Effect of p53 activity on the sensitivity of human glioblastoma cells to PARP-1 inhibitor in combination with topoisomerase i inhibitor or radiation (original) (raw)
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Oncogene, 2004
A critical challenge in cancer research is to identify genetic lesions that sensitize patients to chemotherapy. p53, which is mutated in nearly one-third to half of glioblastomas, may be such a lesion. In this paper, we demonstrate that p53 disruption dramatically sensitizes glioblastoma cells to DNA topoisomerase I inhibitormediated apoptosis. Using 19 glioblastoma cell lines, including 15 low-passage ex vivo cell lines derived from patients, as well as isogenic glioblastoma cells varying in p53 status, we show that clinically relevant levels of SN-38 potently induce cell cycle arrest and temporary senescence in glioblastoma cells with wild-type p53 while causing massive apoptosis in p53-deficient cells (Po0.0002). We demonstrate that glioblastoma cells with wild-type p53 proliferate when recultured in drug-free medium, whereas p53-deficient cells do not. We also show that p16 protein expression is neither necessary nor sufficient for initiation and/or maintenance of SN-38-induced arrest/senescence. These results indicate that p53 disruption has a dramatic effect on how glioblastoma cells process topoisomerase I inhibitor-mediated DNA damage.
Repair of Potentially Lethal Damage does not Depend on Functional TP53 in Human Glioblastoma Cells
Radiation Research, 2004
The functionality of G 1-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after ␥ irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G 1 arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G 1 arrest.
Acta biochimica Polonica, 2002
We here report the influence of the cell cycle abrogator UCN-01 on RKO human colon carcinoma cells differing in p53 status following exposure to two DNA damaging agents, the topoisomerase inhibitors etoposide and camptothecin. Cells were treated with the two drugs at the IC90 concentration for 24 h followed by post-incubation in drug-free medium. RKO cells expressing wild-type, functional p53 arrested the cell cycle progression in both the G1 and G2 phases of the cell cycle whereas the RKO/E6 cells, which lack functional p53, only arrested in the G2 phase. Growth-arrested cells did not resume proliferation even after prolonged incubation in drug-free medium (up to 96 h). To evaluate the importance of the cell cycle arrest on cellular survival, a non-toxic dose of UCN-01 (100 nM) was added to the growth-arrested cells. The addition of UCN-01 was accompanied by mitotic entry as revealed by the appearance of condensed chromatin and the MPM-2 phosphoepitope, which is characteristic for ...
International Journal of Radiation Oncology*Biology*Physics, 1996
Purpose: Loss of the ~53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p5fmediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells.
Journal of Biological Chemistry, 2011
Background: PARP inhibitors and topoisomerase I poisons (Top1p) synergize by an unknown mechanism. Results: Although Parp1 deletion fails to increase Top1p sensitivity, transfection with catalytically inactive PARP1 or its isolated DNA binding domain does sensitize. Conclusion: PARP inhibitors poison PARP1 to diminish repair of topoisomerase I-triggered DNA damage. Significance: These results predict that tumors with elevated PARP1 will be particularly sensitive to Top1p/PARP inhibitor combinations. Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1 ؊/؊ MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1 ؊/؊ MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair. Topoisomerase I (topo I) 3 is an abundant nuclear enzyme (1, 2) that catalyzes unwinding of supercoiled DNA. Topo I facilitates this process through a well defined catalytic cycle (for review, see Refs. 3 and 4) that involves the following sequential steps: binding of topo I to sites of supercoiling (5, 6), nucleophilic attack on the DNA backbone to produce a 3Ј-phosphotyrosine linkage between topo I and DNA (topo I-DNA covalent complex; Top1cc) and a single-strand DNA nick (7), unwinding of DNA around this nick, and nucleophilic attack of the free 5Ј-hydroxyl of DNA on the phosphotyrosine bond to reseal the DNA backbone (8). Topo I is a target for a number of anticancer drugs (9), including camptothecin (CPT; Ref. 10) and its water-soluble derivatives topotecan and 7-ethyl-10-hydroxycamptothecin (SN-38). These agents bind to the interface between the enzyme and cleaved DNA (11), thereby stabilizing the covalent complex (10) and turning the normal enzyme into an agent of DNA damage (a process called "poisoning"; Ref. 12). These stabilized Top1cc are vulnerable to collisions with replication or transcription machinery (13-15), leading to replication fork stalling (16, 17) and, if forks are not restarted, toxic DNA double-strand breaks (14, 15, 18). To prevent the formation of these toxic lesions, Top1cc must be recognized and repaired. One molecule implicated in the repair of these lesions is poly(ADPribose) polymerase 1 (PARP1). PARP1 is a nuclear enzyme that binds to and is activated by damaged DNA (19, 20). Once activated, PARP1 synthesizes long polymers of poly(ADP-ribose) (pADPr) attached to hundreds of protein acceptors, including PARP1 itself (21). The formation of pADPr appears to play a critical role in coordinating the DNA damage response by regulating base excision * This work was supported, in whole or in part, by National Institutes of Health Grants P50 CA136393 and R01 CA73709. □ S This article contains supplemental Figs. S1-S9.