carlsson et al. Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al, Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al, Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al, J. Am. Chem. Soc.

110:4470 (1988); Letsinger et al, Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al, Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al, J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and

5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al, Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occuring nucleic acids and analogs may be made. The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occuring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.

In one aspect it is understood that when identifying agents which bind to Rad51, the agent either is exclusive of or in addition to the DNA on which Radδl normally binds to in the process of recombinational activity.

In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.

In another preferred embodiment, the candidate agent is a small molecule. The small molecule is preferably 4 kilodaltons (kd) or less. In another embodiment, the compound is less than 3 kd, 2kd or 1kd. In another embodiment the compound is less than 800 daltons (D), 500 D, 300 D or 200 D.

The candidate agents are added to a sample of Radδl protein. As is outlined above, all or part of a full-length Radδl protein can be used, or derivatives thereof. Generally, the addition is done under conditions which will allow the binding of candidate agents to the Radδl protein, with physiological conditions being preferred. The binding of the candidate agent to the Radδl sample is determined. As will be appreciated by those in the art, this may be done using any number of techniques.

In one embodiment, the candidate bioactive agent is labelled, and binding determined directly.

Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescent molecules, enzyme reporters, colorimetric reporters, chemiluminescers, specific binding molecules, particles, e.g. magnetic or gold particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxygenin and antidigoxygenin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures.

In some embodiments, only one of the components is labeled. For example, the Rad51 may be labeled at tyrosine positions using ¹²⁵l. Alternatively, more than one component may be labeled with different labels; using ¹²⁵l for the Rad51, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agent is determined directly. For example, the Rad51 may be attached to a solid support such as a microtiter plate or other solid support surfaces, and labelled candidate agents added under conditions which favor binding of candidate agents to the Rad51 protein. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagents are washed off, the system is evaluated for the presence of the label, which is indicative of an agent which will bind to the Rad51. The agent which binds can then be characterized or identified as needed.

In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is can be any molecule known to bind to Radδl , for example an antibody to Radδl , or one of the proteins known to interact with Radδl , including Radδ2, Rad54, Radδδ, DMC 1 , BRCA1 , BRCA2, p53, UBC9, RNA polymerase II, and Radδl itself, any or all of which may be used in competitive assays. Either the candidate agents or the competitor may be labeled, or both may be labeled with different labels. In this embodiment, either the candidate bioactive agent, or the competitor, is added first to the Rad51 sample for a time sufficient to allow binding, if present, as outlined above. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

In addition, filament formation assays may be done. In this embodiment, nucleic acid

(generally single stranded) is assayed in the presence of candidate agents, Radδl and dyes (particularly fluorescent dyes). In the absence of Radδl, the dye binds to the nucleic acid. However, upon addition of Radδl , the dye is displaced, and the signal changes.

In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the activity of Rad51 comprise the steps of adding a candidate bioactive agent to a sample of Rad51, as above, and determining an alteration in the biological activity of Rad51. "Modulating the activity of Rad51" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to Rad51 (although this may not be necessary), and alter its biological or biochemical activity as defined above.

Thus, in this embodiment, the methods comprise combining a Rad51 sample and a candidate bioactive agent, and testing the Rad51 biological activity as is known in the art to evaluate the effect of the agent on the activity of Rad51 , including ATPase activity, ATP binding, strand exchange, etc.

In a preferred embodiment, methods for screening for a bioactive agent which modulates the strand exchange activity of Rad51 are done. In a preferred embodiment, FRET assays that exhibit changes in fluorescence, as outlined in the examples. The method comprises providing a preformed double stranded nucleic acid comprising a first nucleic acid single strand comprising a first fluor and a second nucleic acid single strand comprising a second fluor. The first and second nucleic acids are hybridized, quenching of one of the fluors occurs. A Rad51 nucleofilament is added comprising Rad51 and a third single stranded nucleic acid substantially complementary to one of the first or second strands. The double stranded nucleic acid is contacted with the nucleofilament in the presence of a candidate , agent to form a mixture, and the mixture is assayed for strand exchange activity.

In a preferred embodiment, the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution or activity of Rad51. Accordingly, in a preferred embodiment, the methods comprise the steps of adding a candidate bioactive agent to a cell, and determining the effect on the formation or distribution of Rad51 foci in the cell. Generally, the process provided herein which determines the effect on foci, excludes candidate bioactive agents already known in the art such as methyl methanesulfonate.

The addition of the candidate agent to a cell will be done as is known in the art, and may include the use of nuclear localization signal (NLS). NLSs are generally short, positively charged (basic) domains that serve to direct the entire protein in which they occur to the cell's nucleus. Numerous NLS amino acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon (1984), et al. Cell, 39:499-509; the human retinoic acid receptor-β nuclear localization signal (ARRRRP); NFKB p50 (EEVQRKRQKL; Ghosh et al. Cell

62:1019 (1990); NFKB p65 (EEKRKRTYE; Nolan et al. Cell 64:961 (1991); and others (see for example Boulikas, J. Cell. Biochem. 55(1):32-δ8 (1994), hereby incorporated by reference) and double basic NLS's exemplified by that of the Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gin Ala Lys Lys Lys Lys Leu Asp), Dingwall, et al. Cell, 30:449-468, 1982 and Dingwall, et al, J. Cell Biol, 107:641-849; 1988). Numerous localization studies have demonstrated that NLSs incorporated in synthetic peptides or grafted onto reporter proteins not normally targeted to the cell nucleus cause these peptides and reporter proteins to be concentrated in the nucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol, 2:367-390, 1986; Bonnerot, et al, Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al, Proc. Natl. Acad. Sci. USA, 87:458-462, 1990. In general, the Rad51 foci will be evaluated as is generally discussed above.

In a preferred embodiment, the methods comprise adding a candidate bioactive agent to a cell, and determining the effect on double strand break repair, homologous recombination, sensitivity to ionizing radiation, and class switch recombination. Assays are detailed in Park, J. Biol. Chem. 270(26):15467 (1995) and Li et al, PNAS USA 93:10222 (1996), Shinohara et al, supra, 1992, all of which are hereby incorporated by reference.

In a preferred embodiment, the cells to which candidate agents are added are subjected to conditions which induce nucleic acid damage, including the addition of radioisotopes (l^12S, Tc, etc, including ionizing radiation and uv), chemicals (Fe-EDTA, bis(1 ,10-phenanthroline), etc.), enzymes (nucleases, etc.).

A variety of other reagents may be included in the screening assays or kits, below. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc, may be used. In general, the mixture of components may be added in any order that provides for the requisite binding.

In a preferred embodiment, the methods and compositions of the invention are utilized in high throughput screening (HTS) systems, generally comprising a robotic system. The systems outlined herein are generally directed to the use of 96 well microtiter plates, but as will be appreciated by those in the art, any number of different plates or configurations may be used. In addition, any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety of components which can be used, including, but not limited to, one or more robotic arms; plate handlers for the positioning of microplates; automated lid handlers to remove and replace lids for wells on non-cross contamination plates; tip assemblies for sample distribution with disposable tips; washable tip assemblies for sample distribution; 96 well loading blocks; cooled reagent racks; microtitler plate pipette positions (optionally cooled); stacking towers for plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism- handling including high throughput pipetting to perform all steps of the screening process, both in vitro and in vivo applications. This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration. These manipulations are cross- contamination-free liquid, particle, cell, and organism transfers. This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates, tubes, magnetic particle, or other solid phase matrices can be used, particularly to bind one or more of the components of the assay. The binding surfaces of microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention to capture the required components. In a preferred embodiment, platforms for multi-well plates, multi-tubes, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity. This modular platform includes a variable speed orbital shaker, electroporator, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems are used for stabilizing the temperature of the heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from 4°C to 100°C.

In a preferred embodiment, Interchangeable pipet heads (single or multi-channel ) with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms. Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.

In some preferred embodiments, the instrumentation will include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluroescence resonance energy transfer (FRET), luminescence, quenching, two- photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation. These will enable the monitoring of the size, growth and phenotypic expression of specific markers on cells, tissues, and organisms; target validation; lead optimization; data analysis, mining, organization, and integration of the high-throughput screens with the public and proprietary databases.

These instruments can fit in a sterile laminar flow or fume hood, or are enclosed, self- contained systems, for cell culture growth and transformation in multi-well plates or tubes and for hazardous operations. The living cells will be grown under controlled growth conditions, with controls for temperature, humidity, and gas for time series of the live cell assays. Automated transformation of cells and automated colony pickers will facilitate rapid screening of desired clones.

Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms. The flexible hardware and software allow instrument adaptability for multiple applications. The software program modules allow creation, modification, and running of methods. The system diagnostic modules allow instrument alignment, correct connections, and motor operations. The customized tools, labware, and liquid, particle, cell and organism transfer δ patterns allow different applications to be performed. The database allows method and parameter storage. Robotic and computer interfaces allow communication between instruments.

In a preferred embodiment, the robotic workstation includes one or more heating or cooling components. Depending on the reactions and reagents, either cooling or heating may be 0 required, which can be done using any number of known heating and cooling systems, including Peltier systems.

In a preferred embodiment, the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. The general interaction between a central processing δ unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.

Once identified, the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The 0 inhibitory agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.

The pharmaceutical compositions can be prepared in various forms, such as granules, 6 aerosols, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic 0 pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

In a preferred embodiment, the modulators of Radδl are inhibitors. A Radδl inhibitor as defined herein inhibits expression or translation of a Rad51 nucleic acid or the biological activity of a Radδl peptide by at least 30%, more preferably 40%, more preferably 50%, more preferably 70%, more preferably 90%, and most preferably by at least 95%. In one embodiment herein, a Rad51 inhibitor inhibits expression or translation of a Rad51 nucleic acid or the activity of a Rad51 protein by 100%.

The biological activity of Rad51 is described above. As shown herein, in one aspect, by inhibiting the biological activity of Rad51, cell proliferation is inhibited. In another aspect, a Rad51 inhibitor is defined as a molecule that disrupts mammalian double stranded break repair. In a further aspect, a Rad51 inhibitor results in the cells containing it to be more sensitive to radiation and/or chemotherapeutic agents.

In one embodiment herein, inhibitors of Rad51 include those identified by the methods provided herein as well as known downregulators or inhibitors of Rad51 as defined above. In another aspect, Rad51 inhibitors can include known inhibitors of RecA and/or known inhibitors that sensitize cells to radiation and also affect aspects of recombination in vivo. Inhibitors of interest also include but are not limited to peptide inhibitors of Rad51 (including but not limited to amino acids 94-160 and 264-315 of p53 and Rad51 antibodies (further described below) including but not limited to single chain antibodies), small molecules, nucleotide analogues (including but not limited to ADP analogues), minor groove DNA binding drugs as inhibitors of Rad51 (including but not limited to distamycin and derivatives thereof), known radiation sensitizers (e.g., xanthine and xanthine derivatives including caffeine) on the biochemical activities of Rad51, antigenes against Rad51, particularly those which inhibit transcription by locked hybrids, and antisense molecules. The inhibitor can inhibit Rad51 directly or indirectly, preferably directly by interacting with at least a portion of the Rad51 nucleic acid or protein. Additionally, the inhibitors herein can be utilized individually or in combination with each other.

Generally, the Rad51 antisense molecule is at least about 10 nucleotides in length, more preferably at least 12, and most preferably at least 15 nucleotides in length. The skilled artisan understands that the length can extend from 10 nucleotides or more to any length which still allows binding to the Rad51 nucleic acid. In a preferred embodiment herein, the length is about 100 nucleotides long, more preferably about 60 nucleotides, more preferably about 2δ nucleotides, and most preferably about 12 to 25 nucleotides in length.

The nucleic acids herein, including antisense nucleic acids, and further described above, are recombinant nucleic acids. A recombinant nucleic acid is distinguished from naturally occurring nucleic acid by at least one or more characteristics. For example, the nucleic acid may be isolated or purified away from some or all of the nucleic acids and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated nucleic acid is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 6% by weight of the total nucleic acid in a given sample. A substantially pure nucleic acid comprises at least about 75% by weight of the total nucleic acid, with at least about 80% being preferred, and at least about 90% being particularly preferred. Alternatively, the recombinant molecule could be made synthetically, i.e., by a polymerase chain reaction, and does not need to have been expressed to be formed. The definition includes the production of a nucleic acid from one organism in a different organism or host cell.

The antisense molecules hybridize under normal intracellular conditions to the target nucleic acid to inhibit Rad51 expression or translation. The target nucleic acid is either DNA or RNA. In one embodiment, the antisense molecules bind to regulatory sequences for Rad51. In one embodiment, the antisense molecules bind to 5' or 3' untranslated regions directly adjacent to the coding region. Preferably, the antisense molecules bind to the nucleic acid within 1000 nucleotides of the coding region, either upstream from the start or downstream from the stop codon. In a preferred embodiment, the antisense molecules bind within the coding region of the Rad51 molecule. In a particularly preferred embodiment, the antisense molecule has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID

NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In one embodiment, the antisense molecules are not directed to the structural gene; this embodiment is particularly preferred when the antisense molecule is not combined with another antisense molecule.

In one embodiment combinations of antisense molecules are utilized. In one embodiment, at least antisense molecule is selected from the 3' untranslated region.

The term "antibody" is used in the broadest sense and specifically covers single anti-Rad51 monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti- Rad51 antibody compositions with polyepitopic specificity. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. In a preferred embodiment, the antibodies are specific for a particular homolog. The anti-Rad51 antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the Rad51 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant

(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

The anti-Rad51 antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature. 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the Rad51 polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol.. 133:3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against Rad51. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supral. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al, supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen- combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

The anti-Rad51 antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al. Nature, 321 : 522-625 (1986); Riechmann et al. Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct Biol, 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain; Humanization can be essentially performed following the method of Winter and co-workers [Jones et al. Nature, 321:522-626 (1986); Riechmann et al. Nature. 332:323-327 (1988); Verhoeyen et al. Science. 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J. Immunol, 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,645,807; 6,645,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks etal., Bio/Technology 10, 779-783 (1992); Lonberg ef al., Nature 368 856-859 (1994); Morrison, Nature 368. 812-13 (1994); Fishwild et al.. Nature Biotechnology 14. 845-51 (1996); Neuberqer, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the Rad51 , the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit. In a preferred embodiment, one of the binding specificities is for the Rad51 , the other one is for a tumor suppressor antigen subunit or a tumor antigen subunit. In one embodiment, one of the binding specificities is for the Rad51 , the other one is for c-abl or ATM (ataxia telangiectasia mutated).

In another embodiment, the antibodies bind to Rad51 only when it is complexed with another protein. The antibody may bind to both Rad51 and the other protein, or it may only bind to Rad51 , wherein the epitope is not exposed on Rad51 in its uncomplexed form. As discussed below, Rad51 complexes include Rad51 complexed with oncogene products such as c-abl, ATM, tumor suppressor gene products, Rad51 homologs, other Rad proteins such as Rad54, 5δ and 57 and all the proteins forming a Rad51 recombisome.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:637-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge,

CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.

DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al. Methods in Enzvmology. 121:210 (1986).

Heteroconjugate antibodies are also within the scope of the present invention.

Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO

92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.

For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.

Patent No. 4,676,980. Phage display methods can be used to identify epitopes.

In an embodiment provided herein, the invention provides methods of treating disease states requiring inhibition of cellular proliferation. In a preferred embodiment, the disease state requires inhibition of at least one of Rad51 expression, translation or the biological activity of Rad51 as described herein. As will be appreciated by those in the art, a disease state means either that an individual has the disease, or is at risk to develop the disease.

Disease states which can be treated by the methods and compositions provided herein include, but are not limited to hyperproliferative disorders. More particular, the methods can be used to treat, but are not limited to treating, cancer (further discussed below), autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. Thus, in one embodiment, the invention herein includes application to cells or individuals afflicted or impending affliction with any one of these disorders.

The compositions and methods provided herein are particularly deemed useful for the treatment of cancer including solid tumors such as skin, breast, brain, cervical carcinomas, testicular carcinomas, pancreas, prostate, colon, etc.. More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofϊbroma, osteoid osteoma and giant cell tumors; Nervous system: skull

(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa- thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma. dermatofibroma, keloids. psoriasis; and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above identified conditions.

The individual, or patient, is generally a human subject, although as will be appreciated by those in the art, the patient may be animal as well. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc, and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of patient. In a preferred embodiment, the individual requires inhibition of cell proliferation. More preferably, the individual has cancer or a hyperproliferative cell condition.

The compositions provided herein may be administered in a physiologically acceptable carrier to a host, as previously described. Preferred methods of administration include systemic or direct administration to a tumor cavity or cerebrospinal fluid (CSF).

In a preferred embodiment, these compositions can be administered to a cell or patient, as is outlined above and generally known in the art for gene therapy applications. In gene therapy applications, the antisense molecules are introduced into cells in order to achieve inhibition of Rad51. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or RNA. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al.. Trends in Biotechnology 11. 206-210 [1993]).

In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu ef a/, J. Biol. Chem. 262. 4429-4432 (1987); and Wagner et al.. Proc. Natl. Acad. Sci. USA 87. 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson ef al., Science 256, 808- 813 (1992).

The antisense molecules can be combined in admixture with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the δ appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon 0 Press, New York 1989, pp. 42-96.

In one aspect, the Rad51 inhibitors herein induce sensitivity to alkylating agents and radiation. Induced sensitivity (also called sensitization or hypersensitivity) can be measured by the cells tolerance to radiation or alkylating agents. For example, sensitivity, which can be measured, i.e., by toxicity, occurs if it is increased by at least 20%, more preferably at 5 least 40%, more preferably at least 60%, more preferably at least 80%, and most preferably by 100% to 200% or more.

In an embodiment herein, the methods comprising administering the Rad51 inhibitors provided herein further comprise administering an alkylating agent or radiation. For the purposes of the present application the term ionizing radiation shall mean all forms of 0 radiation, including but not limited to alpha, beta and gamma radiation and ultra violet light, which are capable of directly or indirectly damaging the genetic material of a cell or virus. The term irradiation shall mean the exposure of a sample of interest to ionizing radiation, and term radiosensitive shall refer to cells or individuals which display unusual adverse consequences after receiving moderate, or medically acceptable (i.e., nonlethal diagnostic or δ therapeutic doses), exposure to ionizing irradiation. Alkylating agents include BCNU and CENU. Particularly preferred are DNA damaging agents. A preferred agent is cisplatin.

In one embodiment herein, the Radδl inhibitors provided herein are administered to prolong the survival time of an individual suffering from a disease state requiring the inhibition of the proliferation of cells. In a preferred embodiment, the individual is further administered a DNA 0 damaging agent, such as radiation or an alkylating agent

In a preferred embodiment, kits are provided. The kits can be utilized in a variety of applications, including determining the distribution of Rad51 foci, diagnosing an individual at risk for a disease state, including cancer, diseases associated with apoptosis, and diseases associated with stress (including oxidative stress, hypoxic stress, osmotic stress or shock, heat or cold stress or shock). The kits include a Radδl binding agent, that will bind to the Radδl with sufficient affinity for assay. Antibodies are preferred binding agents. The kits further include a detectable label such as is outlined above. In one embodiment, the Radδl binding agent is labeled; in an additional embodiment, a secondary binding agent or label is used. Thus for example, the binding agent may include biotin, and the secondary agent can include streptavidin and a fluorescent label. Additional reagents such as outlined above can also be included. Furthermore, the kit may include packaging and instructions, as required.

The identification of the crucial role of Radδl in a number of cellular processes and disease states also identifies a number of methods and compositions relating to combinations of Radδl and other tumor suppressor genes. Thus, Radδl may function interactively with a number of tumor suppressor genes and thus compositions comprising combinations of these genes may be useful in methods of gene therapy treatment and diagnosis.

Accordingly, in a preferred embodiment, compositions comprising a nucleic acid encoding a Rad51 protein and at least one nucleic acid encoding a tumor suppressor gene are provided. Suitable tumor suppressor genes include, but are not limited to, pδ3, and the BRCA genes, including BRCA1 and BRCA2 genes. Thus, preferred embodiments include compositions of nucleic acids encoding a) a Rad51 gene and a pδ3 gene; b) a Rad51 gene and a BRCA1 gene; c) a Radδl gene and a BRCA2 gene; d) a Radδl gene, a pδ3 gene, and a BRCA gene; and e), a Rad51 gene, a p53 gene, a BRCA1 gene and a BRCA2 gene.

Other compositions provided herein are either the nucleic acids encoding a complex comprising Radδl or a Radδl complex. A Radδl complex as defined herein is a composition which Radδl associates with in vivo. Examples include but are not limited to Radδl in a complex with Radδ4, Radδδ, Radδ7, any of the tumor suppressor genes described herein or oncogene gene products. In one embodiment the composition comprises the epitope to which an antibody binds to the Rad51 complex.

In an additional embodiment, the compositions comprise recombinant proteins. By "recombinant" herein is meant a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above, a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.6%, more preferably at least about 5% by weight of the total protein in a given sample, a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. The definition includes the production of a protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.

In a preferred embodiment, these compositions can be administered to a cell or patient, as is outlined above and generally known in the art for gene therapy applications.

The following examples serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are specifically incorporated by reference in their entirety.

EXAMPLES

Example 1

Immunofluorescent Staining of Human Breast Cancer Cells

Breast tumour cells have mutated p53 and have various types of chromosomal aberrations like insertions, deletions, rearrangements, amplifications etc. Recombination proteins such as Rad51 could evidently participate in such processes. In order to better understand the role of uncontrolled recombination and its role in tumour formation and progression, the status of Radδl protein in breast tumour cells by staining them with anti Rad51 antibodies was done.

Detailed methods of cloning and expression of HsRadδl gene in E.coli, purification of recombinant HsRadδl protein with six histidine residues at it's aminoterminal end and preparation of ployclonal antibodies against HsRadδl protein were described previously by Haaf, Golub et al. 1996, supra, which is expressly incorporated herein by reference.

Immunofluorescent staining with anti-Rad51 protein antibodies. Monolayer cultures of different cell substrates (see table 1) were grown in Dulbecco's MEM medium supplemented with 10% fetal bovine serum and antibiotics. The cells were detached from culture flasks by gentle trypsinization, pelleted and resuspended in phosphate buffered saline (PBS; 136 mM NaCI, 2 mM KCI, 10.6 mM Na₂HP0₄, 1.5 mM KH₂P0₄ [pH 7.3]) prewarmed at 37°C. For immunofluorescence staining standard protocols were used (Haaf 1995, supra). Cultured cells were washed and resuspended in PBS. The density of somatic cells was adjusted to about 10⁵ cells per ml in PBS. Aliquots (0.5 ml) of the cell suspension were centrifuged onto clean glass slides at 800 rpm for 4 min, in a Cytospin (Shandon, Pittsburg). Immediately after cytocentrifugation, the slides were fixed in -20°C methanol for 30 min and then immersed in ice-cold acetone for a few seconds to permealize the cells for antibody staining. Following three washes with PBS, the preparations were incubated at 37°C with rabbit anti-HsRadδl antiserum, diluted 1:50 with PBS containing 0.5% bovine serum albumin, in a humidified incubator for 30 min. The slides were washed three times for 10 min each and then incubated for 30 min with fluorescein-isothiocyanate (FITC)- conjugated anti-rabbit IgG diluted 1:20 with PBS. After three washes with PBS, the preparations were counterstained with 4',6-diamidino-2- phenylindole (DAPI; 0.1 ug/ml for 1 min) and mounted in antifade {90% (vol/vol) glycerol/0.1 m tris-HCI pH 8.0)/2.3% 1 ,4-diazabicyclo[2.2.2]octane (DABCO)}.

Digital Imaging Microscopy. Images were taken with a Zeiss epifluorescence microscope with a thermo-electronically cooled charge coupled device (CCD) camera (model PM512; Photometries, Tucson, AZ) which was controlled by an Apple Macintosh computer. Grey scale source images were captured separately with filter sets for fluorescein and DAPI. Gray scale source images were pseudocolored and merged using ONCOR Image and ADOBE Photoshop software. It is worth emphasizing that although a CCD imaging system was used, all antibody signals were clearly visible by eye through the microscope.

To study the possible involvement of Radδl in tumorigenesis we compared the the in situ localization of of Rad51 protein homologs in different cell substrates i.e. mortal fibroblast strains, virus-transformed non-malignant cell lines and tumor cell lines (see table), a specific rabbit antiserum raised against human Radδl protein was used in these studies. These antibodies reacted mainly with Radδl protein in mammalian cell extracts as judged by Western blotting (see fig No 2 in (Haaf, Golub et al. 1995). Immunostaining of different cells showed that HsRadδl is concentrated in small and discrete sites (foci) through out nucleoplasm and is largely excluded from nucleoli and cytoplasm. At least 260 nuclei of exponentially growing cultures were analyzed for each experiment. As reported earlier (Haaf, Golub et al. 1996) immunostaining revealed three kinds of nuclei: 1) nuclei that did not show any staining at all ( no foci), 2) nuclei that showed weak to medium staining and showed only a few foci (Type I nuclei) 3) nuclei that showed strong staining and showed many foci (Type II nuclei). In normal fibroblast control cells, we found type I nuclei in about 10% of cells and type II nuclei in less than 0.4 to 1% of cells and about 90%of the cells showed no foci. Use of preimmune serum, as well as omission of either the primary or secondary antibody, resulted in the absence of focally concentrated nuclear immunofluorescence.

As reported earlier (Haaf, Golub et al. 1996) in normal (mortal) fibroblast control cells (Hs68) we found type I nuclei in 7% -10% of cells and type II nuclei in less than 0.4% of cells, where as 90% or more of the cells showed no foci (Table 1). In contrast all breast tumor cell lines tested (BT20, SrBr3, McF7) exhibited 1-5% of type II nuclei and 10-38% of type I nuclei (Table 1). Transformed but non-malignant human cells, i.e. SV 40 transformed fibroblasts (LNL8, 63L7), EBV-transformed lymphoblasts (GM 01194), and adenovirus-transformed kidney cells (293) also showed an increased percentage of Radδl -positive cells (compared to normal fibroblasts), however the numbers observed were lower than in tumor cells. Interestingly, some tumor substrates i.e. the ovarian cancer line Hey; did not show a significant increase of Radδl -positive cells.

As demonstrated earlier (Haaf, Golub et al. 1996), when the normal fibroblast cells were exposed to DNA damaging agents like 137Cs, there was a significant increase of cells containing type I and type II nuclei (Table 2). It is worth emphasizing that non-irradiated breast tumor cells show approximately the same percentage of Rad 51 -positive nuclei as Hs68 fibroblasts exposed to 900 rad Cs137 which kills 99% of cells (Table 2). The immunofluorescent patterns of (non-irradiated) breast cancer cells (Figure 1) and fibroblasts that were exposed to DNA damaging agents are identical.

When the breast cancer cells were exposed to Cs137, the increase in the number of cells with type I and type II nuclei was even more dramatic than in normal (Hs68) or transformed (LNL8) fibroblasts (Table 2). Up to 40% of irradiated breast cancer cells showed type I nuclei and 11%-18% showed type II nuclei.

In order to rule out any artifacts that would arise due to the examination of cultured breast cancer cells, we then examined the breast tissue obtained directly from the patient for Radδl positive staining. Immunohistochemical evaluation revealed definite nuclear staining of invasive breast carcinoma cells. Specifically, nuclear reactivity could be demonstrated in sections obtained from three paraffin-embedded samples. The nuclear staining appeared granular in some areas, and in others, occupied the entire nucleus. The actual number of invasive carcinoma cells that fluoresced was quite small, and estimated to be less than δ% of the nuclei seen in three samples with definite reactivity Figure 2). Nuclear staining was not identical in normal breast epithelium or lactating breast tissue. Bright nuclear reactivity was seen in positive control testicular tissue, specifically, in the cells lining the seminiferous tubules. Background staining did not appear to be problematic.

Increase in immunofluorescence of HsRadδl in breast cancer cells can result from either increase in the amount of Hsradδl in these cells or it could be seen as a result of re-organization of Hsradδl in these nuclei in response to damage related activities. We think that the latter is true because there was no apparent increase in the amount HsRadδl in breast cancer cells as shown by the Western blots (data not shown).

The molecular basis and the consequence of the increase in HsRadδl in breast cancer cells is not clear. Since Radδl protein interacts with other proteins of the Rad52 epistasis group and these muitiprotein complexes are involved in the recombinational repair of double-strand breaks (Hays, et al, (1995). Proc. Natl. Acad. Sci. USA 92: 6925-6929; Johnson, R. D. and L. S. Symington (1995). Mol. Cell. Biol. 15: 4843-4850), it is tempting to speculate that these foci are the sites where repair/recombination events are taking place. Since p53 is known to interact with Rad51 it iwill be interesting to see the colocalization of pδ3 and Radδl protein in these complexes. It is quite possible that these foci contain either wild type or mutant pδ3 and other breast cancer related proteins like BRCA1, BRCA2 or the newly discovered STG1 protein. We propose that the increase in the immunofluorescence of Rad51 in the breast cells can be used as an important cytological marker for cell proliferation and malignant cell growth. Further experimentation will be done to validate this proposal and to understand the role of increase in Radδl foci and carcinogenesis. Table 1 : Percentage of nuclei containing discrete foci enriched with HsRadδl protein.

Cell Substrate No foci Type I Type II

Hs68 Normal fibroblasts 90% 10% 0% 93% 7% 0%

LNL8 (Nl 00847) Transformed fibroblasts 90% 9% 1%

(SV 40) 90% 8% 2%

63L7 Transformed fibroblasts 94% 6% 0%

(SV40) 94% 3% 3%

GM01194 Transformed lymphoblasts 91% 7% 2%

(EBV) 90% 9% 1%

92% 8% 0%

80% 18% 2%

80% 19% 1%

293 Cells Transformed kidney cells 75% 23% 2% (Adenovirus) 83%> 15% 2% 82% 17% 1%

BT20 Breast cancer line 86%> 10% 4%

82% 13% 5%

78% 17% 5%

SrBr3 Breast cancer line 74% 25% 1 % McF7 Breast cancer line 67% 38% 5% 88% 10% 2%

Tera2 Testicular teratoma 76% 23% 1% 77% 22%o 1%)

Hey Ovarian cancer line 94% 5% 1% 98% 2% 0%

HeLa Cervix (?) tumor cells 67% 31% 2%

Table 2: Percentage of nuclei containing discrete foci enriched with HsRadδl protein. Cell substrate Treatment No foci Type I Type II

Hs68 None 90% 10% 0% (normal None 93%o 7% 0% fibroblasts) 6 hrs after 10 rad Cs137 96% 4% 0%

6 hrs after 50 rad Cs137 96% 4% 0%

6 hrs after 150 rad Cs137 92% 7% 1%

6 hrs after 450 rad Cs137 88% 8% 4%

6 hrs after 900 rad Cs 137 91% 4% 5%

LNL8(NI 00847) None 90% 9% 1%

(SV40-transformed None 90% 8% 2% fibroblasts) ^' 6 hrs after 150 rad Cs 137 88% 11% 1%

6 hrs after 300 rad Cs137 76% 19%> 5% 6 hrs after 900 rad Cs137 78% 17% δ%

BT20 None 86% 10% 4%

(breast cancer None 82% 13% 5% cells) None 78% 17% δ%

6 hrs after 300 rad Cs137 44% 41% 11%

6 hrs after 900 rad Cs137 52% 30% 18%

Example 2

Nuclear foci of human recombination protein Radδl in nucleotide excision repair defective cells

Eurkaryotic cells have several different mechanisms for repairing damaged DNA (for review see R. Wood, 1996). One of the major pathway is nucleotide excision repair (NER), which excises damage within oligomers that are 25-32 nucleotides long. Patients with recessive heredity disorder XP have defects in one of several enzymes, which participate in ER. There are seven XP groups (XP-A to XP-G), which have defects in the initial steps of the DNA excision repair.

DNA damage is removed several-fold faster from transcribed genes than from non- transcribed, mainly due to preferential NER of the transcribed strand (for review see Hanawalt, 1994). This mechanism does not function in Cockayne's syndrome (CS) patients.

NER defective cells, evidently, sustain increased amount of DNA damage. Thus we evaluated NER defective cells from XP and CS cells for an increased amount of Rad51 protein foci.

To study possible effect of NER on localization of HsRadδl in somatic tissue culture cells, we compare in situ localization of the protein in normal fibroblasts, different XP cells and CS- B cells. A policlonal rabbit antiserum raised against human Radδl protein was used in this study. These antibody reacted in mammalian cell extract mainly with Radδl protein as judged by Western Blotting (see FIG. 2 in Haaf et al, 1995). Immunostaining of different cell lines showed that HsRadδl is concentrated in small and discrete sites (foci) throughout nucleoplasm and is largely excluded from nucleoli and cytoplasm. As discussed above, immunostaining revealed three kinds of nuclei, types I, II and III. The results are shown in Table 3.

Table 3: Percentage of nuclei containing discrete foci enriched with HsRadδl protein

*Type I nuclei show only few (<15) foci and/or weak to medium HsRadδl immunofluorescence, whereas Type II cells show many and/or strongly fluorescing foci. 250 nuclei were analyzed for each experiment.

In normal (mortal) fibroblast control cells, LNL8 and NF, we found type I nuclei in δ-9% cells and type II nuclei in 1-7% cells, where as 88-90% of the cells showed no foci (Table 3). Use of preimmune serum, as well as omission of either the primary or secondary antibody, resulted in the absence of focally concentrated nuclear immunofluorescence.

δ XP-V cells are normal in NER, but have defect in postreplication repair process (Boyer et al, 1990; Griffiths et al, 1991; Wang et al, 1991, 1993). As we expected, these cells showed the same distribution pattern of nuclear HsRadδl as control cell lines (Table 3).

Distribution of HsRadδl foci in CS-B cells also was similar to the cells with normal NER (Table 3). This result was also anticipated. CS-B cells are defective in NER which is 0 coupled with transcription (Venema et al, 1990). Transcribed genes, evidently, comprise only a small part of the whole genomic DNA and damage in transcribed genes, therefore, should be accounted for only a very small fraction of the damage in genomic DNA.

XP-A, XP-B, XP-F and XP-G cells are all defective in NER. XP-A cells have defect in XPA protein, which carries out a crucial rate-limiting step in NER-recognition of DNA lesion (Jones 6 and Wood, 1993). The protein makes a ternary complex with ERCC1 protein and XPF protein, which is defective in XP-F cells (Park and Sankar, 1994). XP-B and XP-G cells are defective in different steps of NER which follow damage recognition (Reviewed in Ma et al, 1995).

XP-A and XP-F cell lines have increased amount of cells with HsRadδl protein foci (Table 0 3). In contrast XP-B and XP-G cells have about the same level of HsRadδl protein foci, as cells with normal NER (Table 3). This result could be easily understood if we assume, that 1) formation of HsRadδl foci is caused by DNA damage, b) DNA lesion is excluded from the pool of damage DNA which cause Radδl foci formation as soon as XPA/XPF/ERCC1 complex binds to the lesion. DNA damage in XP-Band XP-G cells is recognized by NER 5 system, but the damage cannot be proceeded and removed by the system. Such unremoved damage, evidently, is not considered as a substrate for Rad51 protein involved repair as soon as the damage is recognized by NER complex XPA/XPF/ERCC1 as a substrate for NER, even if defect in subsequent steps of NER makes its removing impossible.

0 Induction of principal DNA repair system (SOS respond) in E. coli is, assumed to be triggered by formation of single-stranded DNA (ssDNA) which results from DNA damage (reviewed in Little and Mount, 1982). DNA damage in XP-A cells is not recognized by NER and, therefore, at least a considerable part of DNA damage is not proceeded to formation of ssDNA regions. Nevertheless, Radδl foci are effectively formed in XP-A cells and their amount could be further increased by UV or -irradiation (Tables 4 and δ). Evidently, ssDNA is not a primary signal for HsRadδl protein foci formation.

Table 4: Percentage of nuclei containing discrete foci enriched with HsRad51 protein

*Type I nuclei show only a few (<15) foci and/or weak to medium HsRadδl immunofluorescence, whereas type II cells show many and/or strongly fluorescing foci. 250 nuclei were analyzed for each experiment.

Table δ: Percentage of nuclei containing discrete foci enriched with HsRadδl protein

^*Type I nuclei show only a few (<15) foci and/or weak to medium HsRadδl immunofluorescence, whereas Type II cells show many and/or strongly fluorescing foci. 150 nuclei were analyzed for each experiment.

^**lnduction of HsRadδl foci in Xeroderma pigmentosum (Type A) implies that single stranded DNA molecules are not the primary signal.

^***lnduction of HsRad51 foci in cells from patients with Cockayne's syndrome implies that the induction is not dependent on transcription.

In conclusion, human recombination protein HsRadδl is concentrated in multiple discrete foci in nucleoplasm of cultured human cells. After treatment of cells with DNA damaging agents, the percentage of cells with HsRadδl protein immunofluorescence increases. Xeroderma pigmentosum (XP) cells XP-A with inactive protein XPA, responsible for lesion recognition by nucleotide excision repair (NER) system have increased percentage of cells with HsRadδl protein foci. XP-F cells, defective in XPF protein, which forms complex with XPA protein, also have increased level of the HsRadδl protein foci. In contrast, XP-B and 5 XP-G cells with defects in different steps ER, which follow the damage recognition, as well as XP-V cells (normal level of NER) and Cockayne's syndrome (CS) cells (defect in NER, responsible for preferential repair of the transcribed DNA strand) have normal level of HsRadδl protein foci. Evidently, formation of HsRadδl protein foci is caused by DNA damages. DNA damages, however, do not participate in causing formation of HsRadδl 0 protein foci, as soon as they are recognized by NER system, even if the system is blocked on one of the step, leading to DNA repair.

Example 3

Higher order nuclear structures of Radδl and its exclusion into micronuclei after cell damage

δ Previous studies have revealed a time- and dose-dependent increase of nuclear HsRadδl protein foci after DNA damage introduced into the genome by various agents (Haaf et al, 1996, supra). Here we show that when the damaged cells are allowed to recover, these Rad51 foci form specific higher-order nuclear structures. Finally, all the focally concentrated Radδl protein is eliminated into micronuclei that undergo apoptotic genome fragmentation. 0 Treatment of cells with clastogens and aneuploidogens implements a mechanism that affects the nuclear distribution of Radδl protein and targets Rad51 foci, most likely along with irreversibly damaged DNA into micronuclei. To examine the role of Radδl protein in DNA repair and cell proliferation, we have analyzed the intranuclear distribution of overexpressed Radδl protein during the cell cycle and in cell populations proceeding 6 through apoptosis.

Experimental Procedures

Cell Culture. The sources of the cell lines were as follows. Rat TGR-1 cells, J. Sedivy, Brown University; mouse 3T3-Swiss cells, ATCC; human 293 kidney cells, ATCC; human teratoma cells, B. King, Yale University; human LNL8 fibroblasts, S. Meyn, Yale; human XPA 0 and XPF fibroblasts, P Glazer, Yale.

Monolayer cultures were grown in D-MEM medium supplemented with 10% fetal bovine serum and antibiotics. The cells were detached from culture flasks by gentle trypsination, pelleted and resuspended in phosphate-buffered saline (PBS; 136 mM NaC1, 2 mM KCI, 10.6 mM Na₂HP0₄, 1.5 mM KH₂P0₄, pH 7.3) prewarmed at 37°C. To induce DSBs in DNA and recombinational repair, cell cultures were exposed to a ¹³⁷Cs irradiator at doses of 900 rad and then allowed to recover for various time spans. In another experiment, cells were treated with 10 μM δ-aza-dC for 24 hrs. This hypomethylating base analog is a potent DNA-strand breaker (Snyder, et al, (1989). Mutation Res. 226, 185-190; Haaf, 1996). Incubation of cells with the spindle poison colcemid (1 μg/ml for 24 hrs) resulted in the formation of multinuclei and micronuclei containing entire chromosomes. Under the experimental conditions chosen, colcemid does not cause chromsome breakage. Treatment with etoposide (Sedivy), a drug that inhibits DNA topoisomerase II, is a classic system for inducing apoptosis in cells (Mizumoto, et al, (1994). Mol. Pharmac. 46, 890-896).

Antibody Probes. HsRadδl protein, expressed in E. coli, was isolated and used for preparation of rabbit polyclonal antibodies. Western blotting experiments revealed that rabbit antiserum does not react significantly with any other proteins in mammalian cells except Radδl (Haaf et al, 1996). Similarly, polydonal antibodies against HsRadS2, a structural homolog of yeast Radδ2, were raised in the rat, as is known in the art. Mouse monoclonal antibody 30T14 recognizes Gadd45, a ubiquitously expressed mammalian protein that is induced by DNA damage (Smith, et al, (1994). Science 266, 1376-1380). Monoclonal antibodies H4 and H14 bind specifically to the large subunit of RNAPII (Bregman et al, (1996) J. Cell Biol. 129, 287-298). Monoclonal antibody Pab246 against amino acids 88-93 of mouse p53 was purchased from Santa Cruz Biotechnology, Inc.

Immunofluorescent Staining. Harvested cells were washed and resuspended in PBS. Cell density was adjusted to ~10⁵ cells/ml. 0.6 ml aliquots of this cell suspension were centrifuged onto clean glass slides at 800 rpm for 4 min, using a Shandon Cytospin. Immediately after cytocentrifugation, the preparations were fixed in absolute methanol for 30 min at -20°C and then rinsed in ice-cold acetone for a few seconds. Following three washes with PBS, the preparations were incubated at 37°C with rabbit anti-HsRad51 antiserum, diluted 1:100 with PBS, in a humidified incubator for 30 min. For some experiments, the slides were simultaneously labeled with rat anti-HsRadδ2 antiserum or mouse monoclonal antibody. The slides were then washed in PBS another three times for 10 min each and incubated for 30 min with fluorescein-isothiocyanate (FITC)-conjugated anti-rabbit laG, appropriately diluted with PBS. Radδ2, Gadd45, pδ3, and RNAPII were detected with rhodamine, conjugated anti-rat IgG or anti-mouse IgG+lgM. After three further washes with PBS, the preparations were counterstained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) in 2xSSC for 5 min. The slides were mounted in 90% glycerol, 0.1 M Tris-HCI, pH 8.0, and 2.3% 1 ,4-diazobicyclo-2,2,2-octane (DABCO). For preparation of chromatin fibers, cells were centrifuged onto a glass slide and covered with 50 μl of 50 mM Tris-HCI, pH 8.0, 1 mM EDTA, and 0.1% SDS. The protein-extracted chromatin was mechanically sheared on the slide with the aid of another slide (Heiskanan, et al, (1994) BioTechniques 17, 928-933) and then fixed in methanol/acetone.

Fluorescence In Situ End Labeling (FISEL). FISEL detects cell death (apoptosis) in situ by quantitating DNA strand breaks in individual nuclei. It uses terminal transferase (TdT) to label the 3'-ends in fragmented genomic DNA with biotinylated nucleotide. 100 μl of reaction mix contain I μl (26 Units) TdT (Boehringer Mannheim), 20 μl δxTdT buffer (supplied with the enyzme), 1 μl O.δ mM biotin-ll-dUTP, 3 μl 0.5 mM dTTP, and 75 μl ddH₂0. Cytological preprations are incubated at 37°C for 1 hr with this reaction mix. Washing the slides for 3x5 min in PBS is sufficient to terminate the reaction. The incorporated biotin-dUTP is detected with rhodamine-conjugated avidin.

In Situ Labeling of DNA-Replication Synthesis. The base analog BrdU is incorporated in place of thymidine into the DNA of replicating cells. In order to mark cycling cells, 10 μg/ml BrdU were added to the culture medium 30 hrs before cell harvesting. Depending on the cell substrate, this corresponds to one or two population doublings. At the end of the labeling period, slides were prepared as described above. After Rad51 -protein staining, the preparations were again fixed in a 3:1 mixture of methanol and acetic acid for several hours at -20°C. Since the anti-BrdU antibody only recognizes BrdU incorporated into chromosomal DNA if the DNA is in the single-stranded form, the slides were denatured in 70% formamide, 2xSSC for 1 min at 80°C and then dehydrated in an alcohol series. BrdU incorporation was visualized by indirect anti-BrdU antibody staining. First, the preparations were incubated with mouse monoclonal anti-BrdU antibody (Boehringer Mannheim), diluted 1:50 with PBS, for 30 min. The slides were washed with PBS and then incubated with rhodamine-conjugated anti-mouse IgG, diluted 1 :20 with PBS, for another 30 min. Only cells with intense BrdU labeling of the entire nucleus were considered BrdU-positive and scored as cycling cells.

Overexpression of HsRad51 Protein in Mammalian Cells. Human kidney cells (line 293, ATCC CRL1573) were stably transformed by plasmid pEG9 16. This plasmid carries the whole coding sequence of the HsRadδl gene inserted in frame with the δ'-end terminal sequence of vector pEBVHisB (Invitrogen). The resulting cell lines 710 and 717 constitutively express Rad51 protein fused to a T7-tag epitope (Haaf et al, 199δ).

Digital Imaging Microscopy. Images were taken with a Zeiss epifluorescence microscope equipped with a thermoelectronically cooled charge coupled device (CCD) camera (Photometries CH260), which was controlled by an Apple Macintosh computer. Gray scale source images were captured separately with filter sets for FITC, rhodamine, and DAPI. Gray scale images were pseudocolored and merged using ONCOR Image and ADOPE Photoshop software. It is worth emphasizing that although a CCD imaging system was used, the immunofluorescent signals described here were clearly visible by eye through the microscope.

Dynamic Nuclear Distribution of Rad51 Protein after DNA Damage Nuclear foci of mammalian Radδl -recombination protein can be induced significantly after irradiation of cell cultures with Cesium (¹³⁷Cs). Since Western blots have not shown a dramatic net increase in Radδl protein in irradiated cells, we conclude that DNA damage mainly affects its nuclear distribution (Haaf et al, 1996). To gain insight into the radiation-induced perturbations in nuclear organization and the possible role of Rad51 protein in repair processes, we have analyzed the topological rearrangements of Radδl-protein foci in rat TGR-I fibroblasts that have sustained DNA damage. TGR-I is an immortal rat cell line with a stably diploid karyotype. After ¹³⁷Cs irradiation with a dose of 900 red which kills 99% of cells, rat Radδl protein was visualized in situ using polyclonal antibodies raised against HsRadδl . The percentage of cells with cytologically detectable Rad: 1-protein foci started to increase in the first three hours (Table 6). Radδl -positive nuclei contained up to several dozen discrete foci throughout their nucleoplasm. Immunofluorescence staining was largely excluded from the cytoplasm. Many of these nuclear Radδl foci had a double-dot appearance, typical of paired DNA segments (Figure 3a).

Table 6. Induction of Radδl Foci after ¹³⁷Cs Irradiation of TGR-1 cells and Their

Elimination into Micronuclei

^aType I nuclei and micronuclei show weak to medium HsRadδl immuno-fluorescence, whereas type II cells show strongly fluorescing foci. 1000 cells were anlayzed for each experiment.

When irradiated cells were then cultured for various times to allow repair of induced DNA damage and apoptosis to occur, significant changes in the distribution of Radδl-protein foci were detected. Nuclear foci coalesced into larger clusters with extremely high immunofluorescence intensity after 6-20 furs. Only a few discrete foci remained singly in the nucleoplasm. In a percentage of nuclei linear strings of δ-10 Radδl -protein foci were formed (Figure 3b). Immediately striking was the somatic association of "homologous" strings of similar length. These strings were always tightly paired at one of their ends. The dynamics of the Radδl-protein foci after induction of DNA damage are clear evidence for a higher-order organization of nuclear structure that accompanies DNA repair and/or programmed cell death.

One to two days after ¹³⁷Cs irradiation with a lethal dose the coalesced Radδl clusters showed a highly non-random localization towards the nuclear periphery (Figure 3c). Finally, the Radδl structures were excluded into micronuclei. The nucleoplasm was virtually cleared of Rad51 protein and only aggregated Radδl foci in MN were remaining (Figure 4;Table 6). Similar to the situation seen earlier in interphase nuclei, many MN displayed paired Rad51 foci and higher-order structures. The highest number of MN (approximately three per cell) as well as the highest number of Radδl -positive MN(approximately 30%) were observed 16 hrs after irradiation (Table 7). However, at each time point analyzed the majority of radiation-induced MN did not show detectable Rad51-protein foci.

Table 7. Rad51 Foci in Micronuclei of Different Cell Substrates

1000 cells were analyzed for each experiment

Segregation of Rad51-Protein Foci into Micronuclei An increased rate of MN is also observed in 5-azadeoxycytidine (δ-aza-dC)-treated cell cultures (Guttenbach, et al, (1994) Exp. Cell Res. 211, 127-132; Stopper et al, 1995, supra). This hypomethylating base analog induces inhibition of chromatin condensation, leading to instability of the affected chromosome regions (Haaf, 1996). Its cytotoxic effects are at least partially due to the induction of single- and double-strand breaks in DNA. Like ¹³⁷Cs irradiation, δ-aza-dC can induce the formation of Radδl -protein foci in nuclei and its elimination into MN. Rat TGR-1 and human LNL8 fibroblast cultures treated with non-lethal doses of δ-aza-dC displayed MN with focally concentrated Radδl protein in 6-10%. of their cells (Table 8). Table 8. Induction of Rad51 Foci by 5-Azadeoxycytidine

^a Type I nuclei and micronuclei show weak to medium HsRad51 immuno-fluorescence, whereas type II cells show strongly fluorescing foci. 500 cells were analyzed for each experiment. ^b 10^"5 M 5-aza-dC were added to the culture medium 24 h rs before cell harvest.

Rapidly dividing cell cultures always exhibit a baseline MN frequency even without exposure to clastogens or aneuploidogens. In five different substrates studied, LNL8, XPA, teratoma, 3T3-Swiss, and TGR-1 cells, 10-30% of these spontaneously occuring, non-induced MN exhibited Radδl-protein foci (Table 7). This further links Rad51-protein foci and MN formation. Rad52 and Other Repair Proteins Are Not Excluded into Micronuclei Studies in yeast (Shinohara et al, 1992, supra; Milne, G, and Weaver, D. (1993). Genes Dev. 7, 1756-1765) and humans (Shen, et al, (1996). J. Biol. Chem. 271, 148-152) have shown physical interaction between Rad51 and Radδ2 proteins both in vitro and in vivo. Double immunofluorescence with rabbit anti-Rad51 and rat anti-Radδ2 antibodies on ¹³⁷Cs irradiated TGR-1 cells showed that both proteins are enriched in nuclear foci but they do not co-localize. Rad52-protein foci remained in the nucleus throughout the entire time course, while Rad51 -protein foci were segregated into MN (data not shown). The same holds true for Gadd45 (data not shown) an inducible DNA-repair protein that is stimulated by p53 (Smith et al, 1994, supra). Biochemical evidence further suggests specific protein-protein association between HsRad51 and p53 (Sturzbecher et al, 1996, supra). However, after anti-p53 antibody staining the RadSl foci were not particularly enriched with p53 protein (data not shown). In addition, HsRadδl was reported to be associated with a RNA polymerase II (RNAPII) holoenyme (Maldonado et al, 1996, supra). Afthough RNAPII was irnmunolocalized in discrete discrete nuclear foci, as reported previously (Bregman et al, 1995, supra), transcription complexes did not coincide with Rad51 foci (data not shown).

Association of Rad51 Protein with DNA Fibers In a few (<1%) cells of irradiated and drug-treated cultures, we observed very elongated Rad51 structures, up to several hundred 5 micrometer io length, that were eliminated from the nuclei. Since these fiber-like structures stained DAPI-positively, they are thought to contain single DNA molecules of several megabases covered with Rad51 (data not shown). Fluorescence in situ end labeling (FISEL) demonstrated that these DNA fibers contain fragmented DNA typical of apoptosis (data not shown). Sometimes the DNA fibers appeared to leak out of the nucleus through 0 holes in the nuclear membrane and coodense into micronuclei. In all cell substrates studied, a high percentage of MN displayed genome fragmentation (data not shown).

The association of Rad51 protein with DNA was also visible on experimentally extended chromatin fibers from irradiated cells. SDS lysis and mechanical stretching of nuclear chromatin across the surface of a glass slide can cause complete deattachment of DNA 5 loops from the nuclear matrix, producing highly elongated, linear chromatin fibers (Haaf, T, and Ward, D.C. (1994). Hum. Mol. Genet. 3, 629-633.; Heiskanen et al, 1994, supra). Immunofluorescence staining revealed linear strings of Rad51 label on these stretched DNA fibers (data not shown). By comparison with YAC hybridizatⁱon signals on similar preparations (Haaf and Ward, 1994, supra), the length ofthe Radδl fibers was estimated 1-2 0 Mb.

Rad51 -Protein Foci and Apoptosis To determine whether Radδl -positive MN specifically detect exposure to clastogens, analyses were performed in rat TGR-I cells with the aneuploidogen colcemid. This mitotic spindle poison causes lagging of whole chromosomes that are excluded into MN. Surprisingly, when colcemid-treated cells were allowed to δ recover for 24 hrs in drug-free medium, over 30% of the induced MN contained very brightly fluorescing Rad51 foci (Table 9). Some MN contained rod-like linear structures (data not shown) similar to those observed in Radδl-overexpressing cells. Most of these Radδl-positive MN, 24 hrs after colcemid, did not contain fragmented DNA, as determined by simultaneous FISEL (Table 9). When cells were grown for one or two more days in the 0 absence of the drug, the percentage of Radδl -containing MN decreased dramatically. In addition, the Radδl protein was no longer concentrated in discrete foci but appeared to disperse throughout the entire MN volume. At the same time most MN became apoptotic and by FISEL their degraded DNA showed incorporation of fluorescent nucleotides. Thus, we conclude that mitotic arrest after colcemid triggers a cascade that induces the elimination δ of Radδl protein into MN and drives apoptotic events. Our results seem to be consistent with the hypothesis that apoptosis is a special form of aberrant mitosis (Ucker, D.S. (1991). New Biologist 3, 103-1009; Shietal, 1994, supra).

Table 9. Radδl Foci and Apoptosis in Colcemid-lnduced Micronuclei of TGR-1 Cells

Treatment Number of Percentage of Cells Showing³ micro- nuclei in Rad51-/ Rad51+/ Rad 51+/ Rad51-/

1000 cells FISEL- FISEL- FISEL+ FISEL+

None 93 75% 12% 2% 11%

Colcemid^b n.d. 85% 6% 0% 9%

1 day of recovery 1293 54% 31% 1% 14%

2 days of recovery 1061 45% 45% ^• 6% 40%>

3 days of recovery 769 43% 7% 4% 46%

^aApoptotic cells show fluorescence in situ end labeling (FISEL+), while cells without genome fragmentation show absence of labeling (FISEL-). "Rad51+" cells with Rad51 foci, "Rad51-+ cells without foci.

TGR-1 cells were grown for 24 hrs in medium contaiing 0.1 μg/ml colcemid to induce micronucleus formation (without inducing DNA damage). 186 of the colcemid-treated cells were arrested at metaphase, 17% showed multinuclei (>10 micronuclei), and 65% had no micronuclei. The cells were then allowed to recover for various times in the absence of the drug. 600 micronuclei were analyzed for each experiment.

Another more classical way for inducing apoptosis in vitro is the exposure of TGR-1 cells to the topoisomerase II inhibitor etoposide. After adding etoposide to the culture medium, the percentage of apoptotic cells steadily increased (Table 10). After 36 hrs half of the cells showed genome fragmentation and stained FISEL-positively. The nuclear events of apoptosis were accompanied by the appearance of Rad51 protein in nuclei and MN. These results indicate that different stimuli (e.g., irradiation and DNA-damaging drugs, topisomerase inhibitors, and aneuploidogens) that condem cells to apoptosis can induce focal concentration of Radδl protein and its exclusion into MN.

Table 10. Induction of Radδl Foci and Apoptosis by Etoposide Treatment of TGR-1 Cells

^aType I nuclei and micronuclei show weak to medium HsRadδl immunofluorescence, whereas type II cells show strongly fluorescing foci. 600 cells were analyzed for each experiment. "Detected by fluorescence in situ end labeling (FISEL+). ^cCells were grown in medium containing etoposide for the indicated times.

Higher-Order Nuclear Organization of Overexpressed Rad51 Protein Human 293 cells were transfected with the HsRadδl gene. The resulting cell lines 710 and 717 constitutively expressed a HsRadδl -fusion protein. This overexpressed protein formed brightly fluorescing linear structures inside the nucleus (Figure 7a). Some nuclei were completely filled with a network of rod-like structures (Figure 7b). Identical Rad51 structures were observed in transformed rat TGR 928.1-9 cells, stably expressing the HsRadδl protein without a tag epitope (data not shown). This suggests that Radδl protein is able to assemble into higher-order structures within the highly ordered interphase nucleus. The linear nature of RadS 1 structures in overexpressing cells is reminscent of the strings of Rad51 -protein foci after DNA damage and colcemid treatment and in meiotic cells (Haaf et al, 1995).

However, in contrast to the situation after DNA damage, the overexpressed HsRadδl protein is not eliminated into MN. The numbers of Radδl -positive MN were not radically different in Radδl-overexpressing human 717 cells versus in 293 control cells and in rat 928.1-9 overexpressers versus in parental TGR-1 cells. This means that Radδl overexpression alone does not cause apoptosis. In exponentionally growing unsynchronized cultures, 14% of both 717 and 293 cells (600 cells were analyzed for each experiment) and 8% of both 928.1-9 and TGR-1 cells showed cleavage of the cell's DNA by FISEL. We conclude that the segregation of Rad51 into MN is a specific behavior of apoptotic cells and precedes genome fragmentation.

Cell-Cycle Arrest of Cells with Focally Concentrated RadSl Protein Simultaneous Radδl-protein immunofluorescence and antibromodeoxyuridine (BrdU) antibody staining demonstrated that nuclei with focally concentrated Radδl protein do not undergo DNA-replication synthesis (data not shown). BrdU was incorporated into replicating DNA of unsynchronized cell cultures for 30 hrs. Rapidly growing transformed cell lines (293, LNL8, XPA, and XPF) which showed detectable Rad51 immunolabiing in a percentage of nuclei even without induction of DNA damage as well as Rad51 overproducers (928.1-9 and 717) were analyzed. For each experiment, 100 nuclei with prominent Rad51 foci and 100 nuclei without detectable Rad51 foci were stained with fluorescent anti-BrdU antibody. In the widely different substrates tested, 80%-100% of the cells with focally concentrated Rad51 protein were found to be BrdU-staining negative (Table 11). In contrast, 30%-90% of the cells without Rad51 foci from the same cultures showed BrdU incorporation, indicative of cycling cells. The BrdU-substituted DNA was located in discrete replication sites throughout the nucleus as reported previously (Nakayasu, H, and Berezney, R. (1989). J. Cell Biol. 108, 1-11 ; Fox, et al, (1991) J. Cell Sci. 99, 247-253). This suggests that even without induction of DNA damage the cells with Rad51 foci are arrested during the cell cycle or enter S phase delayed of the Radδ1-foci negative cells.

Table 11 Induction of Rad51 Foci after ³⁷Cs Irradiation of TGR-1 cells and Their Elimination into Micronuclei

None 93% 6% 0% 1% 0%

3 hrs after

900 rad 8% 0.4% 11 % 0.6%

¹³⁷Cs 80%

16 hrs after 9% 8% 1% 9%

900 rad

¹³⁷Cs 73%

1% 13% 1% 13%

30 hrs after

900 rad

¹³⁷Cs 72% 0% 4% 0% 6%

4 days after

900 rad

¹³⁷Cs 90% ^aType I nuclei and micronuclei show weak to medium HsRadδl immuno-fluorescence, whereas type II cells show strongly fluorescing foci. 1000 cells were anlayzed for each experiment.

Rat TGR-1 cells are capable of normal physiological withdrawal into the quiescent (Go) phase of the cell cycle as well as resumption of growth following the appropriate stimuli (Prouty, et al, (1993). Oncogene 8, 899-907). In TGR 928.1-9 cells o~erexpressing a HsRadδl transgene(s), Go arrest upon serum starvation dramatically induced HsRad:

1 -protein foci (Table 12). Synchronous re-entry into the cell cycle after feeding reduced the percentage of HsRadδl -foci positive cells to very low levels. However, new Go arrest upon contact inhibition following three population doublings increased the number of cells with nuclear Radδl foci again. We therefore conclude that cells with prominent nuclear Radδl foci are most likely in Go or G1 phase of the cell cycle.

Table 12. Radδl Foci in Micronuclei of Different Cell Substrates

1000 cells were analyzed for each experiment

(On ward)Example 4 Rad51 Biochemical Assays Compatible for High Throughput Screening

As discussed above, homologous pairing and DNA strand exchange are unique properties of recombination proteins like Rad51 and RecA protein. Several assays are available to detect the homologous pairing and strand exchange activity of Rad51. Strand exchange reactions catalyzed by human Rad51 are monitored with oligonucleotide substrates. These substrates are very convenient and easy to use because of machine synthesis and labeling of oligonucleotides either with fluorophores or with biotin. Radδl (or RecA) protein carries out strand exchange in three distinct phases: I) presynapsis, during which RecA protein binds cooperatively and stoichiometrically to single-stranded DNA and forms a right handed helical nucleoprotein filament; II) synapsis, in which duplex DNA is taken up into the nucleoprotein filament and homologously aligns; and III) DNA strand displacement, which produces a recombinant (heteroduplex) double-stranded DNA molecule and a displaced single-stranded DNA molecule (Figure 8).

Intermediates of the reaction known as joint molecules (also referred to as D-loops) and the final products can be monitored either by filter assays or by gel electrophoresis.

A given test inhibitor can inactivate Radδl by interfering with any one of these three steps. Hence, test Radδl inhibitors can be added at the stage of presynapsis, synapsis or strand displacement stage of DNA strand exchange. These methods can be used to determine whether the inhibitors are acting by a) interfering with the cooperative polymerization of Radδl on single-stranded DNA, b) affecting the pairing of the filament to the homologous DNA target or, c) affecting the process of strand exchange by inhibiting hydrolysis of ATP.

Filter binding DNA strand exchange assays (solid phase-based) of Rad51 activity which are compatible with high throughput screening. Filter binding assays are based on single-stranded DNA binding to nitrocellulose membranes under the appropriate salt conditions. The linear duplex DNA is labeled with bases linked to fluorophores, ³²P, or biotin and the single-stranded DNA substrates are unlabeled. After uptake of the double-stranded DNA into the nucleoprotein filament, DNA base pair switching displaces the complementary strand of the parental duplex DNA. As a result of DNA strand displacement, hybrid DNA intermediates of the DNA strand exchange reaction contain single-stranded DNA tails, and one of the products of strand exchange is single-stranded DNA; both of which can be trapped on nitrocellulose filters. The unreacted linear double-stranded DNA cannot bind to the membrane and is washed away in the filtrate. Since the initial single-stranded DNA used to make the nucleoprotein filament is not labeled, it is not detected.

The filter binding assays are easy to use and extremely reliable. Typical results of DNA strand exchange by RecA protein as monitored by the filter assays is shown in Figure 9. These assays can be performed in high throughput mode, for example, using a 96 well format on manifolds fitted with nitrocellulose membranes.

Fluorescence spectroscopy-based assays for monitoring the DNA strand exchange activity of human Rad51 are highly specific and compatible with high throughput screening. Assays based on fluorescence to measure DNA pairing and DNA strand exchange by human Rad51 protein have been developed. This approach enables one to distinguish homologous DNA pairing from subsequent DNA strand exchange. Homologous pairing of a single-stranded oligonucleotide with a duplex oligonucleotide is measured by fluorescence resonance energy transfer (FRET). Energy transfer between two fluorescent dyes indicates their proximity. In the case of DNA, the proximity of two complementary strands labeled with dyes can be determined by FRET. For example, when a δ'-Watson strand labeled with fluorescein comes into proximity with a complementary 3'-Crick strand labeled with rhodamine, the overlap between its emission spectrum and the excitation spectrum of rhodamine allows the nonradiative transfer of energy to rhodamine by fluorescence resonance energy transfer (Bazemore et al, J. Biological Chem, 272(23): 14672-14682 (1997) and Bazemore, et al, PNAS USA, 94:11863-11868 (1997)).

Homologous DNA pairing assay by FRET. A test oligonucleotide labeled at its 3' end with fluorescein is used to form the nucleoprotein filament with Rad51. Rhodamine is attached to the δ' end of the complementary strand in duplex DNA. Homologous pairing between the two DNA molecules juxtaposes the two fluorescent molecules, resulting in nonradiative energy transfer from fluorescein to rhodamine when fluorescein is excited at 493 nm, near its excitation peak. As a result of the energy transfer, the fluorescence emission from fluorescein is quenched and that from rhodamine is enhanced (Figure 10).

To measure homologous pairing of a test single-stranded oligonucleotide with its homologous duplex oligonucleotide, an 83-mer oligonucleotide (minus strand) labeled at its 3' end with fluroescein is preincubated with 1.2 μM Radδl protein in a reaction mixture containing 1 mM MgCI₂, 26 mM HEPES (pH 7.4), 1 mM DTT, 2 mM ATP and 100 μg of BSA per ml for 4 minutes at 37°C. The concentration of MgCI₂ is increased to 30 mM, and finally 3 μM duplex DNA (labeled with rhodamine at the 5' end of the plus strand) is added.

DNA strand exchange assay by FRET. When the fluorescein and rhodamine are juxtaposed by 20 A on opposite complementary strands, the emission from the fluorescein is quenched and that from rhodamine is enhanced as a result of energy transfer. To measure the strand exchange activity, both fluorophores are present in the duplex where they are juxtaposed. When strand exchange is completed, the two labeled strands are separated from each other as monitored by the enhanced emission from fluorescein (Figure 11).

To measure DNA strand exchange by FRET, Rad51 protein is added to unlabeled single- strand oligonucleotide for 4 min. at 37°C followed by the addition of the filament to a reaction mixture containing 30 mM MgCI₂ and 3 μM duplex oligonucleotide (labeled on the 3' end of the minus strand with fluorescein and on the 5' end of the plus strand with rhodamine). The final concentrations of ssDNA and protein are 3 μM and 1.2 μM, respectively.

Fluorescence emission spectra are recorded from 502 to 620 nm upon excitation at 493 nm on an SLM 8000C (SLM Aminco, Urbana, IL) or similar spectrofluorimeter. Example 5 Determination of Lead Compound Specificity

As discussed above, modulators for Radδl biological activity can be assayed in a number of ways. The following assays can be used to assay for a change in biological activity to δ initially identify inhibitors, or to determine the specificity of identified inhibitors: D-loop assay, DNA dependent ATPase assay, nucleoprotein filament assay, and complementary single- strand hybridization assay. These assays are unique features of the Rad51 protein and determine the specificity of, for example, small molecules that inhibit Radδl protein activity.

D-loop assay. The non-enzymatic uptake of a homologous single-stranded DNA by a 0 negatively supercoiled DNA leads to the formation of a DNA displacement loop (D-loop, Figure 12) (Holloman et al, PNAS, USA, 76:1638-1642 (1975)). However, nonenzymatic formation of D-loops is seen only at elevated temperatures (65°C).

RecA and Rad51 enzymes both catalyze D-loop formation under physiological conditions. Negative superhelicity is not required in these reactions catalyzed by RecA or Rad51. Only 5 members of RecA and Rad51 protein families can catalyze the formation of D-loops under physiological conditions.

Several standard assays monitor D-loop formation by DNA recombination enzymes. Most commonly used assays use duplex DNA (either supercoiled DNA or a linear duplex DNA) and a linear single-stranded DNA (either an oligonucleotide or a relatively longer linear 0 single-stranded DNA) as substrates. The D-loop products are analyzed either by filter assays or by gel electrophoresis. Filter assays are simple, fast and samples can be analyzed in a plate format. In these experiments, the target duplex DNA is labeled and the single-stranded DNA substrate is unlabeled. After uptake of the single-stranded DNA into duplex DNA, the tails of the unincorporated single-stranded DNA of the hybrid molecules are δ trapped on the filter and only the D-loops can be detected. Unreacted double-stranded DNA (superhelical DNA or linear duplex DNA) do not bind to the membrane. If necessary, the D- loop products can also be monitored by following the separation of hybrids by gel electrophoresis.

DNA dependent ATPase assay. The ATP binding domain is highly conserved throughout 0 evolution in the homologues of RecA protein (Heyer, Experentia, 50:223-233 (1994)). The unique feature of the ATPase activity of RecA and Rad51 is that this activity is DNA dependent. Radδl hydrolyzes ATP only in the presence of single-stranded DNA and has no ATPase activity in the absence of DNA. To monitor ATP hydrolysis, labeled ATP is incubated with single-stranded DNA and Radδl. The reaction mixture is incubated at 37°C for 30 min. and an aliquot of the reaction is applied directly onto CEL 300 PEI/UV_2S4 thin layer chromatography plates to separate the product of hydrolysis (ADP) from the substrates. Non-DNA dependent ATPases are used as controls δ for these reactions. Small molecule compounds that inhibit the ATPase activity of Radδl would not be expected to affect the activity of other ATPase enzymes.

Assay of nucleoprotein filament formation. Radδl protein binds cooperatively to DNA to form a right-handed helix. The resulting protein-DNA complex is an active nucleoprotein filament which catalyzes DNA pairing and DNA strand exchange reactions. The DNA helix 0 inside the filament is extended 1.6 times the size of B-form duplex length. This structure of the nucleoprotein filament is a hallmark feature of RecA and Radδl proteins and is DNA sequence independent. The DNA inside the filament is completely protected from phosphodiesterases, as RecA and Radδl proteins bind to and protect the phosphate backbone from cleavage. Formation of nucleoprotein filaments is easily monitored by 6 protein-based filter binding assays. Another DNA binding protein is used as a control.

Complementary single-strand DNA renaturation assay. Radδl protein promotes the hybridization of complementary strands of DNA under specific conditions in which the spontaneous renaturation of complementary strands does not occur. Hybridization activity is easy to monitor in a DNA micro array format or by filter binding assays. Other single-strand 0 annealing proteins, such as SSB, will be used as controls.

Example 6 Down-regulation of Human Rad51 Protein by Antisense Oligodeoxynucleotides in Human

Breast, Brain and Prostate Cells

An essentially complete reduction in the expression of Radδl protein by using specific δ human Radδl antisense oligodeoxynucleotides in a variety of human tumor cell lines has been achieved herein. The human Radδl mRNA sequence is shown in Figure 15A and 15B wherein the regions complementary to the antisense molecules SEQ ID NOS: 1-9 are underlined. Figure 16 shows the antisense oligonucleotides.

Specific antisense oligonucleotides targeted against the 5' untranslated region (AS4 (SEQ ID 0 NO:2), AS5 (SEQ ID NO:3)), the 3' untranslated region (AS6, 7, 8, and 9 (SEQ ID NO:4, 5, 9 and 6, respectively) and the coding region (AS3 (SEQ ID NO:1) of Radδl mRNA were used herein. Also independently tested herein were AS1 (SEQ ID NO:7) and AS2 (SEQ ID NO:8) as indicated in Ohnishi, et al. Biochemical and Biophysical Res. Comm, 246:319-324 (1998), incorporated by reference herein in its entirety. AS1 (SEQ ID NO:7), AS2 (SEQ ID NO:8) and AS3 (SEQ ID NO:1) target the coding sequence of both mouse and human Radδl mRNA.

These antisense oligonucleotides were tested against human cell lines derived from breast, brain and prostate tumors. Combinations of two antisense oligonucleotides reduced the levels of Radδl protein 30% to 96% compared to sense, scrambled and untreated controls. Figure 13 shows the levels of Radδl protein after treatment of MDA-MB-231 cells with some of the most effective antisense oligonucleotides used. Quantitation is shown in Figure 14. Combination of AS3 and AS6 induces a near complete reduction of Radδl.

Similar results were obtained in other human breast (MCF-7), brain (U87 and U261), and prostate tumor cell lines (LnCAP and DU146) tumor cell lines.

The degree of reduction of Rad51 protein with antisense oligonucleotides in MDA-MB-231 breast cancer cells are as follows: AS3 (SEQ ID NO:1), 70%; AS4 (SEQ ID NO:2), 70%;

ASδ (SEQ ID NO:3), 40%>; AS6 (SEQ ID NO:4), 80%; AS7 (SEQ ID NO:5), 90%; AS9 (SEQ ID NO:6), 50%. AS8 (SEQ ID NO:9) was lethal, Rad51 protein levels were undetectable.

SEQ ID NOS:7, 8, 10, 11 and 12 had no determinable reduction. Similar results ocurred with

MCF-7 breast cancer cells and U87 glioblastoma cells.

Further experiments will include the following cell lines, WT pδ3 and p3, BT20, BT649, MCF7, MDA-MB-231 , MDA-MB-468, and HMEC-4144-2.

Example 7

Cytotoxicity and Cell Growth Assays and Sensitization of Human Tumor Cells to Radiation and DNA-damaging Chemicals

A sulforhodamine B-based optical density assay of protein in cultured cells will be used (Skenen, et al, J. Natl. Cancer Inst, 82:1107-12 (1990); Skehan, et al. Cell Biol. Toxicol. 2:357-368 (1986) as a cell-based high throughput drug screen for inhibitors of Rad51 activity. The phenotypes screened for are cytotoxicity and growth inhibition in target tumor (breast, brain and prostate) and control (non tumorigenic) cell lines. Cells are placed in 96- well microtiter plates. Next, these drugs are introduced after one day of culture, and treat for an additional 96 hours. Assays begin at the beginning of drug treatment, at 48 hours and at 96 hours. Qualitative changes are monitored by comparing the amount of cellular protein present at the beginning of the drug incubation period with the amount of protein present in control and test cultures at day 3 and day 5 of growth. Other time points will be added if necessary. Quantitative drug-induced changes in culture growth will be evaluated using the doubling time and fractional growth rate (Skehan, Assays of cell growth and cytotoxicity, Cell Growth and apoptosis: A practical approach, G. Studzinski, ed, 2nd Ed, pp 169-191).

Also determined is Rad51 activity in these samples using methods outlined above to determine whether the biological effects measured are specific to alterations in Radδl activity. This can be done by comparing the results to antisense oligodeoxynucleotides that specifically down-regulate Radδl protein.

Sensitization of human tumor cells to radiation and DNA-damaging chemicals.

Following DNA damage, cells may survive by undergoing transient cell arrest and regrowth, or may remain in permanent cell cycle arrest; or the cells may simply die. The extent to which Radδl down-regulation sensitizes cells to DNA damage can be determined by assaying how Radδl down-regulation shifts the dose response curves for DNA damaging agents (radiation, BCNU, cyclophosphamide, cis-platin) in systems that measure growth, survival and death. The effects on cell growth are screened using the sulforhodamine B assay described above. Assayed is the effect on cell survival using a clonigenic assay to determine the surviving fraction of clonigenic cells. Apoptosis is assayed using either a flow cytometric assay for subdiploid fractions or by using the TUNEL method, which utilizes terminal deoxynucleotide transferase to incorporate fluorescein-conjugated deoxyuridine triphosphate into DNA nicks formed in apoptotic cells. As noted above, parallel experiments with antisense oligodeoxynucleotides will assure the specificity.

Example 8 Human Tumor Xenografts Implanted in Nude Mice

Human tumors in a human host and human tumors transplanted into athymic mice respond similarly to antineoplastic drugs, and therefore xenografts grown in athymic mice provide an invaluable preclinical model to validate candidate agents identified in cell-based assays.

There are two aims to these experiments. One is to determine whether Radδl down- regulation has a growth inhibitory effect. The second evaluates Radδl down-regulation as a sensitizer to DNA damage. The experimental blueprint for these studies in human tumor xenografts is: a) define growth curves for the untreated xenograft and for xenografts treated with inhibitors of Radδl . This aim will also determine how growth is related to Radδl down- regulation; b) choose appropriate doses of DNA damaging agents that will allow us to compare the responses of the control and Radδl down-regulated cell lines; c) quantitate the amount of sensitization to DNA damage. Implantation and growth of human tumor xenografts in nude mice. The target tumor cell lines chosen herein can all form tumors in nude mice. 1 x 10⁵, 1 x 10⁶, and 1 x 10⁷ cells taken from cell culture in the log phase of growth will be injected into the flanks of nude mice, and the tumor will be measured daily for 50 days, until the tumor reaches 2000mm³, or until the animal becomes debilitated. Each group will have δ mice. Generally tumors are treated when they reach a volume of 60-100mm³.

Determination of lethal dose. This is a simple determination of lethality of the lead compound found by escalating single doses in mice which are indexed to intracellular concentrations that down-regulate Rad51 in culture. Toxicity will be investigated by physical exam and organ histopathology.

Treatment of tumors with lead compounds. Once tumors reach a treatable size, animals will be injected with escalating doses of the lead compound. Tested will be both systemic and local routes of administration. The tumors will be examined for Radδl activity and for volume. Toxicity of the compound in these animals will be assayed by weight gain, serum chemistries and organ histopathology (including liver, lung, kidneys, heart, gastrointestinal tract and brain).

Dose finding for DNA damage. The growth characteristics of the cell lines are established and then various doses of DNA damaging agents (for example, 0, 5, 10 and 16 Gy for radiation) will be used to define a series of growth curves that describe response. This information will allow selection of doses to compare control cell lines to the Rad51 down- regulated cell lines. Each group will have at least 5 animals and appropriately matched controls.

Sensitization of radiation and chemotherapy treatments by lead compounds. Control and Rad51 down-regulated cell lines will be grown and treated with DNA damaging agents at times and doses determined above. If the growth rates of control and down-regulated lines are similar, direct comparisons can be made between growth delays caused by radiation in sets of modified and unmodified cell lines. However, if their growth rates differ, a dose will be chosen that will allow us to measure the average size of tumors at a specific point in time. If the difference between the average sizes of control (i.e., not down-regulated) tumors treated and not treated with DNA damaging agents is ΔU and the difference between the average sizes of down-regulated tumors treated and not treated with DNA damaging agents is ΔM, our hypothesis would predict that ΔU - ΔM > 0. Studies include 4 groups of 15 animals for each lead compound. Assuming a normal distribution, this would provide 90%> power to detect a difference ΔU - ΔM of 1.5 times the standard deviation, assuming a one- tailed hypothesis test is α = 0.05.

Other references specifically incorporated by reference are Haaf, T. (1996) Pharmac. Ther. 65, 19-46; Haaf, T, and Schmid.M. (1991) Exp. Cell Res. 792, 325-332; and Owaga, et al, (1993) Science 259, 1896-1899.

Claims

CLAIMS We claim:

1. A method for inhibiting cell proliferation comprising administering to a cell a Rad51 antibody or antibody fragment.

2. A method for screening for a bioactive agent which modulates the strand exchange activity of Rad51, said method comprising: a) providing a double stranded nucleic acid comprising: i) a first nucleic acid single strand comprising a first fluor; and ii) a second nucleic acid single strand comprising a second fluor, such that when said first and second nucleic acids are hybridized, quenching of one of said fluors occurs; b) providing a Rad51 nucleofilament comprising: i) Rad51; and ii) a third single stranded nucleic acid substantially complementary to one of said first or second strands; c) contacting said double stranded nucleic acid and said nucleofilament in the presence of a candidate agent to form a mixture; and d) assaying said mixture for strand exchange activity.

3. A method according to claim 2 wherein said Rad51 is a Rad51 homolog.

4. A method according to claim 2 wherein a library of candidate agents are used to form a plurality of mixtures.

5. A method according to claim 2 wherein said nucleofilament further comprises a protein selected from the group consisting of Radδ2, Radδ4 and RPA.