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.

Claims

1. 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.

2. The use as claimed m claim 1 wherein the enzyme is poly(ADP-ribose) polymerase (PARP).

3. The use as claimed in claim 2 wherein the agent is a PARP inhibitor.

4. The use as claimed in claim 3 wherein the PARP inhibitor is selected from the group consisting of PARP-1, PARP-2, PARP-3, PARP-4, tankyrase 1 and tankyrase 2.

5. The use as claimed in claim 4 wherein the PARP is PARP-1.

6. The use as claimed in claim 1 or claim 2 wherein the agent is an RNAi molecule specific to a PARP gene.

7. The use as claimed in claim 6 wherein the RNAi molecule is derived from a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence as represented by the sequence in FIG. 9, 10, 11, 12, 13 or 14, or a fragment thereof; 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; or c) a nucleic acid sequence which comprises sequences which are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) and (b).

8. The use as claimed in claim 6 or 7 wherein the RNAi molecule comprises the nucleic acid sequence aaa agc cau ggu gga gua uga.

9. The use as claimed in claim 6 or 7 wherein the RNAi molecule consists of the nucleic acid sequence aag acc aau cuc ucc agu uca ac.

10. The use as claimed in claim 6 or 7 wherein the RNAi molecule consists of the nucleic acid sequence aag acc aac auc gag aac aac.

11. The use as claimed in any preceding claim wherein the defect is a mutation in a gene encoding a protein involved in HR.

12. The use as claimed in any of claims 1 to 10 wherein the defect is the absence of a gene encoding a protein involved in HR.

13. The use as claimed in any of claims 1 to 10 wherein the defect is in the expression of a gene encoding a protein involved in HR.

14. The use as claimed in any preceding claim wherein the gene that mediates HR is selected from the group consisting of 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, 20 RAD54, RAD50, MRE11, NBS1, WRN, BLM, Ku70, Ku80, ATM, ATR, chk1, chk2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, RADS, FEN-1, Mus81, Eme1, DDS1 and BARD.

15. The use as claimed in any preceding claim in the treatment of cancer.

16. The use as claimed in claim 15 wherein the cancer is selected from the group consisting of lung, colon, pancreatic, gastric, ovarian, cervical, breast and prostate cancer.

17. The use as claimed in claim 15 or 16 wherein the cancer is in a human.

18. The use as claimed in any of claims 15 to 17 wherein the cancer is gene-linked hereditary cancer.

19. The use as claimed in claim 18 wherein the cancer is breast cancer.

20. The use as claimed in any of claims 15 to 19 wherein the cancer cells to be treated are defective in BRCA1 expression.

21. The use as claimed in any of claims 15 to 19 wherein the cancer cells to be treated are defective in BRCA2 expression.

22. The use as claimed in claim 20 or 21 wherein the cancer cells are partially deficient in BRCA1 and/or BRCA2 expression.

23. The use as claimed in claim 20 or 21 wherein the cancer cells are totally deficient in BRCA1 and/or BRCA2 expression.

24. The use as claimed in any preceding claim wherein the gene that mediates HR is a tumour suppressor gene.

25. The use as claimed in claim 24 wherein the tumour suppressor gene is BRCA1.

26. The use as claimed in claim 24 wherein the tumour suppressor gene is BRCA2.

27. Use of a PARP inhibitor in the manufacture of a medicament for inducing apoptosis in HR. defective cells.

28. The use as claimed in claim 27 wherein the HR. defective cells are cancer cells.

29. The use as claimed in claim 28 wherein the cancer cells defective in HR are partially deficient in HR.

30. The use as claimed in claim 28 wherein the cancer cells defective in HR are totally deficient in HR.

31. A method of treatment of a disease or condition in a mammal, including human, which is caused by a genetic defect in a gene that mediates homologous recombination, which method comprises administering to the mammal a therapeutically effective amount of an agent that inhibits the activity of an enzyme that mediates repair of DNA strand breaks.

32. 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.

Description

[0051] 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.

[0052] 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;

[0053] 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;

[0054] FIG. 3 is a histogram showing the percentage of the cells in apoptosis following a 72 hour incubation with NU1025;

[0055] FIG. 4A-4C. (FIG. 4A) Western blot analysis of protein lysates isolated from MCF-7 (p53′) or MDA-MB-231 (p53′) 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.

[0056] 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 μM). 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.

[0057] 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.

[0058] 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 μM). (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.

[0059] 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.

[0060] FIG. 9A-9B is the human cDNA sequence of PARP-1;

[0061] FIG. 10 is the human cDNA sequence of PARP-2;

[0062] FIG. 11 is the human cDNA sequnce of PARP-3;

[0063] FIG. 12A-12B is the human gDNA sequnce of Tankyrase 1;

[0064] FIG. 13A-13C is the human mRNA sequnce of Tankyrase 2;

[0065] FIG. 14A-14B is the human mRNA sequnce of VPARP.

MATERIALS AND METHODS

[0066] Cytotoxicity of PARP Inhibitors to HR-Defective Cells: XRCC2, XRCC3 or BRCA2

[0067] Cell Culture

[0068] 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].

[0069] 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 μ.Math.g/mL) at 37° C. under an atmosphere containing 5% CO2.

[0070] Toxicity Assay—Colony Outgrowth Assay

[0071] 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.

[0072] Apoptosis Experiments

[0073] 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.

[0074] Immunofluorescence

[0075] 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° 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′)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 μg/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 μm apart and with a section thickness of 1.0 μm. 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.

[0076] PARP Activity Assays

[0077] 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 μl normal growth media for 4 h at 37° C.K8 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 (OD450) 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.

[0078] 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 μl of NU1025-treated (15 min) permeabilised cells were incubated at 26° C. with oligonucleotide (final conc. 2.5 μg/ml), 75imMNAD+[.sup.32P] NAD (Amersham Pharmacia, Amersham, UK) in a total volume of 400 μl. 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.

[0079] Pulse-Field Gel Electrophoresis

[0080] 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° angle, 60 to 240 s switch time, 4 V/cm). The gel was subsequently stained with ethidium bromide for analysis.

[0081] siRNA Treatment

[0082] 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

[0083] Homologous Recombination Deficient Cells are Hypersensitive to PARP-1 Inhibition

[0084] 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).

[0085] BRCA2 Deficient Cells are Hypersensitive to PARP-1 Inhibition

[0086] 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.

[0087] 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).

[0088] BRCA2 Deficient Breast Cancer Cells are Hypersensitive to PARP-1 Inhibition

[0089] 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.

[0090] BRCA2 Deficient Cells Die from PARP-1 Inhibition in Absence of DNA Double-Strand Breaks (DSBs) but in Presence of yH2Ax

[0091] 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.

[0092] PARP-1 and not PARP-2 is Important in Preventing Formation of a Recombinogenic Lesion

[0093] 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.

[0094] PARP-1 is Activated by Replication Inhibitors

[0095] 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.

[0096] 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.

[0097] PARP-1 and HR have Separate Roles at Stalled Replication Forks

[0098] 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).

[0099] 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.

[0100] 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 AA8 [41] in HR CXR3 XRCC3.sup.− + Wt irs1SF [41] hXRCC3 V79-4 Wt Wt V79 [40] irs1 XRCC2.sup.− XRCC2.sup.−, deficient V79-4 [40] in HR irs1X2.2 XRCC2.sup.− + Wt irs1 [40] hXRCC2 V79-Z Wt Wt V79 [42] VC8 BRCA2.sup.− BRCA2.sup.−, deficient V79-Z [42] in HR VC8#13 BRCA2.sup.− + Wt VC8 [42] hBRCA2 VC8 + B2 BRCA2.sup.− + Wt VC8 [42] hBRCA2

REFERENCES

[0101] [1] A. R. Venkitaraman Cancer susceptibility and the functions of BRCA1 and BRCA2, Cell 108 (2002) 171-182. [0102] [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. [0103] [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. [0104] [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. [0105] [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. [0106] [6] M. S. Satoh and T. Lindahl Role of poly(ADP-ribose) formation in DNA repair, Nature 356 (1992) 356-358. [0107] [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. [0108] [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. [0109] [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. [0110] [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. [0111] [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. [0112] [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. [0113] [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. [0114] [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. [0115] [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. [0116] [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. [0117] [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. [0118] [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. [0119] [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. [0120] [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. [0121] [21] L. Tentori, I. Portarena and G. Graziani Potential clinical applications of poly(ADP-ribose) polymerase (PARP) inhibitors, Pharmacol Res 45 (2002) 73-85. [0122] [22] T. Lindahl and R. D. Wood Quality control by DNA repair, Science 286 (1999) 1897-1905. [0123] [23] K. W. Caldecott DNA single-strand break repair and spinocerebellar ataxia, Cell 112 (2003) 7-10. [0124] [24] D. D'Amours and S. P. Jackson The Mrell complex: at the crossroads of dna repair and checkpoint signalling, Nat Rev Mol Cell Biol 3 (2002) 317-327. [0125] [25] A. D. D'Andrea and M. Grompe The Fanconi anaemia/BRCA pathway, Nat Rev Cancer 3 (2003) 23-34. [0126] [26] S. P. Jackson Sensing and repairing DNA double-strand breaks, Carcinogenesis 23 (2002) 687-696. [0127] [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. [0128] [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. [0129] [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. [0130] [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. [0131] [31] A. Chiarugi Poly(ADP-ribose) polymerase: killer or conspirator? The ‘suicide hypothesis’ revisited, Trends Pharmacol Sci 23 (2002) 122-129. [0132] [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. [0133] [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. [0134] [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. [0135] [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. [0136] [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. [0137] [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. [0138] [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. [0139] [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. [0140] [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. [0141] [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. [0142] [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. [0143] [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. [0144] [44] H. Halldorsson, D. A. Gray and S. Shall Poly (ADP-ribose) polymerase activity in nucleotide permeable cells, FEBS Lett 85 (1978) 349-352. [0145] [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. [0146] [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. [0147] [47] Schreider et al., Journal of Biological Chemistry 277: 23028-23036 (2002).