Poly(ADP-ribose) polymerase-1 and ionizing radiation: sensor, signaller and therapeutic target

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that binds rapidly to single and double-strand breaks in DNA and consequently modifies a range of nuclear proteins involved in the cellular response to ionising radiation. PARP-1 knockout mice are highly sensitive to ionising radiation, and inhibition or depletion of PARP-1 brings about modest sensitisation of cells in culture to radiation doses of 2 Gy and above. In certain cell lines, chemical inhibition of PARP activity is also associated with marked sensitisation to very low doses of radiation (<0.5 Gy). The mechanisms underlying these effects are discussed, and possible therapeutic applications of PARP-1 manipulation in combination with ionising radiation are considered.     

Preferential radiosensitization to glioblastoma cancer stem cell-like cells by a Hsp90 inhibitor,N-vinylpyrrolidone-AUY922

The present study examined the radiosensitization induced by a heat shock protein 90 inhibitor, N-vinylpyrrolidone (NVP)-AUY922, in CD133-positive cells in a hypoxic area of T98G spheroids. CD133-positive cells that are induced in the hypoxic microenvironment of spheroids have previously been reported to exhibit cancer stem cell-like properties. The present study used CD133-positive cells from a glioblastoma cell line (T98G) as cancer stem cell-like cells. CD133-positive and negative cells were sorted from T98G spheroids using fluorescence-activated cell sorting and used for colony formation assay. Colony formation assay results indicated that NVP-AUY922 enhanced radiosensitivity more strongly in CD133-positive cells compared with CD133-negative cells. This result showed that NVP-AUY922 was a preferential radiosensitization candidate targeting glioblastoma cancer stem cells. The mechanisms underlying radiosensitization by NVP-AUY922 are discussed in relation to the properties of cancer stem cells. Overall, HIF-1α inhibition by NVP-AUY922 may induce higher sensitization of cancer stem cells to radiation.     

Preferential radiosensitization of G1 checkpoint-Deficient cells by methylxanthines

To develop a checkpoint-based strategy for preferential radiosensitization of human tumors with deficient and/or mutant p53.

A 549 human lung adenocarcinoma cell lines differing in their expression of the p53 tumor suppressor gene were produced by transduction with the E6 oncogene from human papilloma virus type 16. The cells expressing E6 (E6+) lack a G1 arrest in response to ionizing radiation, are deficient in p53 and p21 expression, and exhibit a fivefold greater clonogenic survival following 10 Gy radiation.

Postirradiation incubation with millimolar concentrations of the methylxanthine pentoxifylline (PTX) results in preferential radiosensitization of the E6+ cells compared to the LXSN+ vector transduced controls. There is a threefold sensitization of the LXSN+ cells and a 15-fold sensitization of the E6+ cells, which results in equal clonogenic survival of the two lines. Flow cytometry reveals PTX abrogation of the radiation induced G2 arrest for both cell lines. PTX also prolongs G1 transit for both cell lines. Preliminary results are presented using a novel methylxanthine, lisofylline (LSF), which has similar cell cycle effects and G1 and G2 and achieves differential radiosensitization at micromolar concentrations that are sustainable in humans.

This checkpoint-based strategy is a promising approach for achieving preferential radiosensitization of p53− tumors relative p53+ normal tissues.     

Radiosensitizing potential of gemcitabine (2′,2′-difluoro-2′-deoxycytidine) within the cell cycle in vitro

Purpose: Gemcitabine (2′,2′-difluorodeoxycytidine; dFdCyd) is a new deoxycitidine analog which exhibits substantial activity against solid tumors and radiosensitizing properties in vitro. To examine cell cycle–specific effects of a combined treatment with gemcitabine and radiation, the in vitro clonogenic survival of two different cell lines was measured for cells from log-phase culture, G1 and S-phase cells.Methods and Materials: Chinese hamster (V79) and human colon carcinoma (Widr) cells were exposed to different radiation doses and for different points of time relative to gemcitabine treatment (2 h). Experiments were also carried out with different cell-cycle populations obtained after mitotic selection (V79) or after serum stimulation of plateau-phase cells (Widr). The resulting survival curves were analyzed according to the LQ model, and mean inactivation doses (MID) and the cell cycle–specific enhancement ratios (ER) were calculated from the survival curve parameters.Results: Effectiveness of combined treatment of log-phase cells was greatest when cells were irradiated at the end of the gemcitabine exposure [ER: 1.28 (V79), 1.24 (Widr)]. For later times after the removal of the drug, radiosensitization declined, approaching independent toxicity. From the time course of interactive-type damage decay half-life values of 75 min (V79) and 92 min (Widr) were derived. Gemcitabine did not radiosensitize G1 Widr cells or V79 cells from the G1/S border, but substantial radiosensitization was observed for the S-phase cell preparations [ER: 1.45 (V79-lateS), 1.57 (Widr)].Conclusions: Treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle that is manifested by radioresistance during S phase. This reversal of S-phase radioresistance could imply that gemcitabine interferes with the potentially lethal damage repair/fixation pathway. Other approaches have been taken to overcome S-phase radioresistance, such as hyperthermia or densely ionizing radiation, and combined treatments with dFdCyd could prove of value to complement such efforts.     

Radiosensitization of malignant glioma cells through overexpression of dominant-negative epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) plays an important role in neoplastic growth control of malignant gliomas. We have demonstrated that radiation activates EGFR Tyr-phosphorylation (EGFR Tyr-P) and the proliferation of surviving human carcinoma cells, a likely mechanism of accelerated cellular repopulation, a major cytoprotective response after radiation. We now investigate the importance of radiation-induced activation of EGFR on the radiosensitivity of the human malignant glioma cells U-87 MG and U-373 MG. The function of EGFR was inhibited through a genetic approach of transducing cells with an Adenovirus (Ad) vector containing dominant-negative (DN) EGFR-CD533 (Ad-EGFR-CD533) at efficiencies of 85-90%. The resulting cells are referred to as U-87-EGFR-CD533 and U-373-EGFR-CD533. After irradiation at 2 Gy, both of the cell lines exhibited a mean 3-fold increase in EGFR Tyr-P. The expression of EGFR-CD533 completely inhibited the radiation-induced activation of EGFR. In clonogenic survival assays after a single radiation exposure, the radiation dose for a survival of 37% (D37) for U-87-EGFR-CD533 cells was 1.4- to 1.5-fold lower, relative to cells transduced with AdLacZ or untransduced U-87 MG cells. This effect was amplified with repeated radiation exposures (3 x 2 Gy) yielding a D37 ratio of 1.8-2.0. In clonogenic survival studies with U-373 MG cells, the radiosensitizing effect of EGFR-CD533 was similar. Furthermore, in vivo studies with U-87 MG xenografts confirmed the effect of EGFR-CD533 on tumor radiosensitization (dose enhancement ratio, 1.8). We conclude that inhibition of EGFR function via Ad-mediated gene transfer of EGFR-CD533 results in significant radiosensitization. As underlying mechanism, we suggest the disruption of a major cytoprotective response involving EGFR and its downstream effectors, such as mitogen-activated protein kinase. The experiments demonstrate for the first time that radiosensitization of malignant glioma cells through disruption of EGFR function may be achieved by genetic therapy approaches.     

Cytoplasmic PARP-1 promotes pancreatic cancer tumorigenesis and resistance

The poly(ADP-ribose) polymerases (PARP) play important roles in repairing damaged DNA during intrinsic cell death. We recently linked PARP-1 to death receptor (DR)-activated extrinsic apoptosis, the present studies sought to elucidate the function of cytoplasmic PARP-1 in pancreatic cancer tumorigenesis and therapy. Using human normal and pancreatic cancer tissues, we analyzed the prevalence of cytoplasmic PARP-1 expression. In normal human pancreatic tissues, PARP-1 expression was present in the nucleus; however, cytoplasmic PARP-1 expression was identified in pancreatic cancers. Therefore, cytoplasmic PARP-1 mutants were generated by site-direct mutagenesis, to determine a causative effect of cytoplasmic PARP-1 on pancreatic cancer tumorigenesis and sensitivity to therapy with TRA-8, a humanized DR5 antibody. PARP-1 cytoplasmic mutants rendered TRA-8 sensitive pancreatic cancer cells, BxPc-3 and MiaPaCa-2, more resistant to TRA-8-induced apoptosis; whereas wild-type PARP-1, localizing mainly in the nucleus, had no effects. Additionally, cytoplasmic PARP-1, but not wild-type PARP-1, increased resistance of BxPc-3 cells to TRA-8 therapy in a mouse xenograft model in vivo. Inhibition of PARP enzymatic activity attenuated cytoplasmic PARP-1-mediated TRA-8 resistance. Furthermore, increased cytoplasmic PARP-1, but not wild-type PARP-1, was recruited into the TRA-8-activated death-inducing signaling complex and associated with increased and sustained activation of Src-mediated survival signals. In contrast, PARP-1 knockdown inhibited Src activation. Taken together, we have identified a novel function and mechanism underlying cytoplasmic PARP-1, distinct from nuclear PARP-1, in regulating DR5-activated apoptosis. Our studies support an innovative application of available PARP inhibitors or new cytoplasmic PARP-1 antagonists to enhance TRAIL therapy for TRAIL-resistant pancreatic cancers.     

Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy

DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA double-strand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Low-dose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNA-PKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70(S6K), a molecule downstream of the mammalian target of rapamycin associated with autophagy in M059J cells but not in M059K cells. The treatment of M059K cells with antisense oligonucleotides against DNA-PKcs caused radiation-induced autophagy and radiosensitized the cells. Furthermore, antisense oligonucleotides against DNA-PKcs radiosensitized other malignant glioma cell lines with DNA-PK activity, U373-MG and T98G, by inducing autophagy. The specific inhibition of DNA-PKcs may be promising as a new therapy to radiosensitize malignant glioma cells by inducing autophagy.     

Susceptibility and radiosensitization of human glioblastoma cells to trichostatin A, a histone deacetylase inhibitor

PURPOSE: Histone deacetylase inhibitors (HDAC-Is) show in vitro and in vivo antitumor activity in various types of cancer cells and are being studied in clinical trials. However, studies addressing the combination of HDAC-I and radiation are lacking. The purpose of this study was to assess the effect of trichostatin A (TSA), an HDAC-I, on the radiosensitivity of U373MG and U87MG (human glioblastoma) cell lines. METHODS AND MATERIALS: Intrinsic TSA toxicity was determined by measuring survival in exponentially growing cells treated with 0-200 nM TSA for 0-24 h. To assay the radiosensitizing effect of TSA, cells were exposed to 0-200 nM TSA for 18 h before irradiation, and radiation survival curves were obtained. Radiation survival of TSA-treated cells was determined by clonogenic assay. RESULTS: The human glioblastoma cells showed a dose-dependent reduction in survival and radiosensitization with TSA treatment in the range of 50-200 nM. Exposure to 200 nM TSA resulted in reduced survival of both cell lines, and survival was further reduced with time. Exposure of these cells to TSA before irradiation led to dose-dependent radiosensitization. CONCLUSIONS: These results suggest that HDAC-Is may be a useful adjunct in the treatment of glioblastoma and merit further investigation. Given the limited efficacy of standard treatments for patients afflicted with glioblastoma, the results reported here provide support for clinical trials integrating HDAC-I with radiation therapy.     

Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor,radiosensitizes human lung and breast cancer cells

The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H₂AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H₂O₂ and converted H₂O₂-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.     

不同浓度MPA对Ishikawa细胞PARP-1表达的影响

目的：研究不同浓度孕激素作用子宫内膜癌Ishikawa细胞后DNA损伤修复蛋白PARP-1的表达变化,探讨PARP-1在子宫内膜腺癌中的作用及PARP-1和孕激素相互关系。方法：免疫组化SP法和实时荧光定量PCR（Real-time PCR）法检测不同浓度的醋酸甲羟孕酮（Medroxyprogesterone acetate,MPA）干预Ish-ikawa细胞后PARP-1蛋白及PARP-1 mRNA表达的变化。结果：免疫组化显示PARP-1蛋白表达于Ish-ikawa细胞核,随着MPA浓度的增加PARP-1的表达呈下降趋势。RT-PCR检测到MPA呈浓度依赖性地抑制PARP-1 mRNA表达。结论：醋酸甲羟孕酮呈浓度依赖性地抑制PARP-1的表达,提示孕激素可能间接或直接通过抑制PARP-1而发挥治疗子宫内膜癌的作用。     

PARP-1 and PARP-2: New players in tumour development

Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 belong to a family of enzymes that, using NAD(+) as a substrate, catalyze poly(ADP-ribosyl)ation of proteins. PARP-1 and PARP-2 catalytic activity is stimulated by DNA-strand breaks targeting mainly proteins involved in chromatin structure and DNA metabolism, providing strong support for a dual role of both PARP-1 and PARP-2 in the DNA damage response as DNA damage sensors and signal transducers to downstream effectors. The DNA damage response has important consequences for genomic stability and tumour development. In order to manipulate DNA damage responses to selectively induce tumour cell death, a considerable effort is centred on defining the molecular mechanisms that allow cells to detect, respond to, and repair DNA damage. PARP inhibitors that compete with NAD+ at the highly conserved enzyme active site are arisen as new potential therapeutic strategies as chemo- and radiopotentiation and for the treatment of cancers with specific DNA repair defects as single-agent therapies. In the present review, we highlight emerging information about the redundant and specific functions of PARP-1 and PARP-2 in genome surveillance and DNA repair pathways. Understanding these roles might provide invaluable clues to design new cancer therapeutic approaches. In addition, we provide an overview of ongoing clinical trials with PARP inhibitors and the value of PARP-1 and PARP-2 expression as prognostic biomarkers in cancer.     

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.     

Combination interferon-alpha2a and 13-cis-retinoic acid enhances radiosensitization of human malignant glioma cells in vitro.

We investigated the individual and combined effects of cis-retinoic acid (CRA) and/or IFN-alpha (IFN) and/or radiation therapy (RT) against a human glioma cell line (American Type Culture Collection; U373MG) to evaluate the possible radiosensitization properties of these agents in vitro. Glioma cells were incubated for 24 h in 96-well plates (2 x 10(2) cells/well) in standard culture medium. Sets of U373 (n = 12) were exposed to CRA (3 x 10(6) microM), IFN (25 units/ml), CRA plus IFN, or standard culture medium. After an additional 24 h of incubation, the U373 cells were subjected to increasing radiation doses (up to 16 Gy). Glioma cells were harvested 92 h after irradiation, and cell survival curves were determined from [3H]thymidine incorporation data (over the last 24 h). The experiment was repeated for both the untreated control group and the combined CRA/IFN group. To verify the [3H]thymidine assays, a clonogenic assay was also performed. Single cell suspensions of U373 cells were plated out in six-well plates (n = 3). After chemical and RT treatment, colonies of 50 cells or more were counted, and cell survival curves were generated as fractions of nonirradiated controls. The amount of RT (in Gy) that would cause a 50% survival fraction (lethal dose 50 or LD50) was calculated from the survival curves by regression analysis. The following LD50s were obtained: [table: see text] The results showed that for both the [3H]thymidine incorporation assay and the clonogenic assay, the combination of IFN/CRA rendered U373 cells more susceptible to ionizing radiation than the untreated control or either single agent alone.     

Poly(ADP-ribose) polymerase (PARP) inhibition or PARP-1 gene deletion reduces angiogenesis

Poly(ADP-ribose) polymerase (PARP)-1 has recently been shown to promote tumour progression. Since angiogenesis is an essential requirement for tumour growth, we examined whether PARP inhibition/deletion might affect endothelial cell functions. To this end, the influence of PARP inhibitors on endothelial cell proliferation, migration, tube formation and angiogenesis in PARP-1 knock-out mice, using an in vivo matrigel plug assay, was investigated. The results indicated that the PARP inhibitor GPI 15427 (IC50 on endothelial PARP: 237 +/- 27 nM), at concentrations devoid of cytotoxic effects (0.5-1 microM), abrogated migration in response to vascular endothelial growth factor or placenta growth factor, hampered formation of tubule-like networks and impaired angiogenesis in vivo. The anti-angiogenic effect of the PARP inhibitor was confirmed in PARP-1 knock-out mice that displayed a defect of angiogenesis induced by growth factors. These results provide evidence for targeting PARP for anti-angiogenesis, adding novel therapeutic implications to the use of PARP inhibitors in cancer treatment.     

Is low-dose hyper-radiosensitivity a measure of G2-phase cell radiosensitivity?

Low-dose hyper-radiosensitivity describes a phenomenon by which cells die from excessive sensitivity to small single doses of ionizing radiation below approximately 20-30 cGy. This review describes experimental data that strongly imply that low-dose hyper-radiosensitivity is exclusively associated with the survival response of cells in the G2 phase of the cycle. This G2-centric concept arose when the characteristic cell survival pattern that denotes low-dose hyper-radiosensitivity was not detected in the radiation survival response of cell populations enriched in G1 or S phase cells. In contrast, an extended or exaggerated low-dose hyper-radiosensitivity response was evident from populations selected to contain only G2 phase cells by flow cytometry cell sorting. The historical data that has defined the field of low-dose hyper-radiosensitivity will be considered with respect to the radiation sensitivity of, and cell cycle checkpoints specific to, G2 phase cells. A working model of the putative mechanism of low-dose hyper-radiosensitivity will be presented within this context. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell-cycle checkpoint that is specific to cells irradiated in the G2 phase. This early G2 phase checkpoint has been demonstrated to exhibit a dose expression profile that is comparable to the cell-survival pattern that defines low-dose hyper-radiosensitivity and is therefore a likely key regulator of the phenomenon.     

Mechanism of vinorelbine-induced radiosensitization of human small cell lung cancer cells

Vinorelbine (Navelbine, KW-2307), a semisynthetic vinca alkaloid, is a potent inhibitor of mitotic microtubule polymerization. The aims of this study were to demonstrate vinorelbine-induced radiosensitization of human small cell lung cancer (SCLC) SBC-3 cells and to elucidate the mechanisms of radiosensitization. A clonogenic assay demonstrated that SBC-3 cells were sensitized to radiation by vinorelbine using different schedules combining exposure to both. The sensitizer enhancement ratios (SERs) at a cell survival level of 10% were 1.42+/-0.21 to 1.33+/-0.06, and 1.22+/-0.07 depending on schedule. Vinorelbine-induced radiosensitization did not depend on the schedule of the combined exposure. Flow cytometric analyses showed that the cells did not accumulate in the radiosensitive G(2)/M phase of the cell cycle after concurrent treatment with vinorelbine and radiation. The results of an alkaline filter elution assay demonstrated that in the presence of vinorelbine at 1 n M radiation-induced DNA strand breaks were not completely repaired at 24 h postradiation. We conclude that human SCLC SBC-3 cells are sensitized to radiation by vinorelbine and that a possible mechanisms of vinorelbine-induced radiosensitization may at least in part be associated with impairment of DNA repair following radiation-induced DNA damage.     

Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinase on enzyme activities and DNA repair

DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.