Use of RNAI Inhibiting PARP Activity for the Manufacture of a Medicament for the Treatment of Cancer

Abstract

The present invention relates to the use of an agent that inhibits the activity of an enzyme that mediates repair of a DNA strand break in the manufacture of a medicament for the treatment of diseases caused by a defect in a gene that mediates homologous recombination.

Description

CROSS REFERENCE

[0001] This application claims benefit and is a Continuation of application Ser. No. 14/322,759 filed Jul. 2, 2014, which is a Continuation of application Ser. No. 10/555,507 filed Sep. 15, 2006, now U.S. Pat. No. 8,859,562, issued Oct. 14, 2014, which is a 371 application and claims the benefit of PCT Application No. PCT/GB2004/003235, filed Jul. 23, 2004. This application also claims benefit to GB0317466.1 filed on Jul. 25, 2003. These applications are incorporated herein by reference in their entirety.

[0002] This invention relates to the use of an agent that inhibits the activity of an enzyme which mediates the repair of DNA strand breaks in the treatment of certain forms of cancer in particular breast cancer.

[0003] Homologous recombination (HR) has been shown to play an important role in repair of damage occurring at DNA replication forks in mammalian cells (2). Thus, cells deficient in HR show retarded growth and exhibit higher level of genetic instability. It is believed that genetic instability due to loss of HR repair in human cancers significantly contributes to the development of cancer in these cells (1).

[0004] Post transcriptional modification of nuclear proteins by poly(ADP-ribosyl)ation (PARP) in response to DNA strand breaks plays an important role in DNA repair, regulation of apoptosis, and maintenance of genomic stability.

[0005] Poly(ADP-ribose) Polymerase (PARP-1) is an abundant nuclear protein in mammalian cells that catalyses the formation of poly(ADP-ribose) (PAR) polymers using NAD.sup.+ as substrate. Upon DNA damage, PARP-1 binds rapidly to a DNA strand break (single strand or double strand) and catalyses the addition of negatively charged PAR chains to itself (automodification) and other proteins (see [3, 4] for reviews). The binding of PARP-1 to DNA strand breaks is believed to protect DNA lesions from further processing until PARP-1 is dissociated from the break by the accumulated negative charge resulting from PAR polymers (5,6).

[0006] Although PARP-1 has been implicated in several nuclear processes, such as modulation of chromatin structure, DNA replication, DNA repair and transcription, PARP-1 knockout mice develop normally (7). Cells isolated from these mice exhibit a hyper recombination phenotype and genetic instability in the form of increased levels of SCE, micronuclei and tetraploidy (8-10). Genetic instability may also occur in these PARP-1 knockout mice through telomere shortening, increased frequency of chromosome fusion and aneuploidy (11), although all of these results could not be repeated in another set of PARP-1 knock-out mice (12). In the former mice knockout, PARP-1 null mutation rescue impaired V(D)J recombination in SCID mice (13). These results support the view suggested by Lindahl and coworkers that PARP-1 has a protective role against recombination (5). They proposed that binding of PARP-1 to DNA strand breaks prevents the recombination machinery from recognizing and processing DNA lesions or, alternatively, that the negative charges accumulated following poly ADP-ribosylation repel adjacent recombinogenic DNA sequences. Only the latter model is consistent with inhibition of PARP-1 itself and expression of a dominant negative mutant PARP-1, inducing SCE, gene amplification and homologous recombination (HR [14-18]).

[0007] Studies based on treating cells with PARP inhibitors or cells derived from PARP-1 or PARP-2 knockout mice indicate that the suppression of PARP-1 activity increases cell susceptibility to DNA damaging agents and inhibits strand break rejoining (3, 4, 8-11, 19, 20, 47).

[0008] Inhibitors of PARP-1 activity have been used in combination with traditional anti-cancer agents such as radio therapy and chemotherapy (21). The inhibitors were used in combination with methylating agents, topoisomerase poisons and ionising radiations and were found to enhance the effectiveness of these forms of treatment. Such treatments, however, are known to cause damage and death to non cancerous or "healthy" cells and are associated with unpleasant side effects.

[0009] There is therefore a need for a treatment for cancer that is both effective and selective in the killing of cancer cells and which does not need to be administered in combination with radio or chemotherapy treatments.

[0010] The present inventors have surprisingly found that cells deficient in homologous recombination (HR) are hypersensitive to PARP inhibitors as compared to wild type cells. This is surprising since PARP-1 knockout mice live normally thereby indicating that PARP-1 is not essential for life. Thus, it could not be expected that cells would be sensitive to PARP inhibition.

[0011] According to a first aspect of the invention there is provided the use of an agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks in the manufacture of a medicament for the treatment of diseases that are caused by a genetic defect in a gene that mediates homologous recombination.

[0012] In a further aspect the invention provides a method of treatment of a disease or condition in a mammal, including human, which is caused by a genetic defect in a gene which mediates homologous recombination, which method comprises administering to the mammal a therapeutically effective amount of an agent which inhibits the activity of an enzyme which mediates repair of DNA strand breaks or other lesions present at replication forks.

[0013] In a preferred aspect said enzyme is PARP. In a further preferred aspect said agent is a PARP inhibitor or an RNAi molecule specific to PARP gene.

In a further preferred aspect, the use is in the treatment of cancer.

[0014] Preferably the medicament is a pharmaceutical composition consisting of the PARP inhibitor in combination with a pharmaceutically acceptable carrier or diluent.

[0015] The specific sensitivity of HR defective tumours to PARP-1 inhibition means that normally dividing cells in the patient will be unaffected by the treatment. Treatment of HR defective cancer cells using a PARP inhibitor also has the advantage that it does not need to be administered as a combination therapy along with conventional radio or chemotherapy treatments thereby avoiding the side effects associated with these conventional forms of treatment.

[0016] A genetic defect in a gene which mediates homologous recombination may be due to a mutation in, the absence of, or defective expression of, a gene encoding a protein involved in HR.

[0017] In a further aspect, the invention further provides the use of a PARP inhibitor in the manufacture of a medicament for inducing apoptosis in HR defective cells.

[0018] In another aspect the invention provides a method of inducing apoptosis in HR defective cells in a mammal which method comprises administering to the mammal a therapeutically effective amount of a PARP inhibitor.

[0019] By causing apoptosis in HR defective cells it should be possible to reduce or halt the growth of a tumour in the mammal.

[0020] Preferably, the HR defective cells are cancer cells.

[0021] Cancer cells defective in HR may partially or totally deficient in HR. Preferably the cancer cells are totally deficient in HR.

[0022] The term "cancer" or "tumour" includes lung, colon, pancreatic, gastric, ovarian, cervical, breast or prostate cancer. The cancer may also include skin, renal, liver, bladder or cerebral cancer. In a preferred aspect, the cancer is in a mammal, preferably human.

[0023] The cancer to be treated may be an inherited form of cancer wherein the patient to be treated has a familial predisposition to the cancer. Preferably, the cancer to be treated is gene-linked hereditary cancer. In a preferred embodiment of the invention the cancer is gene-linked hereditary breast cancer.

[0024] In a preferred aspect, the PARP inhibitor is useful in the treatment of cancer cells defective in the expression of a gene involved in HR. Genes with suggested function in HR include XRCC1, ADPRT (PARP-1), ADPRTL2 (PARP-2), CTPS, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, BRCA1, BRCA2, RAD52, RAD54, RAD50, MRE11, NBS1, WRN, BLM, Ku70, Ku80, ATM, ATR, chk1, chk2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, RAD9, FEN-1, Mus81. Eme1, DDS1, BARD (see (2, 3, 5, 22-28) for reviews).

[0025] A gene involved in HR may be a tumour suppressor gene. The invention thus provides for the treatment of cancer cells defective in the expression of a tumour suppressor gene. Preferably, the tumour suppressor gene is BRCA1 or BRCA2.

[0026] Breast cancer is the most common cancer disease among women in the Western world today. Certain families have strong predisposition for breast cancer, which is often owing to an inherited mutation in one allele of either BRCA1 or BRCA2. However, these patients still maintain one functional allele. Thus, these patients develop normally and have no phenotypic consequence from this mutation. However, in one cell, the functional allele might be lost, making this cell cancerous and at the same time deficient in homologous recombination (HR). This step is critical for the onset of a tumour (1).

[0027] The present inventors have surprisingly found that BRCA2 deficient cells are 100 times more sensitive to the cytotoxicity of the PARP inhibitor, NU1025, than wild type cells.

[0028] Thus in a preferred aspect, the invention provides the use of a PARP inhibitor in the manufacture of a medicament for the treatment of cancer cells defective in HR, e.g due to the loss of BRCA1 and/or BRCA2 expression.

[0029] The cancer cells to be treated may be partially or totally deficient in BRCA1 or BRCA2 expression. BRCA1 and BRCA2 mutations can be identified using multiplex PCR techniques, array techniques (29, 30) or using other screens known to the skilled person.

[0030] PARP inhibitors useful in the present invention may be selected from inhibitors of PARP-1, PARP-2, PARP-3, PARP-4, tankyrase 1 or tankyrase 2 (see 31 for a review). In a preferred embodiment, the PARP inhibitor useful in the present invention is an inhibitor of PARP-1 activity.

[0031] PARP inhibitors useful in the present invention include benzimidazole-carboxamides, quinazolin-4-[3H]-ones and isoquinoline derivatives (e.g. 2-(4-hydroxyphenyl)benzimidazole-4-carboxamide (NU1085), 8-hydroxy-2-methylquinazolin-4-[3H]one (NU1025); 6(5H)phenanthridinone; 3 aminobenzamide; benzimidazole-4-carboxamides (BZ1-6) and tricyclic lactam indoles (TI1-5) [32]. Further inhibitors of PARP may be identified either by design [33] or the novel FlashPlate assay [34].

[0032] The PARP inhibitor formulated as a pharmaceutical composition may be administered in any effective, convenient manner effective for targeting cancer cells including, for instance, administration by oral, intravenous, intramuscular, intradermal, intranasal, topical routes among others. Carriers or diluents useful in the pharmaceutical composition may include, but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.

[0033] In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion. The inhibitor may be administered directly to a tumour or may be targeted to the tumour via systemic administration.

[0034] A therapeutically effective amount of the inhibitor is typically one which is sufficient to achieve the desired effect and may vary according to the nature and severity of the disease condition, and the potency of the inhibitor. It will be appreciated that different concentrations may be employed for prophylaxis than for treatment of an active disease.

[0035] For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be up to 100 mg/kg, for example from 0.01 mg/kg to 50 mg/kg body weight, typically up to 0.1, 0.5, 1.0, 2.0 5.0, 10, 15, 20 or 30 mg/kg body weight. Ultimately, however, the amount of inhibitor administered and the frequency of administration will be at the discretion of a physician.

[0036] A therapeutic advantage of using PARP inhibitors to treat cancer cells is that only very low doses are needed to have a therapeutic effect in treating cancer thereby reducing systemic build up of the inhibitors and any associated toxic effects.

[0037] A preferred aspect of the invention provides an agent which is an inhibitory RNA (RNAi) molecule.

[0038] A technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as inhibitory RNA (RNAi), into a cell which results in the destruction of mRNA complementary to the sequence included in the RNAi molecule. The RNAi molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The RNAi molecule is typically derived from exonic or coding sequence of the gene which is to be ablated.

[0039] Preferably said RNAi molecule is derived from the nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

[0040] a) a nucleic acid sequence as represented by the sequence in FIG. 9, 10, 11, 12, 13 or 14 or fragment thereof;

[0041] b) a nucleic acid sequence which hybridises to the nucleic acid sequences of FIG. 9, 10, 11, 12, 13 or 14 and encodes a gene for PARP;

[0042] c) a nucleic acid sequence which comprise sequences which are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) and (b).

[0043] Recent studies suggest that RNAi molecules ranging from 100-1000 bp derived from coding sequence are effective inhibitors of gene expression. Surprisingly, only a few molecules of RNAi are required to block gene expression which implies the mechanism is catalytic. The site of action appears to be nuclear as little if any RNAi is detectable in the cytoplasm of cells indicating that RNAi exerts its effect during mRNA synthesis or processing.

[0044] More preferably said RNAi molecule according has a length of between 10 nucleotide bases (nb)-1000 nb. Even more preferably said RNAi molecule has a length of 10 nb; 20 nb; 30 nb; 40 nb; 50 nb; 60 nb; 70 nb; 80 nb; 90 nb; or 100 bp. Even more preferably still said RNAi molecule is 21 nb in length.

[0045] Even more preferably still the RNAi molecule comprises the nucleic acid sequence aaa age cau ggu gga gua uga (PARP-1)

[0046] Even more preferably still the RNAi molecule consists of the nucleic acid sequence aag acc aau cuc ucc agu uca ac (PARP-2)

[0047] Even more preferably still the RNAi molecule consists of the nucleic acid sequence aag acc aac auc gag aac aac (PARP-3)

[0048] The RNAi molecule may comprise modified nucleotide bases.

[0049] Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis.

[0050] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0051] FIG. 1A-1C is a graph demonstrating that HR deficient cells are hypersensitive to the toxic effect caused by inhibition of PARP-1. Colony outgrowth of the Chinese hamster cell lines AA8 (wild-type), irs1SF (deficient in HR[4]), CXR3 (irs1SF complemented with XRCC3 [2]), V79 (wild-type), irs1 (deficient in HR[5]) or irs1X2.2 (irs1 complimented with XRCC2 [1]) upon exposure to 3-AB (FIG. 1A), ISQ (FIG. 1B) or NU1025 (FIG. 1C). The means (symbols) and standard deviation (bars) of at least three experiments are shown. Colony outgrowth assay was used;

[0052] FIG. 2 is a graph showing cell survival in the presence of PARP inhibitor NU1025 in wt V79 cells, BRCA2 deficient VC-8 cells and VC-8 cells complimented with functional BRCA2 gene (VC-8#13, VC-8+B2). Colony outgrowth assay was used;

[0053] FIG. 3 is a histogram showing the percentage of the cells in apoptosis following a 72 hour incubation with NU1025;

[0054] FIG. 4A-4C. (FIG. 4A) Western blot analysis of protein lysates isolated from MCF-7 (p53.sup.wt) or MDA-MB-231 (p53.sup.mut) breast cancer cells following 48 hours transfection with siRNA. (FIG. 4B) Colony outgrowth of siRNA-treated MCF-7 cells or (FIG. 4C) MDA-MB-231 cells following exposure to the PARP inhibitor NU1025. The means (symbols) and standard deviation (bars) of at least three experiments are shown.

[0055] FIG. 5A-5E. BRCA2 deficient cells fail to repair a recombination lesion formed at replication forks by inhibitors of PARP. (FIG. 5A) Visualization of double strand breaks (DSBs) in BRCA2 proficient or deficient cells following a 24-hour treatment with NU1025 (0.1 mM) by pulse-field gel electrophoresis. Hydroxyurea 2 mM was used as a positive control. (FIG. 5B) Visualisation of .gamma.H2Ax foci in untreated V-C8+B2 and V-C8 cells. Number of cells containing .gamma.H2Ax foci (FIG. 5C) or RAD51 foci (FIG. 5D) visualised in V-C8+B2 and V-C8 cells following a 24-hour treatment with NU1025 (10 The means (symbols) and standard errors (bars) of three to nine experiments are shown. (FIG. 5E) A suggested model for cell death induced in BRCA2 deficient cells.

[0056] FIG. 6A-6C. PARP-1 and not PARP-2 is important in preventing formation of a recombinogenic lesion, causing death in absence of BRCA2. (FIG. 6A) RT-PCR on RNA isolated from SW480SN.3 cells treated with BRCA2, PARP-1 and PARP-2 siRNA in combinations as shown for 48 hours. (FIG. 6B) Clonogenic survival following 48-hours depletion of BRCA2, PARP-1 and PARP-2. The means (symbols) and standard deviation (bars) of at least three experiments are shown. Two and three stars designate statistical significance in t-test p<0.01 and p<0.001, respectively. (FIG. 6C) Western blot for PARP-1 in SW480SN.3 cells treated with different siRNA.

[0057] FIG. 7A-7F. (FIG. 7A) Visualisation of PAR polymers in untreated and (FIG. 7B) thymidine treated V79 cells (5 mM for 24 hours). (FIG. 7C) Percentage cells containing >10 sites of PARP activity following treatment with hydroxyurea (0.2 mM) and thymidine (5 mM). At least 300 nuclei were counted for each treatment and experiment. (FIG. 7D) Survival of V-C8+B2 cells following co-treatment with hydroxyurea or (FIG. 7E) thymidine and NU1025 (10 .mu.M). (FIG. 7F) The activity of PARP was measured by the level of free NAD(P)H.sup.11, following treatment with MMS, hydroxyurea (0.5 mM) or thymidine (10 mM). The means (symbol) and standard deviation (error bars) from at least three experiments are depicted.

[0058] FIG. 8A-8F. (FIG. 8A) Visualisation of PAR polymers in untreated V-C8 and (FIG. 8B) V-C8+B2 cells. (FIG. 8C) Quantification of percentage cells containing >10 sites of PARP activity in untreated V-C8 and V-C8+B2 cells. (FIG. 8D) Level of NAD(P)H measured in untreated V-C8 and V-C8+B2 cells. Three stars designate p<0.001 in t-test. (FIG. 8E) Visualization of RAD51 and sites of PARP activity in V79 cells following a 24-hour thymidine treatment (5 mM). (FIG. 8F) A model for the role of PARP and HR at stalled replication forks.

[0059] FIG. 9 is the human cDNA sequence of PARP-1;

[0060] FIG. 10 is the human cDNA sequence of PARP-2;

[0061] FIG. 11 is the human cDNA sequence of PARP-3;

[0062] FIG. 12 is the human gDNA sequence of Tankyrase 1;

[0063] FIG. 13 is the human mRNA sequence of Tankyrase 2;

[0064] FIG. 14 is the human mRNA sequence of VPARP.

Materials and Methods

[0065] Cytotoxicity of PARP Inhibitors to HR-Defective Cells: XRCC2, XRCC3 or BRCA2

[0066] Cell Culture

[0067] The irs1, irs1X2.1 and V79-4 cell lines were a donation from John Thacker [40] and the AA8, irs1SF and CXR3 cell lines were provided by Larry Thompson [41].

[0068] The VC-8, VC-8+B2, VC-8#13 were a gift from Malgorzata Zdzienicka [42]. All cell lines in this study were grown in Dulbecco's modified Eagle's Medium (DMEM) with 10% Foetal bovine serum and penicillin (100 U/ml) and streptomycin sulphate (100 .mu.g/mL) at 37.degree. C. under an atmosphere containing 5% CO.sub.2.

[0069] Toxicity Assay--Colony Outgrowth Assay

[0070] 500 cells suspended in medium were plated onto a Petri dish 4 hours prior to the addition of 3-AB, ISQ or NU1025. ISQ and NU1025 were dissolved in DMSO to a final concentration of 0.2% in treatment medium. 7-12 days later, when colonies could be observed, these colonies were fixed and stained with methylene blue in methanol (4 g/l). Colonies consisting of more than 50 cells were subsequently counted.

[0071] Apoptosis Experiments

[0072] 0.25.times.10.sup.6 cells were plated onto Petri dishes and grown for 4 hours before treatment with NU1025. After 72 hours, cells were trypsinized and resuspended with medium containing any floating cells from that sample. The cells were pelleted by centrifugation and resuspended for apoptosis analysis with FITC-conjugated annexin-V and propidium iodine (PI) (ApoTarget, Biosource International) according to manufacturer's protocol. Samples were analysed by flow cytometry (Becton-Dickenson FACSort, 488 nm laser), and percentage of apoptotic cells was determined by the fraction of live cells (PI-negative) bound with FITC-conjugated annexin-V.

[0073] Immunofluorescence

[0074] Cells were plated onto coverslips 4 h prior to 24-h treatments as indicated. Following treatments the medium was removed and coverslips rinsed once in PBS at 37.degree. C. and fixed as described elsewhere [2]. The primary antibodies and dilutions used in this study were; rabbit polyclonal anti PAR (Trevigen; 1:500), goat polyclonal anti Rad51 (C-20, Santa Cruz; 1:200) and rabbit polyclonal anti Rad51 (H-92, Santa Cruz; 1:1000). The secondary antibodies were Cy-3-conjugated goat anti-rabbit IgG antibody (Zymed; 1:500), Alexa 555 goat anti-rabbit F(ab').sub.2 IgG antibody (Molecular Probes; 1:500), Alexa 546 donkey anti-goat IgG antibody (Molecular Probes; 1:500) and Alexa 488 donkey anti-rabbit IgG antibody (Molecular Probes; 1:500). Antibodies were diluted in PBS containing 3% bovine serum albumin. DNA was stained with 1 .mu.g/ml To Pro (Molecular Probes). Images were obtained with a Zeiss LSM 510 inverted confocal microscope using planapochromat 63X/NA 1.4 oil immersion objective and excitation wavelengths 488, 546 and 630 nm. Through focus maximum projection images were acquired from optical sections 0.50 .mu.m apart and with a section thickness of 1.0 .mu.m. Images were processed using Adobe PhotoShop (Abacus Inc). At least 300 nuclei were counted on each slide and those containing more than 10 RAD51 foci or sites of PARP activity were classified as positive.

[0075] PARP Activity Assays

[0076] A water-soluble tetrazolium salt (5 mM WST-8) was used to monitor the amount of NAD(P)H through its reduction to a yellow coloured formazan dye [43]. 5000 cells were plated in at least triplicate into wells of a 96 well plate and cultured in 100 .mu.l normal growth media for 4 h at 37.degree. CK8 buffer (Dojindo Molecular Technology, Gaithersburg, USA), containing WST-8, was then added either with or without treatment with DNA damaging agents at concentrations indicated. Reduction of WST-8 in the presence of NAD(P)H was determined by measuring visible absorbance (OD.sub.450) every 30 min. A medium blank was also prepared containing just media and CK8 buffer. Changes in NAD(P)H levels were calculated by comparing the absorbance of wells containing cells treated with DNA damaging agents and those treated with DMSO alone. Alternately relative levels of NAD(P)H in different cells lines were calculated after 4 h incubation in CK8 buffer.

[0077] The ability of NU1025 to inhibit PARP-1 activity was also assayed in permeabilised cells using a modification of the method of Halldorsson et al [44], and described in detail elsewhere [45]. Briefly: 300 .mu.l of NU1025-treated (15 min) permeabilised cells were incubated at 26.degree. C. with oligonucleotide (final conc. 2.5 .mu.g/ml), 75 .mu.m MNAD+[.sup.32P] NAD (Amersham Pharmacia, Amersham, UK) in a total volume of 400 .mu.l. The reaction was terminated after 5 min by adding ice cold 10% TCA 10% Na Ppi for 60 min prior to filtering through a Whatman GF/C filter (LabSales, Maidstone, UK), rinsed 6.times. with 1% TCA 1% NaPPi, left to dry and incorporated radioactivity was measured to determine PARP-1 activity. Data are expressed as pmol NAD incorporated/10.sup.6 cells by reference to [.sup.32P] NAD standards.

[0078] Pulse-Field Gel Electrophoresis

[0079] 1.5.times.10.sup.6 cells were plated onto 100 mm dishes and allowed 4 h for attachment. Exposure to drug was for 18 h after which cells were trypsinsied and 10.sup.6 cells melted into each 1% agarose insert. These inserts were incubated as described elsewhere (8) and separated by pulse-field gel electrophoresis for 24 h (BioRad; 120.degree. angle, 60 to 240 s switch time, 4 V/cm). The gel was subsequently stained with ethidium bromide for analysis.

[0080] siRNA Treatment

[0081] Predesigned BRCA2 SMARTpool and scrambled siRNAs were purchased (Dharmacon, Lafayette, Colo.). 10000 cells seeded onto 6 well plates and left over night before transfected with 100 nM siRNA using Oligofectamine Reagent (Invitrogen) according to manufacturers instructions. Cells were then cultured in normal growth media for 48 h prior to trypsinisation and replating for toxicity assays. Suppression of BRCA2 was confirmed by Western blotting (as described previously [46]) of protein extracts treated with siRNA with an antibody against BRCA2 (Oncogene, Nottingham, UK).

EXAMPLES

[0082] Homologous Recombination Deficient Cells are Hypersensitive to PARP-1 Inhibition

[0083] To investigate the involvement of HR in cellular responses to inhibition of PARP-1, the effects of PARP-1 inhibitors on the survival of HR repair deficient cell lines were studied. It was found that cells deficient in HR (i.e., irs1SF which is defective in XRCC3 or irs1 which is defective in XRCC2 [see Table 1] were very sensitive to the toxic effect of 3-aminobenzamide (3-AB) and to two more potent inhibitors of PARP-1: 1,5-dihydroxyisoquinoline (ISQ; [37]) or 8-hydroxy-2-methylquinazolinone (NU1025 [38, 39]) (FIG. 1). The sensitivity in irs1SF cells to 3-AB, ISQ or NU1025 was corrected by the introduction of a cosmid containing a functional XRCC3 gene (CXR3). Similarly, the sensitivity in irs1 cells to 3-AB, ISQ or NU1025 was corrected by the introduction of a cosmid containing a functional XRCC2 gene (irs1X2.2).

[0084] BRCA2 Deficient Cells are Hypersensitive to PARP-1 Inhibition

[0085] The survival of BRCA2 deficient cells (VC8) and wild type cells (V79Z) in the presence of inhibitors of PARP-1 was investigated. It was found that VC8 cells are very sensitive to the toxic effect of NU1025 (FIG. 2). The sensitivity in VC8 cells was corrected by the introduction of a functional BRCA2 gene either on chromosome 13 (VC8#13) or on an overexpression vector (VC8+B2). This result demonstrates that the sensitivity to PARP-1 inhibitors is a direct consequence of loss of the BRCA2 function.

[0086] To investigate if inhibition of PARP-1 triggers apoptosis in BRCA2 deficient cells, the level of apoptosis 72 hours following exposure to NU1025 was investigated. It was found that NU1025 triggered apoptosis only in VC8 cells, showing that loss of PARP-1 activity in BRCA2 deficient cells triggers this means of death (FIG. 3).

[0087] BRCA2 Deficient Breast Cancer Cells are Hypersensitive to PARP-1 Inhibition

[0088] It was examined whether the MCF7 (wild-type p53) and MDA-MB-231 (mutated p53) breast cancer cell lines displayed a similar sensitivity to NU1025 upon depletion of BRCA2. It was found that PARP inhibitors profoundly reduced the survival of MCF7 and MDA-MB-231 cells only when BRCA2 was depleted with a mixture of BRCA2 siRNA (FIG. 4). This shows that BRCA2 depleted breast cancer cells are sensitive to PARP inhibitors regardless of p53 status.

[0089] BRCA2 Deficient Cells Die from PARP-1 Inhibition in Absence of DNA Double-Strand Breaks (DSBs) but in Presence of .gamma.H2Ax

[0090] HR is known to be involved in the repair of DSBs and other lesions that occur during DNA replication [2]. To determine whether the sensitivity of BRCA2 deficient cells is the result of an inability to repair DSBs following NU1025 treatment, the accumulation of DSBs in V79 and V-C8 cells was measured following treatments with highly toxic levels of NU1025. It was found that no DSBs were detectable by pulsed field gel electrophoretic analysis of DNA obtained from the treated cells (FIG. 5A), suggesting that low levels of DSBs or other recombinogenic substrates accumulated following PARP inhibition in HR deficient cells, which trigger .gamma.H2Ax FIG. 5B). The reason why BRCA2 deficient cells die following induction of these recombinogenic lesions is likely to be due to an inability to repair such lesions. To test this, the ability of BRCA2 deficient V-C8 cells and BRCA2 complimented cells to form RAD51 foci in response to NU1025 was determined. It was found that RAD51 foci were indeed induced in V-C8+B2 cells following treatment with NU1025 (statistically significant in t-test p<0.05; FIG. 5D). This indicates that the recombinogenic lesions trigger HR repair in these cells allowing them to survive. In contrast, the BRCA2 deficient V-C8 cells were unable to form RAD51 foci in response to NU1025 treatment (FIG. 5D) indicating no BR, which would leave the recombinogenic lesions unrepaired and thus cause cell death.

[0091] PARP-1 and not PARP-2 is Important in Preventing Formation of a Recombinogenic Lesion

[0092] There are two major PARPs present in the nucleus in mammalian cells, PARP-1 and PARP-2 and all reported PARP inhibitors inhibit both. In order to distinguish which PARP was responsible for the effect, we tested if the absence of PARP-1 and/or PARP-2 results in accumulation of toxic lesions, by depleting these and BRCA2 with siRNA in human cells (FIG. 6a). We found that the clonogenic survival was significantly reduced when both PARP-1 and BRCA2 proteins were co-depleted from human cells (FIG. 6b). Depletion of PARP-2 with BRCA2 had no effect on the clonogenic survival and depletion of PARP-2 in PARP-1 and BRCA2 depleted cells did not result in additional toxicity. These results suggest that PARP-1 and not PARP-2 is responsible for reducing toxic recombinogenic lesions in human cells. The cloning efficiency was only reduced to 60% of control in PARP-1 and BRCA2 co-depleted cells, while no HR deficient cells survived treatments with PARP inhibitors. This is likely to do with incomplete depletion of the abundant PARP-1 protein by siRNA (FIG. 6c), which might be sufficient to maintain PARP-1 function in some of the cells.

[0093] PARP-1 is Activated by Replication Inhibitors

[0094] HR is also involved in repair of lesions occurring at stalled replication forks, which may not involve detectable DSBs [2]. To test if PARP has a role at replication forks, PARP activation in cells treated cells with agents (thymidine or hydroxyurea) that retard or arrest the progression of DNA replication forks was examined. Thymidine depletes cells of dCTP and slows replication forks without causing DSBs. Hydroxyurea depletes several dNTP and block the replication fork, which is associated with the formation of DSBs at replication forks [2]. Both of these agents potently induce HR [2]. V79 hamster cells treated for 24 hours with thymidine or hydroxyurea were stained for PAR polymers. This revealed a substantial increase in the number of cells containing sites of PARP activity (FIG. 7C). This result suggests a function for PARP at stalled replication forks. It was also shown that inhibition of PARP with NU1025 enhances the sensitivity to thymidine or hydroxyurea in V-C8+B2 cells (FIG. 7D,E). This result suggests that PARP activity is important in repair of stalled replication forks or alternatively that it prevents the induction of death in cells with stalled replication forks.

[0095] PARP is rapidly activated at DNA single-strand breaks (SSB) and attracts DNA repair enzymes [3-6]. Methylmethane sulphonate (MMS) causes alkylation of DNA, which is repaired by base excision repair. PARP is rapidly activated by the SSB-intermediate formed during this repair, which depletes the NAD(P)H levels (FIG. 7F). We found that the activation of PARP and reduction of NAD(P)H levels is much slower following thymidine or hydroxyurea treatments. This slow PARP activation can be explained by the indirect action of thymidine and hydroxyurea and the time required to accumulate stalled replication forks as cells enter the S phase of the cell cycle.

[0096] PARP-1 and HR have Separate Roles at Stalled Replication Forks

[0097] The number sites of PARP activity in untreated BRCA2 deficient V-C8 cells was determined. It was found that more V-C8 cells contain sites of PARP activity compared to V-C8+B2 cells (FIG. 8A,B,C). Also, the V-C8 cells have lower free NAD(P)H levels than the corrected cells (FIG. 8D), as a likely result of the increased PARP activity. Importantly these sites of PARP activity do not overlap with RAD51 foci (FIG. 8E).

[0098] The results herein suggest that PARP and HR have separate roles in the protection or rescue of stalled replication forks (FIG. 8F). A loss of PARP activity can be compensated by increased HR while a loss of HR can be compensated by increased PARP activity. However, loss of both these pathways leads to accumulation of stalled replication forks and to death, as in the case of PARP inhibited BRCA2 deficient cells.

[0099] As shown in the model outlined in FIG. 8F PARP and HR have complementary roles at stalled replication forks. (i) Replication forks may stall when encountering a roadblock on the DNA template. In addition, they may also stall temporarily, due to lack of dNTPs or other replication co-factors. (ii) PARP binds stalled replication forks or other replication-associated damage, triggering PAR polymerization. Resulting negatively charged PAR polymers may protect stalled replication forks, by repelling proteins that normally would process replication forks (e.g., resolvases), until the replication fork can be restored spontaneously when dNTPs or other co-factors become available. Alternatively, PAR polymers or PARP may attract proteins to resolve the replication block by other means. (iii) In absence of PARP activity, HR may be used as an alternative pathway to repair stalled replication forks. This compensatory model explains the increased level of HR and RAD51 foci found in PARP deficient cells.sup.3-5 and higher PARP activity found in HR deficient cells (i.e. V-C8). Spontaneous replication blocks/lesions are only lethal in the absence of both PARP and HR.

TABLE-US-00001 TABLE 1 Genotype and origin of cell lines used in this study. Cell line Genotype Defect Origin Reference AA8 Wt Wt CHO [41] irs1SF XRCC3.sup.- XRCC3.sup.-, deficient in HR AA8 [41] CXR3 XRCC3.sup.- + Wt irs1SF [41] hXRCC3 V79-4 Wt Wt V79 [40] irs1 XRCC2.sup.- XRCC2.sup.-, deficient in HR V79-4 [40] irs1X2.2 XRCC2.sup.- + Wt irs1 [40] hXRCC2 V79-Z Wt Wt V79 [42] VC8 BRCA2.sup.- BRCA2.sup.-, deficient in HR V79-Z [42] VC8#13 BRCA2.sup.- + Wt VC8 [42] hBRCA2 VC8 + B2 BRCA2.sup.- + Wt VC8 [42] hBRCA2

REFERENCES

[0100] [1] A. R. Venkitaraman Cancer susceptibility and the functions of BRCA1 and BRCA2, Cell 108 (2002) 171-182.

[0101] [2] C. Lundin, K. Erixon, C. Arnaudeau, N. Schultz, D. Jenssen, M. Meuth and T. Helleday Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells, Mol Cell Biol 22 (2002) 5869-5878.

[0102] [3] D. D'Amours, S. Desnoyers, I. D'Silva and G. G. Poirier Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions, Biochem J 342 (1999) 249-268.

[0103] [4] Z. Herceg and Z. Q. Wang Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death, Mutat Res 477 (2001) 97-110.

[0104] [5] T. Lindahl, M. S. Satoh, G. G. Poirier and A. Klungland Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks, Trends Biochem Sci 20 (1995) 405-411.

[0105] [6] M. S. Satoh and T. Lindahl Role of poly(ADP-ribose) formation in DNA repair, Nature 356 (1992) 356-358.

[0106] [7] S. Shall and G. de Murcia Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model?, Mutat Res 460 (2000) 1-15.

[0107] [8] Z. Q. Wang, L. Stingl, C. Morrison, M. Jantsch, M. Los, K. Schulze-Osthoff and E. F. Wagner PARP is important for genomic stability but dispensable in apoptosis, Genes Dev 11 (1997) 2347-2358.

[0108] [9] C. M. Simbulan-Rosenthal, B. R. Haddad, D. S. Rosenthal, Z. Weaver, A. Coleman, R. Luo, H. M. Young, Z. Q. Wang, T. Ried and M. E. Smulson Chromosomal aberrations in PARP(-/-) mice: genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA, Proc Natl Acad Sci USA 96 (1999) 13191-13196.

[0109] [10] J. M. de Murcia, C. Niedergang, C. Trucco, M. Ricoul, B. Dutrillaux, M. Mark, F. J. Oliver, M. Masson, A. Dierich, M. LeMeur, C. Walztinger, P. Chambon and G. de Murcia Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells, Proc Natl Acad Sci USA 94 (1997) 7303-7307.

[0110] [11] F. d'Adda di Fagagna, M. P. Hande, W. M. Tong, P. M. Lansdorp, Z. Q. Wang and S. P. Jackson Functions of poly(ADP-ribose) polymerase in controlling telomere length and chromosomal stability, Nat Genet 23 (1999) 76-80.

[0111] [12] E. Samper, F. A. Goytisolo, J. Menissier-de Murcia, E. Gonzalez-Suarez, J. C. Cigudosa, G. de Murcia and M. A. Blasco Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase-deficient mice and primary cells despite increased chromosomal instability, J Cell Biol 154 (2001) 49-60.

[0112] [13] C. Morrison, G. C. Smith, L. Stingl, S. P. Jackson, E. F. Wagner and Z. Q. Wang Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis, Nat Genet 17 (1997) 479-482.

[0113] [14] V. Schreiber, D. Hunting, C. Trucco, B. Gowans, D. Grunwald, G. De Murcia and J. M. De Murcia A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage, Proc Natl Acad Sci USA 92 (1995) 4753-4757.

[0114] [15] J. H. Kupper, M. Muller and A. Burkle Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates carcinogen induced gene amplification in SV40-transformed Chinese hamster cells, Cancer Res 56 (1996) 2715-2717.

[0115] [16] J. Magnusson and C. Ramel Inhibitor of poly(ADP-ribose)transferase potentiates the recombinogenic but not the mutagenic action of alkylating agents in somatic cells in vivo in Drosophila melanogaster, Mutagenesis 5 (1990) 511-514.

[0116] [17] A. S. Waldman and B. C. Waldman Stimulation of intrachromosomal homologous recombination in mammalian cells by an inhibitor of poly(ADP-ribosylation), Nucleic Acids Res 19 (1991) 5943-5947.

[0117] [18] A. Semionov, D. Cournoyer and T. Y. Chow Inhibition of poly(ADP-ribose)polymerase stimulates extrachromosomal homologous recombination in mouse Ltk-fibroblasts, Nucleic Acids Res 27 (1999) 4526-4531.

[0118] [19] F. Dantzer, V. Schreiber, C. Niedergang, C. Trucco, E. Flatter, G. De La Rubia, J. Oliver, V. Rolli, J. Menissier-de Murcia and G. de Murcia Involvement of poly(ADP-ribose) polymerase in base excision repair, Biochimie 81 (1999) 69-75.

[0119] [20] F. Dantzer, G. de La Rubia, J. Menissier-De Murcia, Z. Hostomsky, G. de Murcia and V. Schreiber Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1, Biochemistry 39 (2000) 7559-7569.

[0120] [21] L. Tentori, I. Portarena and G. Graziani Potential clinical applications of poly(ADP-ribose) polymerase (PARP) inhibitors, Pharmacol Res 45 (2002) 73-85.

[0121] [22] T. Lindahl and R. D. Wood Quality control by DNA repair, Science 286 (1999) 1897-1905.

[0122] [23] K. W. Caldecott DNA single-strand break repair and spinocerebellar ataxia, Cell 112 (2003) 7-10.

[0123] [24] D. D'Amours and S. P. Jackson The Mre11 complex: at the crossroads of dna repair and checkpoint signalling, Nat Rev Mol Cell Biol 3 (2002) 317-327.

[0124] [25] A. D. D'Andrea and M. Grompe The Fanconi anaemia/BRCA pathway, Nat Rev Cancer 3 (2003) 23-34.

[0125] [26] S. P. Jackson Sensing and repairing DNA double-strand breaks, Carcinogenesis 23 (2002) 687-696.

[0126] [27] R. Kanaar, J. H. Hoeijmakers and D. C. van Gent Molecular mechanisms of DNA double strand break repair, Trends Cell Biol 8 (1998) 483-489.

[0127] [28] D. C. van Gent, J. H. Hoeijmakers and R. Kanaar Chromosomal stability and the DNA double-stranded break connection, Nat Rev Genet 2 (2001) 196-206.

[0128] [29] S. L. Neuhausen and E. A. Ostrander Mutation testing of early-onset breast cancer genes BRCA1 and BRCA2, Genet Test 1 (1997) 75-83.

[0129] [30] G. Kuperstein, W. D. Foulkes, P. Ghadirian, J. Hakimi and S. A. Narod A rapid fluorescent multiplexed-PCR analysis (FMPA) for founder mutations in the BRCA1 and BRCA2 genes, Clin Genet 57 (2000) 213-220.

[0130] [31] A. Chiarugi Poly(ADP-ribose) polymerase: killer or conspirator? The suicide hypothesis' revisited, Trends Pharmacol Sci 23 (2002) 122-129.

[0131] [32] C. R. Calabrese, M. A. Batey, H. D. Thomas, B. W. Durkacz, L. Z. Wang, S. Kyle, D. Skalitzky, J. Li, C. Zhang, T. Boritzki, K. Maegley, A. H. Calvert, Z. Hostomsky, D. R. Newell and N. J. Curtin Identification of Potent Nontoxic Poly(ADP-Ribose) Polymerase-1 Inhibitors: Chemopotentiation and Pharmacological Studies, Clin Cancer Res 9 (2003) 2711-2718.

[0132] [33] D. Ferraris, Y. S. Ko, T. Pahutski, R. P. Ficco, L. Serdyuk, C. Alemu, C. Bradford, T. Chiou, R. Hoover, S. Huang, S. Lautar, S. Liang, Q. Lin, M. X. Lu, M. Mooney, L. Morgan, Y. Qian, S. Tran, L. R. Williams, Q. Y. Wu, J. Zhang, Y. Zou and V. Kalish Design and synthesis of poly ADP-ribose polymerase-1 inhibitors. 2. Biological evaluation of aza-5[H]-phenanthridin-6-ones as potent, aqueous-soluble compounds for the treatment of ischemic injuries, J Med Chem 46 (2003) 3138-3151.

[0133] [34] K. J. Dillon, G. C. Smith and N. M. Martin A FlashPlate assay for the identification of PARP-1 inhibitors, J Biomol Screen 8 (2003) 347-352.

[0134] [35] A. J. Pierce, R. D. Johnson, L. H. Thompson and M. Jasin XRCC3 promotes homology-directed repair of DNA damage in mammalian cells, Genes Dev 13 (1999) 2633-2638.

[0135] [36] R. D. Johnson, N. Liu and M. Jasin Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination, Nature 401 (1999) 397-399.

[0136] [37] G. M. Shah, D. Poirier, S. Desnoyers, S. Saint-Martin, J. C. Hoflack, P. Rong, M. ApSimon, J. B. Kirkland and G. G. Poirier Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H10T1/2 cells from oxidative stress, Biochim Biophys Acta 1312 (1996) 1-7.

[0137] [38] R. J. Griffin, S. Srinivasan, K. Bowman, A. H. Calvert, N. J. Curtin, D. R. Newell, L. C. Pemberton and B. T. Golding Resistance-modifying agents. 5. Synthesis and biological properties of quinazolinone inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), J Med Chem 41 (1998) 5247-5256.

[0138] [39] S. Boulton, L. C. Pemberton, J. K. Porteous, N. J. Curtin, R. J. Griffin, B. T. Golding and B. W. Durkacz Potentiation of temozolomide-induced cytotoxicity: a comparative study of the biological effects of poly(ADP-ribose) polymerase inhibitors, Br J Cancer 72 (1995) 849-856.

[0139] [40] C. S. Griffin, P. J. Simpson, C. R. Wilson and J. Thacker Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation, Nat Cell Biol 2 (2000) 757-761.

[0140] [41] R. S. Tebbs, Y. Zhao, J. D. Tucker, J. B. Scheerer, M. J. Siciliano, M. Hwang, N. Liu, R. J. Legerski and L. H. Thompson Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene, Proc Natl Acad Sci USA 92 (1995) 6354-6358.

[0141] [42] M. Kraakman-van der Zwet, W. J. Overkamp, R. E. van Lange, J. Essers, A. van Duijn-Goedhart, I. Wiggers, S. Swaminathan, P. P. van Buul, A. Errami, R. T. Tan, N. G. Jaspers, S. K. Sharan, R. Kanaar and M. Z. Zdzienicka Brca2 (XRCC11) deficiency results in radioresistant DNA synthesis and a higher frequency of spontaneous deletions, Mol Cell Biol 22 (2002) 669-679.

[0142] [43] J. Nakamura, S. Asakura, S. D. Hester, G. de Murcia, K. W. Caldecott and J. A. Swenberg Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time, Nucleic Acids Res 31 (2003) e104.

[0143] [44] H. Halldorsson, D. A. Gray and S. Shall Poly (ADP-ribose) polymerase activity in nucleotide permeable cells, FEBS Lett 85 (1978) 349-352.

[0144] [45] K. Grube, J. H. Kupper and A. Burkle Direct stimulation of poly(ADP ribose) polymerase in permeabilized cells by double-stranded DNA oligomers, Anal Biochem 193 (1991) 236-239.

[0145] [46] C. Lundin, N. Schultz, C. Arnaudeau, A. Mohindra, L. T. Hansen and T. Helleday RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells, J Mol Biol 328 (2003) 521-535.

[0146] [47] Schreider et al., Journal of Biological Chemistry 277: 23028-23036 (2002).

Sequence CWU 1

1

9121RNAHomo sapiens 1aaaagccaug guggaguaug a 21223RNAHomo sapiens 2aagaccaauc ucuccaguuc aac 23321RNAHomo sapiens 3aagaccaaca ucgagaacaa c 2145468DNAHomo sapiens 4cgcccgccca gccccggggg cagggaaagc ctaaattacg gaattaccgc gagcaaggag 60cgcggaatcg gggagcgtcc ggagctagct ggatcctcta ggcaggatgg tgatgggaat 120ctttgcaaat tgtatcttct gtttgaaagt gaagtactta cctcagcagc agaagaaaaa 180gctacaaact gacattaagg aaaatggcgg aaagttttcc ttttcgttaa atcctcagtg 240cacacatata atcttagata atgctgatgt tctgagtcag taccaactga attctatcca 300aaagaaccac gttcatattg caaacccaga ttttatatgg aaatctatca gagaaaagag 360actcttggat gtaaagaatt atgatcctta taagcccctg gacatcacac cacctcctga 420tcagaaggcg agcagttctg aagtgaaaac agaaggtcta tgcccggaca gtgccacaga 480ggaggaagac actgtggaac tcactgagtt tggtatgcag aatgttgaaa ttcctcatct 540tcctcaagat tttgaagttg caaaatataa caccttggag aaagtgggaa tggagggagg 600ccaggaagct gtggtggtgg agcttcagtg ttcgcgggac tccagggact gtcctttcct 660gatatcctca cacttcctcc tggatgatgg catggagact agaagacagt ttgctataaa 720gaaaacctct gaagatgcaa gtgaatactt tgaaaattac attgaagaac tgaagaaaca 780aggatttcta ctaagagaac atttcacacc tgaagcaacc caattagcat ctgaacaatt 840gcaagcattg cttttggagg aagtcatgaa ttcaagcact ctgagccaag aggtgagcga 900tttagtagag atgatttggg cagaggccct gggccacctg gaacacatgc ttctcaagcc 960agtgaacagg attagcctca acgatgtgag caaggcagag gggattctcc ttctagtaaa 1020ggcagcactg aaaaatggag aaacagcaga gcaattgcaa aagatgatga cagagtttta 1080cagactgata cctcacaaag gcacaatgcc caaagaagtg aacctgggac tattggctaa 1140gaaagcagac ctctgccagc taataagaga catggttaat gtctgtgaaa ctaatttgtc 1200caaacccaac ccaccatccc tggccaaata ccgagctttg aggtgcaaaa ttgagcatgt 1260tgaacagaat actgaagaat ttctcagggt tagaaaagag gttttgcaga atcatcacag 1320taagagccca gtggatgtct tgcagatatt tagagttggc agagtgaatg aaaccacaga 1380gtttttgagc aaacttggta atgtgaggcc cttgttgcat ggttctcctg tacaaaacat 1440cgtgggaatc ttgtgtcgag ggttgctttt acccaaagta gtggaagatc gtggtgtgca 1500aagaacagac gtcggaaacc ttggaagtgg gatttatttc agtgattcgc tcagtacaag 1560tatcaagtac tcacacccgg gagagacaga tggcaccaga ctcctgctca tttgtgacgt 1620agccctcgga aagtgtatgg acttacatga gaaggacttt tccttaactg aagcaccacc 1680aggctacgac agtgtgcatg gagtttcaca aacagcctct gtcaccacag actttgagga 1740tgatgaattt gttgtctata aaaccaatca ggttaaaatg aaatatatta ttaaattttc 1800catgcctgga gatcagataa aggactttca tcctagtgat catactgaat tagaggaata 1860cagacctgag ttttcaaatt tttcaaaggt tgaagattac cagttaccag atgccaaaac 1920ttccagcagc accaaggccg gcctccagga tgcttctggg aacttggttc ctctggagga 1980tgtccacatc aaagggagaa tcatagacac tgtagcccag gtcattgttt ttcagacata 2040cacaaataaa agtcacgtgc ccattgaggc aaaatatatc tttcctttgg atgacaaggc 2100cgctgtgtgt ggcttcgaag ccttcatcaa tgggaagcac atagttggag agattaaaga 2160gaaggaagaa gcccagcaag agtacctaga agccgtgacc cagggccatg gcgcttacct 2220gatgagtcag gatgctccgg acgtttttac tgtaagtgtt ggaaacttac cccctaaggc 2280taaggttctt ataaaaatta cctacatcac agaactcagc atcctgggca ctgttggtgt 2340ctttttcatg cccgccaccg tagcaccctg gcaacaggac aaggctttga atgaaaacct 2400tcaggataca gtagagaaga tttgtataaa agaaatagga acaaagcaaa gcttctcttt 2460gactatgtct attgagatgc cgtacgtgat tgaattcatt ttcagtgata ctcatgaact 2520gaaacaaaag cgcacagact gcaaagctgt cattagcacc atggaaggca gctccttaga 2580cagcagtgga ttttctctcc acatcggttt gtctgctgcc tatctcccaa gaatgtgggt 2640tgaaaaacat ccagaaaaag aaagcgaggc ttgcatgctt gtctttcaac ccgatctcga 2700tgtcgacctc cctgacctag ccaatgagag cgaagtgatt atttgtcttg actgctccag 2760ttccatggag ggtgtgacat tcttgcaagc caaggaaatc gccttgcatg cgctgtcctt 2820ggtgggtgag aagcagaaag taaatattat ccagttcggc acaggttaca aggagctatt 2880ttcgtatcct aagcatatca caagcaatac cgcggcagca gagttcatca tgtctgccac 2940acctaccatg gggaacacag acttctggaa aacactccga tatcttagct tattgtaccc 3000tgctcgaggg tcacggaaca tcctcctggt gtctgatggg cacctccagg atgagagcct 3060gacattacag ctcgtgaaga ggagccgccc gcacaccagg ttattcgcct gcggtatcgg 3120ttctacagca aatcgtcacg tcttaaggat tttgtcccag tgtggtgccg gagtatttga 3180atattttaat gcaaaatcca agcatagttg gagaaaacag atagaagacc aaatgaccag 3240gctatgttct ccgagttgcc actctgtctc cgtcaaatgg cagcaactca atccagatgc 3300gcccgaggcc ctgcaggccc cagcccaggt gccatccttg tttcgcaatg atcgactcct 3360tgtctatgga ttcattcctc actgcacaca ggcaactctg tgtgcactaa ttcaagagaa 3420agaattttgt acaatggtgt cgactactga gcttcagaag acaactggaa ctatgatcca 3480caagctggca gcccgagctc taatcagaga ttatgaagat ggcattcttc acgaaaatga 3540aaccagtcat gagatgaaaa aacaaacctt gaaatctctg attattaaac tcagtaaaga 3600aaactctctc ataacacaat ttacaagctt tgtggcagtt gagaaaaggg atgagaatga 3660gtcacctttt cctgatattc caaaagtttc tgaacttatt gccaaagaag atgtagactt 3720cctgccctac atgagctggc agggggaacc ccaagaagcc gtcaggaacc agtctctttt 3780agcatcctct gagtggccag aattacgttt atccaaacga aaacatagga aaattccatt 3840ttccaaaaga aaaatggaat tatctcagcc agaagtttct gaagattttg aagaggatgc 3900cttaggtgta ctaccagctt tcacatcaaa tttggaacgt ggacgtgtgg aaaagctatt 3960ggatttaagt tggacagagt catgtaaacc aacagcaact gaaccactat ttaagaaagt 4020cagtccatgg gaaacatcta cttctagctt ttttcctatt ttggctccgg ccgttggttc 4080ctatcttacc ccgactaccc gcgctcacag tcctgcttcc ttgtcttttg cctcatatcg 4140tcaggtagct agtttcggtt cagctgctcc tcccagacag tttgatgcat ctcaattcag 4200ccaaggccct gtgcctggca cttgtgctga ctggatccca cagtcggcgt cttgtcccac 4260aggacctccc cagaacccac cttctgcacc ctattgtggc attgtttttt cagggagctc 4320attaagctct gcacagtctg ctccactgca acatcctgga ggctttacta ccaggccttc 4380tgctggcacc ttccctgagc tggattctcc ccagcttcat ttctctcttc ctacagaccc 4440tgatcccatc agaggttttg ggtcttatca tccctctgct tactctcctt ttcattttca 4500accttccgca gcctctttga ctgccaacct taggctgcca atggcctctg ctttacctga 4560ggctctttgc agtcagtccc ggactacccc agtagatctc tgtcttctag aagaatcagt 4620aggcagtctc gaaggaagtc gatgtcctgt ctttgctttt caaagttctg acacagaaag 4680tgatgagcta tcagaagtac ttcaagacag ctgcttttta caaataaaat gtgatacaaa 4740agatgacagt atcccgtgct ttctggaagt aaaagaagag gatgaaatag tgtgcacaca 4800acactggcag gatgctgtgc cttggacaga actcctcagt ctacagacag aggatggctt 4860ctggaaactt acaccagaac tgggacttat attaaatctt aatacaaatg gtttgcacag 4920ctttcttaaa caaaaaggca ttcaatctct aggtgtaaaa ggaagagaat gtctcctgga 4980cctaattgcc acaatgctgg tactacagtt tattcgcacc aggttggaaa aagagggaat 5040agtgttcaaa tcactgatga aaatggatga cccttctatt tccaggaata ttccctgggc 5100ttttgaggca ataaagcaag caagtgaatg ggtaagaaga actgaaggac agtacccatc 5160tatctgccca cggcttgaac tggggaacga ctgggactct gccaccaagc agttgctggg 5220actccagccc ataagcactg tgtcccctct tcatagagtc ctccattaca gtcaaggcta 5280agtcaaatga aactgaattt taaacttttt gcatgcttct atgtagaaaa taatcaaatg 5340ataatagata cttataatga aacttcatta aggtttcatt cagtgtagca attactgtct 5400ttaaaaatta agtggaagaa gaattacttt aatcaactaa caagcaataa taaaatgaaa 5460cttaaaat 546851910DNAHomo sapiens 5ctagaattca gcggccgctg aattctaggc ggcgcggcgg cgacggagca ccggcggcgg 60cagggcgaga gcattaaatg aaagcaaaag agttaataat ggcaacacgg ctccagaaga 120ctcttcccct gccaagaaaa ctcgtagatg ccagagacag gagtcgaaaa agatgcctgt 180ggctggagga aaagctaata aggacaggac agaagacaag caagatggta tgccaggaag 240gtcatgggcc agcaaaaggg tctctgaatc tgtgaaggcc ttgctgttaa agggcaaagc 300tcctgtggac ccagagtgta cagccaaggt ggggaaggct catgtgtatt gtgaaggaaa 360tgatgtctat gatgtcatgc taaatcagac caatctccag ttcaacaaca acaagtacta 420tctgattcag ctattagaag atgatgccca gaggaacttc agtgtttgga tgagatgggg 480ccgagttggg aaaatgggac agcacagcct ggtggcttgt tcaggcaatc tcaacaaggc 540caaggaaatc tttcagaaga aattccttga caaaacgaaa aacaattggg aagatcgaga 600aaagtttgag aaggtgcctg gaaaatatga tatgctacag atggactatg ccaccaatac 660tcaggatgaa gaggaaacaa aaaaagagga atctcttaaa tctcccttga agccagagtc 720acagctagat cttcgggtac aggagttaat aaagttgatc tgtaatgttc aggccatgga 780agaaatgatg atggaaatga agtataatac caagaaagcc ccacttggga agctgacagt 840ggcacaaatc aaggcaggtt accagtctct taagaagatt gaggattgta ttcgggctgg 900ccagcatgga cgagctctca tggaagcatg caatgaattc tacaccagga ttccgcatga 960ctttggactc cgtactcctc cactaatccg gacacagaag gaactgtcag aaaaaataca 1020attactagag gctttgggag acattgaaat tgctattaag ctggtgaaaa cagagctaca 1080aagcccagaa cacccattgg accaacacta tagaaaccta cattgtgcct tgcgccccct 1140tgaccatgaa agttacgagt tcaaagtgat ttcccagtac ctacaatcta cccatgctcc 1200cacacacagc gactatacca tgaccttgct ggatttgttt gaagtggaga aggatggtga 1260gaaagaagcc ttcagagagg accttcataa caggatgctt ctatggcatg gttccaggat 1320gagtaactgg gtgggaatct tgagccatgg gcttcgaatt gcccaccctg aagctcccat 1380cacaggttac atgtttggga aaggaatcta ctttgctgac atgtcttcca agagtgccaa 1440ttactgcttt gcctctcgcc taaagaatac aggactgctg ctcttatcag aggtagctct 1500aggtcagtgt aatgaactac tagaggccaa tcctaaggcc gaaggattgc ttcaaggtaa 1560acatagcacc aaggggctgg gcaagatggc tcccagttct gcccacttcg tcaccctgaa 1620tgggagtaca gtgccattag gaccagcaag tgacacagga attctgaatc cagatggtta 1680taccctcaac tacaatgaat atattgtata taaccccaac caggtccgta tgcggtacct 1740tttaaaggtt cagtttaatt tccttcagct gtggtgaatg ttgatcttaa ataaaccaga 1800gatctgatct tcaagcaaga aaataagcag tgttgtactt gtgaattttg tgatatttta 1860tgtaataaaa actgtacagg tctaaaaaaa aaaaaaaaaa aaaaaaaaaa 191062263DNAHomo sapiens 6tgggactggt cgcctgactc ggcctgcccc agcctctgct tcaccccact ggtggccaaa 60tagccgatgt ctaatccccc acacaagctc atccccggcc tctgggattg ttgggaattc 120tctccctaat tcacgcctga ggctcatgga gagttgctag acctgggact gccctgggag 180gcgcacacaa ccaggccggg tggcagccag gacctctccc atgtccctgc ttttcttggc 240catggctcca aagccgaagc cctgggtaca gactgagggc cctgagaaga agaagggccg 300gcaggcagga agggaggagg accccttccg ctccaccgct gaggccctca aggccatacc 360cgcagagaag cgcataatcc gcgtggatcc aacatgtcca ctcagcagca accccgggac 420ccaggtgtat gaggactaca actgcaccct gaaccagacc aacatcgaga acaacaacaa 480caagttctac atcatccagc tgctccaaga cagcaaccgc ttcttcacct gctggaaccg 540ctggggccgt gtgggagagg tcggccagtc aaagatcaac cacttcacaa ggctagaaga 600tgcaaagaag gactttgaga agaaatttcg ggaaaagacc aagaacaact gggcagagcg 660ggaccacttt gtgtctcacc cgggcaagta cacacttatc gaagtacagg cagaggatga 720ggcccaggaa gctgtggtga aggtggacag aggcccagtg aggactgtga ctaagcgggt 780gcagccctgc tccctggacc cagccacgca gaagctcatc actaacatct tcagcaagga 840gatgttcaag aacaccatgg ccctcatgga cctggatgtg aagaagatgc ccctgggaaa 900gctgagcaag caacagattg cacggggttt cgaggccttg gaggcgctgg aggaggccct 960gaaaggcccc acggatggtg gccaaagcct ggaggagctg tcctcacact tttacaccgt 1020catcccgcac aacttcggcc acagccagcc cccgcccatc aattcccctg agcttctgca 1080ggccaagaag gacatgctgc tggtgctggc ggacatcgag ctggcccagg ccctgcaggc 1140agtctctgag caggagaaga cggtggagga ggtgccacac cccctggacc gagactacca 1200gcttctcaag tgccagctgc agctgctaga ctctggagca cctgagtaca aggtgataca 1260gacctactta gaacagactg gcagcaacca caggtgccct acacttcaac acatctggaa 1320agtaaaccaa gaaggggagg aagacagatt ccaggcccac tccaaactgg gtaatcggaa 1380gctgctgtgg catggcacca acatggccgt ggtggccgcc atcctcacta gtgggctccg 1440catcatgcca cattctggtg ggcgtgttgg caagggcatc tactttgcct cagagaacag 1500caagtcagct ggatatgtta ttggcatgaa gtgtggggcc caccatgtcg gctacatgtt 1560cctgggtgag gtggccctgg gcagagagca ccatatcaac acggacaacc ccagcttgaa 1620gagcccacct cctggcttcg acagtgtcat tgcccgaggc cacaccgagc ctgatccgac 1680ccaggacact gagttggagc tggatggcca gcaagtggtg gtgccccagg gccagcctgt 1740gccctgccca gagttcagca gctccacatt ctcccagagc gagtacctca tctaccagga 1800gagccagtgt cgcctgcgct acctgctgga ggtccacctc tgagtgcccg ccctgtcccc 1860cggggtcctg caaggctgga ctgtgatctt caatcatcct gcccatctct ggtaccccta 1920tatcactcct ttttttcaag aatacaatac gttgttgtta actatagtca ccatgctgta 1980caagatccct gaacttatgc ctcctaactg aaattttgta ttctttgaca catctgccca 2040gtccctctcc tcccagccca tggtaaccag catttgactc tttacttgta taagggcagc 2100ttttataggt tccacatgta agtgagatca tgcagtgttt gtctttctgt gcctggctta 2160tttcactcag cataatgtgc accgggttca cccatgtttt cataaatgac aagatttcct 2220cctttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 226374491DNAHomo sapiens 7cgaagatggc ggcgtcgcgt cgctctcagc atcatcacca ccatcatcaa caacagctcc 60agcccgcccc aggggcttca gcgccgccgc cgccacctcc tcccccactc agccctggcc 120tggccccggg gaccacccca gcctctccca cggccagcgg cctggccccc ttcgcctccc 180cgcggcacgg cctagcgctg ccggaggggg atggcagtcg ggatccgccc gacaggcccc 240gatccccgga cccggttgac ggtaccagct gttgcagtac caccagcaca atctgtaccg 300tcgccgccgc tcccgtggtc ccagcggttt ctacttcatc tgccgctggg gtcgctccca 360acccagccgg cagtggcagt aacaattcac cgtcgtcctc ttcttccccg acttcttcct 420catcttcctc tccatcctcc cctggatcga gcttggcgga gagccccgag gcggccggag 480ttagcagcac agcaccactg gggcctgggg cagcaggacc tgggacaggg gtcccagcag 540tgagcggggc cctacgggaa ctgctggagg cctgtcgcaa tggggacgtg tcccgggtaa 600agaggctggt ggacgcggca aacgtaaatg caaaggacat ggccggccgg aagtcttctc 660ccctgcactt cgctgcaggt tttggaagga aggatgttgt agaacactta ctacagatgg 720gtgctaatgt ccacgctcgt gatgatggag gtctcatccc gcttcataat gcctgttctt 780ttggccatgc tgaggttgtg agtctgttat tgtgccaagg agctgatcca aatgccaggg 840ataactggaa ctatacacct ctgcatgaag ctgctattaa agggaagatc gatgtgtgca 900ttgtgctgct gcagcacgga gctgacccaa acattcggaa cactgatggg aaatcagccc 960tggacctggc agatccttca gcaaaagctg tccttacagg tgaatacaag aaagacgaac 1020tcctagaagc tgctaggagt ggtaatgaag aaaaactaat ggctttactg actcctctaa 1080atgtgaattg ccatgcaagt gatgggcgaa agtcgactcc tttacatcta gcagcgggct 1140acaacagagt tcgaatagtt cagcttcttc ttcagcatgg tgctgatgtt catgcaaaag 1200acaaaggtgg acttgtgcct cttcataatg catgttcata tggacattat gaagtcacag 1260aactgctact aaagcatgga gcttgtgtta atgccatgga tctctggcag tttactccac 1320tgcacgaggc tgcttccaag aaccgtgtag aagtctgctc tttgttactt agccatggcg 1380ctgatcctac gttagtcaac tgccatggca aaagtgctgt ggatatggct ccaactccgg 1440agcttaggga gagattgact tatgaattta aaggtcattc tttactacaa gcagccagag 1500aagcagactt agctaaagtt aaaaaaacac tcgctctgga aatcattaat ttcaaacaac 1560cgcagtctca tgaaacagca ctgcactgtg ctgtggcctc tctgcatccc aaacgtaaac 1620aagtgacaga attgttactt agaaaaggag caaatgttaa tgaaaaaaat aaagatttca 1680tgactcccct gcatgttgca gccgaaagag cccataatga tgtcatggaa gttctgcata 1740agcatggcgc caagatgaat gcactggaca cccttggtca gactgctttg catagagccg 1800ccctagcagg ccacctgcag acctgccgcc tcctgctgag ttacggctct gacccctcca 1860tcatctcctt acaaggcttc acagcagcac agatgggcaa tgaagcagtg cagcagattc 1920tgagtgtgag ttacggctct gacccctcca tcatctcctt acaaggcttc acagcagcac 1980agatgggcaa tgaagcagtg cagcagattc tgagtggtca ttcgtagata gtgatcattc 2040tacttcagcc ttaatggtga tcttgagacg ggaagattta gaaggaaatc tatccagcat 2100gtcttcactg tcaacatgaa gagtacacct atacgtactt ctgatgttga ttatcgactc 2160ttagaggcat ctaaagctgg agacttggaa actgtgaagc aactttgcag ctctcaaaat 2220gtgaattgta gagacttaga gggccggcat tccacgccct tacacttcgc agcaggctac 2280aacagagtac acctatacgt acttctgatg ttgattatcg actcttagag gcatctaaag 2340ctggagactt ggaaactgtg aagcaacttt gcagctctca aaatgtgaat tgtagagact 2400tagagggccg gcattccacg cccttacact tcgcagcagg ctacaaccgc gtgtctgttg 2460tagagtacct gctacaccac ggtgccgatg tccatgccaa agacaagggt ggcttggtgc 2520cccttcataa tgcctgttca tatggacact atgaggtggc tgagctttta gtaaggcatg 2580gggcttctgt caatgtggcg gacttatgga aatttacccc tctccatgaa gcagcagcta 2640aaggaaagta tgaaatctgc aagctccttt taaaacatgg agcagatcca actaaaaaga 2700acagagatgg aaatacacct ttggatttgg taaaggaagg agacacagat attcaggact 2760tactgaaagg ggatgctgct ttgttggatg ctgccaagaa gggctgcctg gcaagagtgc 2820agaagctctg taccccagag aatatcaact gcagagacac ccagggcaga aattcaaccc 2880ctctgcacct ggcagcaggc tataataacc tggaagtagc tgaatatctt ctagagcatg 2940gagctgatgt taatgcccag gacaagggtg gtttaattcc tcttcataat gcggcatctt 3000atgggcatgt tgacatagcg gctttattga taaaatacaa cacgtgtgta aatgcaacag 3060ataagtgggc gtttactccc ctccatgaag cagcccagaa aggaaggacg cagctgtgcg 3120ccctcctcct agcgcatggt gcagacccca ccatgaagaa ccaggaaggc cagacgcctc 3180tggatctggc aacagctgac gatatcagag ctttgctgat agatgccatg cccccagagg 3240ccttacctac ctgttttaaa cctcaggcta ctgtagtgag tgcctctctg atctcaccag 3300catccacccc ctcctgcctc tcggctgcca gcagcataga caacctcact ggccctttag 3360cagagttggc cgtaggagga gcctccaatg caggggatgg cgccgcggga acagaaagga 3420aggaaggaga agttgctggt cttgacatga atatcagcca atttctaaaa agccttggcc 3480ttgaacacct tcgggatatc tttgaaacag aacagattac actagatgtg ttggctgata 3540tgggtcatga agagttgaaa gaaataggca tcaatgcata tgggcaccgc cacaaattaa 3600tcaaaggagt agaaagactc ttaggtggac aacaaggcac caatccttat ttgacttttc 3660actgtgttaa tcagggaacg attttgctgg atcttgctcc agaagataaa gaatatcagt 3720cagtggaaga agagatgcaa agtactattc gagaacacag agatggtggt aatgctggcg 3780gcatcttcaa cagatacaat gtcattcgaa ttcaaaaagt tgtcaacaag aagttgaggg 3840agcggttctg ccaccgacag aaggaagtgt ctgaggagaa tcacaaccat cacaatgagc 3900gcatgttgtt tcatggttct cctttcatta atgccattat tcataaaggg tttgatgagc 3960gacatgcata cataggagga atgtttgggg ccgggattta ttttgctgaa aactcctcaa 4020aaagcaacca atatgtttat ggaattggag gaggaacagg ctgccctaca cacaaggaca 4080ggtcatgcta tatatgtcac agacaaatgc tcttctgtag agtgaccctt gggaaatcct 4140ttctgcagtt tagcaccatg aaaatggccc acgcgcctcc agggcaccac tcagtcattg 4200gtagaccgag cgtcaatggg ctggcatatg ctgaatatgt catctacaga ggagaacagg 4260catacccaga gtatcttatc acttaccaga tcatgaagcc agaagcccct tcccagaccg 4320caacagccgc agagcagaag acctagtgaa tgcctgctgg tgaaggccag atcagatttc 4380aacctgggac tggattacag aggattgttt ctaataacaa catcaatatt ctagaagtcc 4440ctgacagcct agaaataagc tgtttgtctt ctataaagca ttgctatagt g 449186189DNAHomo sapiens 8cgcgccgcct cgctagccga aacctgccca gccggtgccc ggccactgcg cacgcgcggg 60acgacgtcac gtgcgctccc ggggctggac ggagctggca ggaggggcct tgccagcttc 120cgccgccgcg tcgtttcagg acccggacgg cggattcgcg ctgcctccgc cgccgcgggg 180cagccggggg gcagggagcc cagcgagggg cgcgcgtggg cgcggccatg ggactgcgcc 240ggatccggtg acagcaggga gccaagcggc ccgggccctg agcgcgtctt ctccgggggg 300cctcgccctc ctgctcgcgg ggccggggct cctgctccgg ttgctggcgc tgttgctggc 360tgtggcggcg gccaggatca tgtcgggtcg ccgctgcgcc ggcgggggag cggcctgcgc 420gagcgccgcg gccgaggccg tggagccggc cgcccgagag ctgttcgagg cgtgccgcaa 480cggggacgtg gaacgagtca agaggctggt

gacgcctgag aaggtgaaca gccgcgacac 540ggcgggcagg aaatccaccc cgctgcactt cgccgcaggt tttgggcgga aagacgtagt 600tgaatatttg cttcagaatg gtgcaaatgt ccaagcacgt gatgatgggg gccttattcc 660tcttcataat gcatgctctt ttggtcatgc tgaagtagtc aatctccttt tgcgacatgg 720tgcagacccc aatgctcgag ataattggaa ttatactcct ctccatgaag ctgcaattaa 780aggaaagatt gatgtttgca ttgtgctgtt acagcatgga gctgagccaa ccatccgaaa 840tacagatgga aggacagcat tggatttagc agatccatct gccaaagcag tgcttactgg 900tgaatataag aaagatgaac tcttagaaag tgccaggagt ggcaatgaag aaaaaatgat 960ggctctactc acaccattaa atgtcaactg ccacgcaagt gatggcagaa agtcaactcc 1020attacatttg gcagcaggat ataacagagt aaagattgta cagctgttac tgcaacatgg 1080agctgatgtc catgctaaag ataaaggtga tctggtacca ttacacaatg cctgttctta 1140tggtcattat gaagtaactg aacttttggt caagcatggt gcctgtgtaa atgcaatgga 1200cttgtggcaa ttcactcctc ttcatgaggc agcttctaag aacagggttg aagtatgttc 1260tcttctctta agttatggtg cagacccaac actgctcaat tgtcacaata aaagtgctat 1320agacttggct cccacaccac agttaaaaga aagattagca tatgaattta aaggccactc 1380gttgctgcaa gctgcacgag aagctgatgt tactcgaatc aaaaaacatc tctctctgga 1440aatggtgaat ttcaagcatc ctcaaacaca tgaaacagca ttgcattgtg ctgctgcatc 1500tccatatccc aaaagaaagc aaatatgtga actgttgcta agaaaaggag caaacatcaa 1560tgaaaagact aaagaattct tgactcctct gcacgtggca tctgagaaag ctcataatga 1620tgttgttgaa gtagtggtga aacatgaagc aaaggttaat gctctggata atcttggtca 1680gacttctcta cacagagctg catattgtgg tcatctacaa acctgccgcc tactcctgag 1740ctatgggtgt gatcctaaca ttatatccct tcagggcttt actgctttac agatgggaaa 1800tgaaaatgta cagcaactcc tccaagaggg tatctcatta ggtaattcag aggcagacag 1860acaattgctg gaagctgcaa aggctggaga tgtcgaaact gtaaaaaaac tgtgtactgt 1920tcagagtgtc aactgcagag acattgaagg gcgtcagtct acaccacttc attttgcagc 1980tgggtataac agagtgtccg tggtggaata tctgctacag catggagctg atgtgcatgc 2040taaagataaa ggaggccttg tacctttgca caatgcatgt tcttatggac attatgaagt 2100tgcagaactt cttgttaaac atggagcagt agttaatgta gctgatttat ggaaatttac 2160acctttacat gaagcagcag caaaaggaaa atatgaaatt tgcaaacttc tgctccagca 2220tggtgcagac cctacaaaaa aaaacaggga tggaaatact cctttggatc ttgttaaaga 2280tggagataca gatattcaag atctgcttag gggagatgca gctttgctag atgctgccaa 2340gaagggttgt ttagccagag tgaagaagtt gtcttctcct gataatgtaa attgccgcga 2400tacccaaggc agacattcaa cacctttaca tttagcagct ggttataata atttagaagt 2460tgcagagtat ttgttacaac acggagctga tgtgaatgcc caagacaaag gaggacttat 2520tcctttacat aatgcagcat cttacgggca tgtagatgta gcagctctac taataaagta 2580taatgcatgt gtcaatgcca cggacaaatg ggctttcaca cctttgcacg aagcagccca 2640aaagggacga acacagcttt gtgctttgtt gctagcccat ggagctgacc cgactcttaa 2700aaatcaggaa ggacaaacac ctttagattt agtttcagca gatgatgtca gcgctcttct 2760gacagcagcc atgcccccat ctgctctgcc ctcttgttac aagcctcaag tgctcaatgg 2820tgtgagaagc ccaggagcca ctgcagatgc tctctcttca ggtccatcta gcccatcaag 2880cctttctgca gccagcagtc ttgacaactt atctgggagt ttttcagaac tgtcttcagt 2940agttagttca agtggaacag agggtgcttc cagtttggag aaaaaggagg ttccaggagt 3000agattttagc ataactcaat tcgtaaggaa tcttggactt gagcacctaa tggatatatt 3060tgagagagaa cagatcactt tggatgtatt agttgagatg gggcacaagg agctgaagga 3120gattggaatc aatgcttatg gacataggca caaactaatt aaaggagtcg agagacttat 3180ctccggacaa caaggtctta acccatattt aactttgaac acctctggta gtggaacaat 3240tcttatagat ctgtctcctg atgataaaga gtttcagtct gtggaggaag agatgcaaag 3300tacagttcga gagcacagag atggaggtca tgcaggtgga atcttcaaca gatacaatat 3360tctcaagatt cagaaggttt gtaacaagaa actatgggaa agatacactc accggagaaa 3420agaagtttct gaagaaaacc acaaccatgc caatgaacga atgctatttc atgggtctcc 3480ttttgtgaat gcaattatcc acaaaggctt tgatgaaagg catgcgtaca taggtggtat 3540gtttggagct ggcatttatt ttgctgaaaa ctcttccaaa agcaatcaat atgtatatgg 3600aattggagga ggtactgggt gtccagttca caaagacaga tcttgttaca tttgccacag 3660gcagctgctc ttttgccggg taaccttggg aaagtctttc ctgcagttca gtgcaatgaa 3720aatggcacat tctcctccag gtcatcactc agtcactggt aggcccagtg taaatggcct 3780agcattagct gaatatgtta tttacagagg agaacaggct tatcctgagt atttaattac 3840ttaccagatt atgaggcctg aaggtatggt cgatggataa atagttattt taagaaacta 3900attccactga acctaaaatc atcaaagcag cagtggcctc tacgttttac tcctttgctg 3960aaaaaaaatc atcttgccca caggcctgtg gcaaaaggat aaaaatgtga acgaagttta 4020acattctgac ttgataaagc tttaataatg tacagtgttt tctaaatatt tcctgttttt 4080tcagcacttt aacagatgcc attccaggtt aaactgggtt gtctgtacta aattataaac 4140agagttaact tgaacctttt atatgttatg cattgattct aacaaactgt aatgccctca 4200acagaactaa ttttactaat acaatactgt gttctttaaa acacagcatt tacactgaat 4260acaatttcat ttgtaaaact gtaaataaga gcttttgtac tagcccagta tttatttaca 4320ttgctttgta atataaatct gttttagaac tgcagcggtt tacaaaattt tttcatatgt 4380attgttcatc tatacttcat cttacatcgt catgattgag tgatctttac atttgattcc 4440agaggctatg ttcagttgtt agttgggaaa gattgagtta tcagatttaa tttgccgatg 4500ggagccttta tctgtcatta gaaatctttc tcatttaaga acttatgaat atgctgaaga 4560tttaatttgt gatacctttg tatgtatgag acacattcca aagagctcta actatgatag 4620gtcctgatta ctaaagaagc ttctttactg gcctcaattt ctagctttca tgttggaaaa 4680ttttctgcag tccttctgtg aaaattagag caaagtgctc ctgtttttta gagaaactaa 4740atcttgctgt tgaacaatta ttgtgttctt ttcatggaac ataagtagga tgttaacatt 4800tccagggtgg gaagggtaat cctaaatcat ttcccaatct attctaatta ccttaaatct 4860aaaggggaaa aaaaaaatca caaacaggac tgggtagttt tttatcctaa gtatattttt 4920tcctgttctt tttacttggt tttattgctg tatttatagc caatctatac atcatgggta 4980aacttaaccc agaactataa aatgtagttg tttcagtccc cttcaggcct cctgaatggg 5040caagtgcagt gaaacaggtg cttcctgctc ctgggttttc tctccatgat gttatgccca 5100attggaaata tgctgtcagt ttgtgcacca tatggtgacc acgcctgtgc tcagtttggc 5160agctatagaa ggaaatgctg tcccataaaa tgccatccct atttctaata taacactctt 5220ttccaggaag catgcttaag catcttgtta cagagacata catccattat ggcttggcaa 5280tctcttttat ttgttgactc tagctccctt caaagtcgag gaaagatctt tactcactta 5340atgaggacat tccccatcac tgtctgtacc agttcacctt tattttacgt tttattcagt 5400ctgtaaatta actggccctt tgcagtaact tgtacataaa gtgctagaaa atcatgttcc 5460ttgtcctgag taagagttaa tcagagtaag tgcatttctg gagttgtttc tgtgatgtaa 5520attatgatca ttatttaaga agtcaaatcc tgatcttgaa gtgcttttta tacagctctc 5580taataattac aaatatccga aagtcatttc ttggaacaca agtggagtat gccaaatttt 5640atatgaattt ttcagattat ctaagcttcc aggttttata attagaagat aatgagagaa 5700ttaatggggt ttatatttac attatctctc aactatgtag cccatattac tcaccctatg 5760agtgaatctg gaattgcttt tcatgtgaaa tcattgtggt ctatgagttt acaatactgc 5820aaactgtgtt attttatcta aaccattgct taatgagtgt gtttttccat gaatgaatat 5880accgtggttc atatgttagc atggcagcat tttcagatag ctttttgttt gttgggaagt 5940tggggttttg gggggagggg gagtattagt acgttgcatg gaatagccta ctttataatg 6000atgggaatgc tttttctttt gttttgggat tttttttttt gaagtgaaat ttaacttttt 6060gtgccagtag tactattata cccatcttca gtgtcttact tgtactgtat caaattccat 6120accctcattt aattcttaat aaaactgttc acttgtaaaa aaaaaaaaaa aaaaaaaaaa 6180aaaaaaaaa 618995490DNAHomo sapiens 9cgcccgccca gccccggggg cagggaaagc ctaaattacg gaattaccgc gagcaaggag 60cgcggaatcg gggagcgtcc ggagctagct ggatcctcta ggcaggatgg tgatgggaat 120ctttgcaaat tgtatcttct gtttgaaagt gaagtactta cctcagcagc agaagaaaaa 180gctacaaact gacattaagg aaaatggcgg aaagttttcc ttttcgttaa atcctcagtg 240cacacatata atcttagata atgctgatgt tctgagtcag taccaactga attctatcca 300aaagaaccac gttcatattg caaacccaga ttttatatgg aaatctatca gagaaaagag 360actcttggat gtaaagaatt atgatcctta taagcccctg gacatcacac cacctcctga 420tcagaaggcg agcagttctg aagtgaaaac agaaggtcta tgcccggaca gtgccacaga 480ggaggaagac actgtggaac tcactgagtt tggtatgcag aatgttgaaa ttcctcatct 540tcctcaagat tttgaagttg caaaatataa caccttggag aaagtgggaa tggagggagg 600ccaggaagct gtggtggtgg agcttcagtg ttcgcgggac tccagggact gtcctttcct 660gatatcctca cacttcctcc tggatgatgg catggagact agaagacagt ttgctataaa 720gaaaacctct gaagatgcaa gtgaatactt tgaaaattac attgaagaac tgaagaaaca 780aggatttcta ctaagagaac atttcacacc tgaagcaacc caattagcat ctgaacaatt 840gcaagcattg cttttggagg aagtcatgaa ttcaagcact ctgagccaag aggtgagcga 900tttagtagag atgatttggg cagaggccct gggccacctg gaacacatgc ttctcaagcc 960agtgaacagg attagcctca acgatgtgag caaggcagag gggattctcc ttctagtaaa 1020ggcagcactg aaaaatggag aaacagcaga gcaattgcaa aagatgatga cagagtttta 1080cagactgata cctcacaaag gcacaatgcc caaagaagtg aacctgggac tattggctaa 1140gaaagcagac ctctgccagc taataagaga catggttaat gtctgtgaaa ctaatttgtc 1200caaacccaac ccaccatccc tggccaaata ccgagctttg aggtgcaaaa ttgagcatgt 1260tgaacagaat actgaagaat ttctcagggt tagaaaagag gttttgcaga atcatcacag 1320taagagccca gtggatgtct tgcagatatt tagagttggc agagtgaatg aaaccacaga 1380gtttttgagc aaacttggta atgtgaggcc cttgttgcat ggttctcctg tacaaaacat 1440cgtgggaatc ttgtgtcgag ggttgctttt acccaaagta gtggaagatc gtggtgtgca 1500aagaacagac gtcggaaacc ttggaagtgg gatttatttc agtgattcgc tcagtacaag 1560tatcaagtac tcacacccgg gagagacaga tggcaccaga ctcctgctca tttgtgacgt 1620agccctcgga aagtgtatgg acttacatga gaaggacttt cccttaactg aagcaccacc 1680aggctacgac agtgtgcatg gagtttcaca aacagcctct gtcaccacag actttgagga 1740tgatgaattt gttgtctata aaaccaatca ggttaaaatg aaatatatta ttaaattttc 1800catgcctgga gatcagataa aggactttca tcctagtgat catactgaat tagaggaata 1860cagacctgag ttttcaaatt tttcaaaggt tgaagattac cagttaccag atgccaaaac 1920ttccagcagc accaaggccg gcctccagga tgcctctggg aacttggttc ctctggagga 1980tgtccacatc aaagggagaa tcatagacac tgtagcccag gtcattgttt ttcagacata 2040cacaaataaa agtcacgtgc ccattgaggc aaaatatatc tttcctttgg atgacaaggc 2100cgctgtgtgt ggcttcgaag ccttcatcaa tgggaagcac atagttggag agattaaaga 2160gaaggaagaa gcccagcaag agtacctaga agccgtgacc cagggccatg gcgcttacct 2220gatgagtcag gatgctccgg acgtttttac tgtaagtgtt ggaaacttac cccctaaggc 2280taaggttctt ataaaaatta cctacatcac agaactcagc atcctgggca ctgttggtgt 2340ctttttcatg cccgccaccg tagcaccctg gcaacaggac aaggctttga atgaaaacct 2400tcaggataca gtagagaaga tttgtataaa agaaatagga acaaagcaaa gcttctcttt 2460gactatgtct attgagatgc cgtatgtgat tgaattcatt ttcagtgata cacatgaact 2520gaaacaaaag cgcacagact gcaaagctgt cattagcacc atggaaggca gctccttaga 2580cagcagtgga ttttctctcc acatcggttt gtctgctgcc tatctcccaa gaatgtgggt 2640tgaaaaacat ccagaaaaag aaagcgaggc ttgcatgctt gtctttcaac ccgatctcga 2700tgtcgacctc cctgacctag ccagtgagag cgaagtgatt atttgtcttg actgctccag 2760ttccatggag ggtgtgacat tcttgcaagc caagcaaatc accttgcatg cgctgtcctt 2820ggtgggtgag aagcagaaag taaatattat ccagttcggc acaggttaca aggagctatt 2880ttcgtatcct aagcatatca caagcaatac cacggcagca gagttcatca tgtctgccac 2940acctaccatg gggaacacag acttctggaa aacactccga tatcttagct tattgtaccc 3000tgctcgaggg tcacggaaca tcctcctggt gtctgatggg cacctccagg atgagagcct 3060gacattacag ctcgtgaaga ggagccgccc gcacaccagg ttattcgcct gcggtatcgg 3120ttctacagca aatcgtcacg tcttaaggat tttgtcccag tgtggtgccg gagtatttga 3180atattttaat gcaaaatcca agcatagttg gagaaaacag atagaagacc aaatgaccag 3240gctatgttct ccgagttgcc actctgtctc cgtcaaatgg cagcaactca atccagatgc 3300gcccgaggcc ctgcaggccc cagcccaggt gccatccttg tttcgcaatg atcgactcct 3360tgtctatgga ttcattcctc actgcacaca agcaactctg tgtgcactaa ttcaagagaa 3420agaattttgt acaatggtgt cgactactga gcttcagaag acaactggaa ctatgatcca 3480caagctggca gcccgagctc taatcagaga ttatgaagat ggcattcttc acgaaaatga 3540aaccagtcat gagatgaaaa aacaaacctt gaaatctctg attattaaac tcagtaaaga 3600aaactctctc ataacacaat ttacaagctt tgtggcagtt gagaaaaggg atgagaatga 3660gtcgcctttt cctgatattc caaaagtttc tgaacttatt gccaaagaag atgtagactt 3720cctgccctac atgagctggc agggggagcc ccaagaagcc gtcaggaacc agtctctttt 3780agcatcctct gagtggccag aattacgttt atccaaacga aaacatagga aaattccatt 3840ttccaaaaga aaaatggaat tatctcagcc agaagtttct gaagattttg aagaggatgg 3900cttaggtgta ctaccagctt tcacatcaaa tttggaacgt ggaggtgtgg aaaagctatt 3960ggatttaagt tggacagagt catgtaaacc aacagcaact gaaccactat ttaagaaagt 4020cagtccatgg gaaacatcta cttctagctt ttttcctatt ttggctccgg ccgttggttc 4080ctatcttacc ccgactaccc gcgctcacag tcctgcttcc ttgtcttttg cctcatatcg 4140tcaggtagct agtttcggtt cagctgctcc tcccagacag tttgatgcat ctcaattcag 4200ccaaggccct gtgcctggca cttgtgctga ctggatccca cagtcggcgt cttgtcccac 4260aggacctccc cagaacccac cttctgcacc ctattgtggc attgtttttt cagggagctc 4320attaagctct gcacagtctg ctccactgca acatcctgga ggctttacta ccaggccttc 4380tgctggcacc ttccctgagc tggattctcc ccagcttcat ttctctcttc ctacagaccc 4440tgatcccatc agaggttttg ggtcttatca tccctctgct tactctcctt ttcattttca 4500accttccgca gcctctttga ctgccaacct taggctgcca atggcctctg ctttacctga 4560ggctctttgc agtcagtccc ggactacccc agtagatctc tgtcttctag aagaatcagt 4620aggcagtctc gaaggaagtc gatgtcctgt ctttgctttt caaagttctg acacagaaag 4680tgatgagcta tcagaagtac ttcaagacag ctgcttttta caaataaagt gtgatacaaa 4740agatgacagt atcccgtgct ttctggaatt aaaagaagag gatgaaatag tgtgcacaca 4800acactggcag gatgctgtgc cttggacaga actcctcagt ctacagacag aggatggctt 4860ctggaaactt acaccagaac tgggacttat attaaatctt aatacaaatg gtttgcacag 4920ctttcttaaa caaaaaggca ttcaatctct aggtgtaaaa ggaagagaat gtctcctgga 4980cctaattgcc acaatgctgg tactacagtt tattcgcacc aggttggaaa aagagggaat 5040agtgttcaaa tcactgatga aaatggatga cccttctatt tccaggaata ttccctgggc 5100ttttgaggca ataaagcaag caagtgaatg ggtaagaaga actgaaggac agtacccatc 5160tatctgccca cggcttgaac tggggaacga ctgggactct gccaccaagc agttgctggg 5220actccagccc ataagcactg tgtcccctct tcatagagtc ctccattaca gtcaaggcta 5280agtcaaatga aactgaattt taaacttttt gcatgcttct atgtagaaaa taatcaaatg 5340ataatagata attataatga aacttcatta aggtttcatt cagtgtagca attactgtct 5400ttaaaaatta agtggaagaa gaattacttt aatcaactaa caagcaataa taaaatgaaa 5460cttaaaataa aaaaaaaaaa aaaaaaaaaa 5490

Claims

1-32. (canceled)

33. A method of treatment of cancer cells defective in homologous recombination (HR) in a human patient, the method comprising; administering to the patient a therapeutically effective amount of a compound which inhibits PARP-1.

34. The method of claim 33 wherein the PARP inhibitor is selected from the group consisting of benzimidazole-carboxamides, quinazolin-4-[3H]-ones and isoquinolone derivatives.

35. The method of claim 34 wherein the PARP inhibitor is selected from the group consisting of 2-(4-hydroxyphenyl)benzimidazole-4-carboxamide, 8-hydroxy-2-methylquinazolin-4-[3H]one, 6(5H)phenanthridinone, 3-aminobenzamide, benzimidazole-4-carboxamides and tricyclic lactam indoles.

36. The method of claim 33 wherein the cancer cells have defect in a gene encoding a protein involved in HR.

37. The method of claim 36 wherein the human patient has one functional allele of said gene, said functional allele being lost in the cancer cells.

38. The method of claim 33 wherein the gene encoding a protein involved in HR is selected from the group consisting of XRCC1, CTPS, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, BRCA1, BRCA2, RAD52, RAD54, RAD50, MRE11, NBS1, WRN, BLM, Ku70, Ku80, ATM, ATR, chkl, chk2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, RAD9, FEN-1, Mus81, Emel, DDS1 and BARD.

39. The method of claim 33 wherein the cancer cells are selected from the group consisting of lung, colon, pancreatic, gastric, ovarian, cervical, breast and prostate cancer.

40. The method of claim 33 wherein the cancer cells are selected from the group consisting of renal, liver, and bladder cancer.

41. The method of claim 33 wherein the cancer is gene-linked hereditary cancer.

42. The method of claim 41 wherein the cancer is breast cancer.

43. The method of claim 33 wherein the cancer cells to be treated are defective in BRCA1 expression.

44. The method of claim 33 wherein the cancer cells to be treated are defective in BRCA2 expression.

45. The method of claim 33 comprising determining the inhibition of PARP-1 in said individual following said administration.

46. The method of claim 33 comprising identifying a patient with a familial predisposition to said cancer and administering said compound to said patient.

47. The method of claim 33 wherein the daily dosage of said compound is sufficient to induce apoptosis in the cancer cells without affecting normally dividing cells in the human patient.

48. The method of claim 33 wherein the daily dosage of said compound is up to 20 mg/Kg body weight.

49. The method of claim 48 wherein the daily dosage of said compound is more than 2 mg/Kg body weight.

50. The method of claim 33 wherein the compound which inhibits PARP-1 is not administered in combination with radio- or chemo-therapy.

51. The method of claim 33 wherein the compound which inhibits PARP-1 is administered in a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

Images