Targeting poly(ADP-ribose) polymerase: a two-armed strategy for cancer therapy.

The DNA repair pathways are protective of the host genome in normal cells; however, in cancer cells, these pathways may be disrupted and predispose to tumorigenesis or their activity may overcome the potentially cytotoxic damage caused by anticancer agents and be a mechanism of resistance. Poly(ADP-ribose) polymerase inhibitors, which block base excision repair of single-strand breaks, have entered the clinic in the last few years. This article discusses the interactions between the pathways of single- and double-strand break repair, which explain the two clinical development strategies for this class of drugs.     

Enhancement of chemotherapeutically-induced apoptosis in vivo by biochemical modulation of poly (ADP-ribose) polymerase

An inhibitor of poly (ADP-ribose) polymerase, 1,5-dihydroxyisoquinoline (DHIQ), evaluated in vivo against a murine advanced breast cancer, significantly improved by 20% the PR rate of tumor-regressing chemotherapy. A detailed sequential biochemical cascade is proposed for chemotherapy-induced apoptosis, and the rationale for the utilization of the inhibitor is explained.     

Correlation of poly(ADP-ribose)polymerase and p53 expression levels in high-grade lymphomas.

The steady-state levels of mRNA for the poly(ADP-ribose)polymerase (PARP), c-myc, p53, and histone H3 genes were investigated in 31 high-grade B-cell lymphomas by northern blot analysis. The panel included 15 nodal large B-cell lymphomas, nine mediastinal large B-cell lymphomas, and seven sporadic Burkitt's lymphomas. The PARP mRNA level was significantly higher in lymphomas than in control tissues and corresponded with the amount of PARP protein, as assessed by immunoblot analysis in six samples. The level of PARP mRNA was positively correlated with that of p53 mRNA. No correlation was found between the mRNA expression levels of PARP and histone H3, suggesting that PARP expression levels are independent of the proliferation rate of neoplastic cells. In this setting, the strong correlation between PARP and p53 suggests that the high expression of PARP may be associated with ongoing DNA damage in high-grade lymphomas.     

MRE11 deficiency increases sensitivity to poly(ADP-ribose) polymerase inhibition in microsatellite unstable colorectal cancers

Microsatellite instability (MSI) is displayed by approximately 15% of colorectal cancers (CRC). Defective DNA mismatch repair generates mutations at repetitive DNA sequences such as those located in the double strand break (DSB) repair gene MRE11. We assessed the mutational status of MRE11 in a panel of 17 CRC cell lines and 46 primary tumors and found a strong correlation with MSI status in both cell lines and tumors. Therefore, we hypothesized that deficiency in MRE11 may sensitize CRC cells to poly(ADP-ribose) polymerase (PARP-1) inhibition based on the concept of synthetic lethality. We further assessed the activity of the PARP-1 inhibitor, ABT-888, in CRC cell lines and observed preferential cytotoxicity in those MSI cell lines harboring mutations in MRE11 compared with both wild-type cell lines and microsatellite stable (MSS) cell lines. A significant correlation between MRE11 expression levels and cytotoxicity to ABT-888 at 10 μM was observed (R2 = 0.915, P < 0.001). Using two experimental approaches, including short hairpin RNA knocking down MRE11 in the wild-type and MSS cell line SW-480 and a second cell line model transfected with mutant MRE11, we experimentally tried to confirm the role of MRE11 in conferring sensitivity to PARP-1 inhibition. Both models led to changes in proliferation in response to ABT-888 at different concentrations, and a drug-response effect was not observed, suggesting a possible contribution of additional genes. We conclude that MSI colorectal tumors deficient in DSB repair secondary to mutation in MRE11 show a higher sensitivity to PARP-1 inhibition. Further clinical investigation of PARP-1 inhibitors is warranted in MSI CRCs.     

Enhancement of melphalan cytotoxicity in vivo and in vitro by inhibitors of poly (ADP-ribose) polymerase

In these preliminary experiments, we have found enhanced cell killing by the bifunctional alkylating agent L-phenylalanine mustard (L-PAM) in the presence of inhibitors of poly (ADP-ribose) polymerase (ADPRP) in vitro. In vivo enhancement of the tumoricidal effects of L-PAM was observed with the ADPRP inhibitor nicotinamide (1000 mg/kg), although enhanced myelosuppression was also demonstrated. Nicotinamide also increased the plasma elimination half-life of L-PAM by a factor of at least 2. This alteration of L-PAm pharmacokinetics makes it difficult to assess the role that ADPRP inhibition plays in the enhancement of L-PAM tumor cell killing in vivo.     

The potential for poly (ADP-ribose) polymerase inhibitors in cancer therapy

The modulation of DNA repair pathways for therapeutic benefit in cancer has now become a reality with the development of poly (ADP-ribose) polymerase inhibitors (PARPi). PARP is involved in single-strand DNA breaks, which in the presence of defective homologous recombination repair lead to double-strand DNA breaks, the most lethal form of DNA damage. These agents therefore may be the drugs of choice for BRCA mutant breast and ovarian cancers. PARPi result in synergistic antitumor effects when combined with cisplatin, temozolomide, topoisomerase inhibitors and ionizing radiation. The indications for PARPi lie beyond BRCA mutations and may include genomic and functional defects in DNA repair and damage response pathways. Several PARPi are in the clinical development phase at this time and, given the recent failure of a phase III clinical trial of iniparib in triple-negative breast cancer, the identification of structural and functional differences between these inhibitors becomes critical. Acquired resistance to PARPi is being noted and represents an important limitation in this field. A concise review of the literature in this field is presented.     

Enhancement of bleomycin activity by 3-aminobenzamide, a poly (ADP-ribose) synthesis inhibitor, in vitro and in vivo

A series of poly (ADP-ribose) synthesis inhibitors was evaluated for their ability to potentiate the cytotoxicity of bleomycin compared with their inhibition of poly(ADP-ribose) synthesis in L1210 cultured cells. Theophylline, nicotinamide, 3-aminobenzamide and thymidine inhibited 70 to 80% of poly(ADP-ribose) synthesis in L1210 cells. The degree of inhibition of poly(ADP-ribose) synthesis by these inhibitors corresponded in general with their potentiating ability of bleomycin cytotoxicity. Among these inhibitors, 3-aminobenzamide significantly potentiated the cytotoxic activity of bleomycin (3.6 fold), but it could not potentiate the cytotoxic activity of other antitumor agents, including nitrosourea and Cis-DDP, in L1210 cells in vitro. Treatment of Ehrlich ascites tumor bearing mice with bleomycin and 3-aminobenzamide produced a synergistic effect. 6-Aminonicotinamide also potentiated the antitumor activity of bleomycin against Ehrlich ascites tumor.     

Systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase-1 inhibitor, increases the antitumor activity of temozolomide against intracranial melanoma, glioma, lymphoma.

PURPOSE: Temozolomide (TMZ) is a DNA methylating agent that has shown promising antitumor activity in recent clinical trials against high grade gliomas, metastatic melanoma, and brain lymphoma. In this study, we tested whether systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase (PARP-1) inhibitor capable of crossing the blood-brain barrier, could enhance the efficacy of TMZ against metastatic melanoma, glioblastoma multiforme, and lymphoma growing in the brain. EXPERIMENTAL DESIGN: Murine B16 melanoma or L5178Y lymphoma cells were injected intracranially in syngeneic mice. An orthotopic xenograft of the human SJGBM2 glioblastoma multiforme was implanted in nude mice. Animals were treated with TMZ + GPI 15427 using a schedule of 40 mg/kg/i.v. GPI 15427 + 100 mg/kg/i.p. TMZ for 3 days. The efficacy of drug treatment was assessed by: (a) the increase of mouse survival and life span; and (b) the suppression of melanoma metastases to lung after i.v. injection of B16 cells. RESULTS: In all models, systemic administration of GPI 15427 shortly before TMZ significantly increased life span of tumor-bearing mice with respect to untreated controls or to groups treated with either GPI 15427 or TMZ only. Moreover, GPI 15427 increased the antimetastatic effect of TMZ. CONCLUSIONS: These data indicate that systemic administration of the poly(ADP-ribose) polymerase-1 inhibitor GPI 15427 significantly enhances TMZ antitumor efficacy against solid or hematological neoplasias even when located at the central nervous system site.     

Pediatric high-grade glioma: identification of poly(ADP-ribose) polymerase as a potential therapeutic target

Pediatric high-grade gliomas (World Health Organization grades III and IV astrocytomas) remain tumors with a very poor prognosis for which novel therapeutic strategies are needed. Poly(ADP-ribose) polymerase (PARP) is known to have multiple functions in tumors, including single-strand DNA repair and induction of caspase-independent apoptosis. PARP has been suggested as a therapeutic target in adult malignancies, and this study examines whether it could also be a potential target in pediatric high-grade glioma. Tissue microarrays containing 150 formalin-fixed pediatric high-grade gliomas were examined by immunohistochemistry for levels of PARP and expression of apoptosis inducing factor (AIF). Full retrospective clinical and survival data were available for this cohort. Stratification and statistical analysis was performed to assess the effect of PARP status on prognosis. The level of PARP immunopositivity had a statistically significant inverse correlation (P = .019) with survival in supratentorial pediatric high-grade glioma. AIF staining was notable for its absence in the majority of tumors but with moderate levels of expression in surrounding normal brain. PARP is expressed at high levels in many pediatric high-grade gliomas, and in these tumors, the ability of PARP to activate AIF appears to have been lost. PARP may therefore represent a promising therapeutic target for these lesions and warrants evaluation in clinical trials.     

High induction of poly(ADP-ribose) polymerase activity in bleomycin-resistant HeLa cells

Poly(ADP-ribose) polymerase activity was measured in bleomycin (BLM)-resistant HeLa (HeLa-BLMr) and the parental HeLa cells after BLM treatment. HeLa-BLMr cells, which had been subcultured in growth medium containing 1 micrograms/ml of BLM, showed a 3.75-fold higher enzyme activity than did HeLa cells, but this activity was decreased to the same level as that of HeLa cells after 48 h of BLM-free cultivation. When HeLa and HeLa-BLMr cells after a 48-h cultivation in BLM-free growth medium were treated with BLM, the enzyme activity was induced at a higher level (2.5-3.8 times) in HeLa-BLMr than in HeLa cells and was inhibited markedly in HeLa-BLMr and slightly in HeLa cells by nicotinamide, an inhibitor of this enzyme. The BLM-induced cell killing by nicotinamide was highly potentiated (about 18 times) in HeLa-BLMr as compared to HeLa cells.     

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers

Background

DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.

Methods

Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.

Results

Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.

Conclusions

It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.     

Current development of clinical inhibitors of poly(ADP-ribose) polymerase in oncology.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that signals the presence of DNA damage by catalyzing the addition of ADP-ribose units to DNA, histones, and various DNA repair enzymes and by facilitating DNA repair. PARP has been gaining increasing interest as a therapeutic target for many diseases and especially for cancer. Inhibition of PARP potentiates the activity of DNA-damaging agents, such as alkylators, platinums, topoisomerase inhibitors, and radiation in in vitro and in vivo models. In addition, tumors with DNA repair defects, such as those arising from patients with BRCA mutations, may be more sensitive to PARP inhibition. At least five different companies have now initiated oncology clinical trials with PARP inhibitors, ranging in stage from phase 0 to phase 2. This review summarizes the preclinical and clinical data currently available for these agents and some of the challenges facing the clinical development of these agents.     

Inhibition of poly(ADP-ribose) polymerase (PARP) induces apoptosis in lung cancer cell lines

We have tested PJ34, a potent inhibitor of poly(ADP-ribose) polymerase (PARP), against various lung cancer cell lines (Calu-6, A549, and H460) and normal human bronchial epithelial cells (HBECs). While using WST1 dye assay, lung cancer cells exhibited LD(50) values of approximately 30 μM PJ34 (72-hr assay). Molecular data showed that the effect of PJ34-induced apoptosis on lung cancer cells occurs via a caspase-dependent pathway. The present study has clearly shown that (a) PARP inhibitor can independently kill tumor cells, (b) caspase-3 has modest influence on PARP-inhibitor-mediated cancer-specific toxicity, and (c) a pan-caspase inhibitor decreases the apoptotic effect of PJ34.     

Functional overexpression of human poly(ADP-ribose) polymerase in transfected rat tumor cells

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a nuclear enzyme possibly involved in DNA base excision repair. The presence of single- or double-strand breaks in DNA stimulates this enzyme to covalently modify acceptor proteins with poly(ADP-ribose) in a reaction that uses NAD+ as substrate. To test the hypothesis that increased PARP activity could promote resistance towards DNA-damaging agents and gamma- radiation, we established stable rat cell transfectants that constitutively express human PARP. A number of subclones that showed different levels of PARP activity were isolated from two primary transfectants of different clonal origin. PARP activity was determined in permeabilized cells after maximal stimulation with a short, double- stranded oligonucleotide. Activity in different human PARP-expressing subclones was increased 1.6- to 3.1-fold compared with non-expressing subclones. In vivo labeling of poly(ADP-ribose) was performed in one of these subclones, revealing that the level of poly(ADP-ribose) accumulation after the same treatment with N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) was four times higher in the human PARP- expressing subclone compared with both non-expressing transfected control cells and parental cells. Clonal survival assays revealed a sensitization upon treatment with gamma-radiation (up to 1.4-fold) or MNNG (up to 2.7-fold) of several subclones expressing human PARP; in some others survival was not changed. Survival after cisplatin (DDP) treatment remained essentially unchanged. A protective effect against DNA-damage was never observed. We conclude that human PARP overexpression in rodent cells leads to increased poly(ADP- ribosyl)ation capacity and does not promote survival after gamma- radiation or treatment with the DNA-damaging agents MNNG or DDP.      

Genetic alteration of poly(ADP-ribose) polymerase-1 in human germ cell tumors

Accumulated evidence suggests that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, cell-death induction, differentiation and tumorigenesis. Parp-1 deficiency also induces trophoblast differentiation from mouse embryonic stem cells during teratocarcinoma-like tumor formation. To understand the relationship of PARP-1 dysfunction and development of germ cell tumors, we conducted a genetic analysis of the PARP-1 gene in human germ cell tumors. Sixteen surgical specimens of germ cell tumors that developed in the brain and testes were used. Two known single nucleotide polymorphisms (SNPs) (Val762Ala and Lys940Arg), which are listed in the SNP database of the NCBI (National Center for Biotechnology Information), were detected. In both cases, cSNPs encoded amino acids located within peptide stretches in the catalytic domain, which are highly conserved among various animal species. Furthermore, another novel sequence alteration, a base change of ATG to ACG, was identified in a tumor specimen, which would result in the amino acid substitution, Met129Thr. This base change was observed in one allele of both tumor and normal tissues, suggesting that it is either a rare SNP or a germline mutation of the PARP-1 gene. Notably, the amino acid Met129 is located within the second zinc finger domain, which is essential for DNA binding and is conserved among animal species. One SNP in intron 2 and one in the upstream 5'-UTR (untranslated region) were also detected.     

Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells

Despite recent advances in the clinical evaluation of various poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer (TNBC) patients, data defining potential anti-tumor mechanisms beyond PARP inhibition for these agents are lacking. To address this issue, we investigated the effects of four different PARP inhibitors (AG-014699, AZD-2281, ABT-888, and BSI-201) in three genetically distinct TNBC cell lines (MDA-MB-468, MDA-MB-231, and Cal-51). Assays of cell viability and colony formation and flow cytometric analysis were used to determine effects on cell growth and cell cycle progression. PARP-dependent and -independent signaling mechanisms of each PARP inhibitor were investigated by western blotting and shRNA approaches. Potential synergistic interactions between PARP inhibitors and cisplatin in suppressing TNBC cell viability were assessed. These PARP inhibitors exhibited differential anti-tumor activities, with the relative potencies of AG-014699 > AZD-2281 > ABT-888 > BSI-201. The higher potencies of AG-014699 and AZD-2281 were associated with their effects on G(2)/M arrest and DNA damage as manifested by γ-H2AX formation and, for AG-014699, its unique ability to suppress Stat3 phosphorylation. Abilities of individual PARP inhibitors to sensitize TNBC cells to cisplatin varied to a great extent in a cell context- and cell line-specific manner. Differential activation of signaling pathways suggests that the PARP inhibitors currently in clinical trials have different anti-tumor mechanisms beyond PARP inhibition and these PARP-independent mechanisms warrant further investigation.     

Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.