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.     

聚腺苷二磷酸-核糖聚合酶抑制剂研究进展

肿瘤基因的不稳定性使其更容易产生并积累DNA损伤,但同时也会导致肿瘤DNA损伤修复功能发生部分丢失,使其更依赖于尚存的DNA修复路径,充分修复放化疗所致的DNA损伤,导致放化疗抵抗。聚腺苷二磷酸-核糖聚合酶[poly-（ADP-ribose）polymerase,PARP]抑制剂可以在同源重组修复缺陷（homologous recombination deficiency,HRD）肿瘤细胞中充分修复DNA损伤,产生协同细胞杀伤的作用。目前,多种PARP抑制剂（PARP inhibitor,PARPi）通过美国食品药品监督管理局（FDA）审批用于晚期卵巢癌患者,多项临床试验也正在评估PARPi单药或联合放化疗是否可以使更多患者获益,以及毒性是否可以耐受,研究对象也从卵巢癌扩大到乳腺癌、前列腺癌、直肠癌、肺癌、胰腺癌、腹膜肿瘤、头颈部肿瘤、脑瘤、鳞状细胞癌及肉瘤等。本文对已应用于临床的PARPi研究情况及面临的问题进行综述。      

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.     

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.     

Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro.

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these two enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.     

PARP抑制剂的临床研究进展

肿瘤细胞对化疗药物耐药是导致化疗失败的重要原因。肿瘤细胞DNA修复基因的过表达引起DNA损伤后修复并最终导致了耐药性的产生。聚腺苷酸二磷酸核糖基聚合酶（PARP）是具有碱基切除修复功能的DNA单链损伤修复酶,由于其在肿瘤细胞内过表达并在肿瘤细胞DNA损伤修复过程中发挥着重要作用,被认为是肿瘤靶向治疗领域的重要靶点。近年来关于PARP抑制剂的研究屡见报道,发现PARP抑制剂不仅对放化疗具有一定的增敏效果,且在特定基因型瘤种中还具有显著的单独抗肿瘤效应。本文旨在对近年来PARP抑制剂的最新临床研究进展进行综述。     

Absence of stimulation of poly(ADP-ribose) polymerase activity in patients predisposed to colon cancer.

Poly(ADP-ribose)polymerase (PARP) has been implicated in DNA repair mechanisms and the associated activity shown to markedly increase after DNA damage in carcinogen-treated cells. A defective DNA repair has been associated to the aetiology of human cancers. In order to assess the potential role of this enzyme in cellular response to DNA damage by gamma-radiation, we studied the activity of PARP in patients with familial adenomatous polyposis (FAP). We compared poly(ADP-ribose)polymerase activity by the rate of incorporation of radioactivity from [3H]adenine-NAD+ into acid-insoluble material in permeabilized leucocytes from FAP patients and healthy volunteers. Concomitantly, the intracellular levels of NAD+--the substrate for the PARP--and the reduced counterpart NADH were determined using an enzymatic cycling assay 30 min after [60Co] gamma-ray cells irradiation. Our results demonstrate that a marked stimulation of PARP activity is produced upon radiation of the cells from healthy subjects but not in the FAP leucocytes, which concomitantly show a marked decrease in total NAD-/NADH content. Our observations point to a role of PARP in the repair of the gamma-radiation-induced DNA lesions through a mechanism that is impaired in the cells from FAP patients genetically predisposed to colon cancer. The differences observed in PARP activation by gamma-radiation in patients and healthy individuals could reflect the importance of PARP activity dependent on treatment with gamma-rays. The absence of this response in FAP patients would seem to suggest a possible defect in the role of PARP in radiation-induced DNA repair in this cancer-prone disease.     

Stochastic modelling of tumorigenesis in p53 deficient mice.

Stochastic models of tumorigenesis have been developed to investigate the implications of experimental data on tumour induction in wild-type and p53-deficient mice for tumorigenesis mechanisms. Conventional multistage models in which inactivation of each p53 allele represents a distinct stage predict excessively large numbers of tumours in p53-deficient genotypes, allowing this category of model to be rejected. Multistage multipath models, in which a p53-mediated pathway co-exists with one or more p53-independent pathways, are consistent with the data, although these models require unknown pathways and do not enable age-specific curves of tumour appearance to be computed. An alternative model that fits the data is the ''multigate'' model in which tumorigenesis results from a small number of gate-pass (enabling) events independently of p53 status. The role of p53 inactivation is as a rate modifier that accelerates the gate-pass events. This model implies that wild-type p53 acts as a ''caretaker'' to maintain genetic uniformity in cell populations, and that p53 inactivation increases the probability of occurrence of a viable cellular mutant by a factor of about ten. The multigate model predicts a relationship between the time pattern of tumour occurrence and tumour genotype that should be experimentally testable. Stochastic modelling may help to distinguish ''gatekeeper'' and ''caretaker'' genes in other tumorigenic pathays.     

Inhibition of poly(ADP-ribose) polymerase increases ({+/-})-anti-benzo[a]pyrene diolepoxide-induced micronuclei formation and p53 accumulation in isolated human peripheral blood lymphocytes

In response to DNA damage, in particular DNA strand breaks,the proposed roles for normal tumour suppressor protein p53are to increase the period of time available for DNA repairprior to replication, or to direct damaged cells into programmedcell-death. Since treatment of mammalian cells with (±)-anti-benzo[a]pyrenediolepoxide [(±)-anti-BPDE]—a mixture of metabolitescomprising the most reactive (+ )-anti-enantiomer of the fullenvironmental carcinogen benzo[a]pyrene—has been shownto result in induction of DNA repair processes and consequentlyin DNA strand break formation, the aim of the present studywas to investigate whether p53 accumulation is induced in (±)-anti-BPDE-treatedphytohaemagglutinin-stimulated human peripheral blood lymphocytes(PBLs). Both immunocytochemical and immunoblot analysis indicatedthat treatment of PBLs with (±)-anti-BPDE results inp53 accumulation. Optimal accumulation was observed at 2.5 uM,while no increase of p53 levels was observed at concentrations<2.5 µM and >10 µM. Further, (±)-anti-BPDE-inducedp53 accumulation in PBLs was found to be time-dependent withaccumulation up to 24 h after the onset of treatment. Treatmentof PBLs with 2.5 µM of (±)-anti-BPDE and 1 mM of3-aminobenzamide, an inhibitor of the DNA strand break-dependentenzyme poly(ADP-ribose) polymerase, resulted in increased p53levels, in comparison to cells treated with (±)-anti-BPDEalone. This combination also potentiated the frequency of (±)-anti-BPDE-inducedmicronuclei. These findings suggest that (±)-anti-BPDE-inducedDNA strand break formation is responsible for the observed p53accumulation. It is unlikely that poly(ADP-ribose) polymer formationis a prerequisite in the process of p53 accumulation, as triggeredby DNA strand-break inducing agents like (±)-anti-BPDE.It is hypothesized that p53-dependent pathways may be activatedin phytohaemagglutinin-stimulated human peripheral blood lymphocytesexposed ex vivo to (±)-anti-BPDE.     

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 enhances cell death and improves tumor growth delay in irradiated lung cancer models.

PURPOSE: Poly(ADP-ribose) polymerase-1 (PARP-1) is the founding member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Ionizing radiation induces DNA strand breaks, suggesting that PARP-1 inhibition may sensitize tumor cells to radiation. EXPERIMENTAL DESIGN: We investigated the combination of PARP-1 inhibition with radiation in lung cancer models. ABT-888, a novel potent PARP-1 inhibitor, was used to explore the effects of PARP-1 inhibition on irradiated tumors and tumor vasculature. RESULTS: ABT-888 reduced clonogenic survival in H460 lung cancer cells, and inhibited DNA repair as shown by enhanced expression of DNA strand break marker histone gamma-H2AX. Both apoptosis and autophagy contributed to the mechanism of increased cell death. Additionally, ABT-888 increased tumor growth delay at well-tolerated doses in murine models. For a 5-fold increase in tumor volume, tumor growth delay was 1 day for ABT-888 alone, 7 days for radiation alone, and 13.5 days for combination treatment. Immunohistochemical staining of tumor sections revealed an increase in terminal deoxyribonucleotide transferase-mediated nick-end labeling apoptotic staining, and a decrease in Ki-67 proliferative staining after combination treatment. Matrigel assay showed a decrease in in vitro endothelial tubule formation with ABT-888/radiation combination treatment, and von Willebrand factor staining of tumor sections revealed decreased vessel formation in vivo, suggesting that this strategy may also target tumor angiogenesis. CONCLUSIONS: We conclude that PARP-1 inhibition shows promise as an effective means of enhancing tumor sensitivity to radiation, and future clinical studies are needed to determine the potential of ABT-888 as a radiation enhancer.     

Genetic ablation of protein tyrosine phosphatase 1B accelerates lymphomagenesis of p53-null mice through the regulation of B-cell development

Protein tyrosine phosphatase 1B (PTP1B) is involved in multiple signaling pathways by down-regulating several tyrosine kinases. For example, gene-targeting studies in mice have established PTP1B as a critical physiologic regulator of metabolism by attenuating insulin signaling. PTP1B is an important target for the treatment of diabetes, because the PTP1B null mice are resistant to diet-induced diabetes and obesity. On the other hand, despite the potential for enhanced oncogenic signaling in the absence of PTP1B, PTP1B null mice do not develop spontaneous tumors. Because the majority of human cancers harbor mutations in p53, we generated p53/PTP1B double null mice to elucidate the role of PTP1B in tumorigenesis. We show that genetic ablation of PTP1B in p53 null mice decreases survival rate and increases susceptibility towards the development of B lymphomas. This suggested a role for PTP1B in lymphopoiesis, and we report that PTP1B null mice have an accumulation of B cells in bone marrow and lymph nodes, which contributed to the increased incidence of B lymphomas. The mean time of tumor development and tumor spectrum are unchanged in p53-/-PTP1B+/- mice. We conclude that PTP1B is an important determinant of the latency and type of tumors in a p53-deficient background through its role in the regulation of B-cell development.     

Deletion in poly(ADP-ribose)polymerase pseudogene and lung cancer risk

The poly(ADP-ribose)polymerase (PADPRP) gene has been implicated in carcinogenesis through its role in DNA repair, replication and recombination. A two-allele polymorphism in the chromosome 13 PADPRP pseudogene has been studied in several racial groups. It has been suggested that the B allele, which results from a 193-bp deletion in the gene, predisposes to myeloma in Blacks. We assessed the association between chromosome 13 PADPRP pseudogene genotype, mutagen sensitivity (a marker reflecting host DNA repair capability), cigarette smoking, and lung cancer risk in a minority lung cancer case-control study. The chromosome 13 PADPRP pseudogene polymorphism was detected by polymerase chain reaction-based analysis. Mutagen sensitivity was measured by an in vitro assay that quantified bleomycin-induced chromatid breaks in peripheral blood lymphocyte cultures. We examined 121 cases (80 African- Americans and 41 Mexican-Americans) with previously untreated lung cancer and 171 matched controls. Our results suggested that the distribution of the PADPRP pseudogene genotype frequencies was significantly different among African-American and Mexican-American controls (P < 0.001). The susceptibility genotype (i.e. at least one B allele) was found in 82.5% of African-American cases, 79.4% of African- American controls, 53.7% of Mexican-American cases, and 32.4% of Mexican-American controls. The odds ratios (OR) and 95% confidence intervals for the PADPRP susceptibility genotypes were 2.3 (95% CI = 0.7-8.0) and 3.2 (95% CI = 1.0-10.3) for African-Americans and Mexican- Americans respectively, after adjustment by age, sex, pack-years and mutagen sensitivity. Patients with the susceptibility genotype appeared to have more mutagen-induced breaks than did patients with the other genotype. Only adenocarcinoma was significantly associated with the PADPRP susceptibility genotype (OR = 3.8). Mutagen sensitivity (> or = 1 break/cell) was significantly associated with lung cancer risk for both ethnic groups with increased ORs of above three-fold. On stratified analysis, synergistic interactions were noted for the PADPRP susceptibility genotype, mutagen sensitivity and smoking status. In Mexican-Americans, the ORs for PADPRP susceptibility genotype, mutagen sensitivity and both risk factors combined were 1.3, 2.7 and 17.1 respectively. The combined OR for the PADPRP susceptibility genotype and smoking status was 15.6. Therefore, this polymorphism appears to be associated with lung cancer risk. However, it is likely that no single genotype is sufficiently predictive of risk and that a panel of susceptibility markers is needed to define the high-risk subgroup.      

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.     

Poly(ADP-ribose) polymerase activity is not affected in ataxia telangiectasia cells and knockout mice

Poly(ADP-ribose) polymerase (PARP) is a constitutive factorof the DNA damage surveillance network in dividing cells. Basedon its capacity to bind to DNA strand breaks, PARP plays a regulatoryrole in their resolution in vivo. ATM belongs to a large familyof proteins involved in cell cycle progression and checkpointsin response to DNA damage. Both proteins may act as sensorsof DNA damage to induce multiple signalling pathways leadingto activation of cell cycle checkpoints and DNA repair. To determinea possible relationship between PARP and ATM, we examined thePARP response in an ATM-null background. We demonstrated thatATM deficiency does not affect PARP activity in human cell linesor Atm-deficient mouse tissues, nor does it alter PARP activityinduced by oxidative damage or -irradiation. Our results supporta model in which PARP and ATM could be involved in distinctpathways, both effectors transducing the damage signal to cellcycle regulators.     

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.     

Some protease inhibitors are also inhibitors of poly(ADP-ribose) polymerase

The low-molecular-weight peptide protease inhibitors, tosyllysine-chloromethylketone, antipain and leupeptin, inhibited poly(ADP-ribose) [poly(ADP-Rib)]polymerase in permeable cells. The concentrations required for50% inhibition were 3.6, 5 and 29 mM, respectively. Two peptideswithout protease inhibitor activity, fibrinopeptide A and phenylalanineleucine-(glutamine)₂-leucine,also inhibited poly(ADP-Rib) synthesis; doses required for 50%inhibition were 0.37 and 11.2 mM, respectively. These concentrationslie within a range bracketed by the 50% inhibition concentrationsof the strong and weak poly(ADP-Rib) synthesis inhibitors, 3-aminobenzamide(0.15 mM) and caffeine (> 100 mM), respectively. N-Ethylmaleimidealso inhibited poly(ADP-Rib) synthesis, at a 50% inhibitorydose of 0.3 mM, in the absence of exogenous thiol reagents.High-molecular-weight protease inhibitors, such as soybean (includingBowman-Birk reagent) and lima bean trypsin inhibitors and human₁-protease inhibitor, had no effect on poly(ADP-Rib) synthesisup to 2 mg/ml. Interference with transformation and other cellulareffects that have been reported in carcinogen-damaged cellstreated with low-molecular-weight peptide protease inhibitorsmay therefore involve common mechanisms with poly(ADP-Rib) inhibitors.Similar effects of high-molecular-weight protease inhibitorspresumably involve different mechanisms.