DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling (original) (raw)
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DNA damage-induced cell death by apoptosis
Trends in Molecular Medicine, 2006
Following the induction of DNA damage, a prominent route of cell inactivation is apoptosis. During the last ten years, specific DNA lesions that trigger apoptosis have been identified. These include O 6 -methylguanine, base N-alkylations, bulky DNA adducts, DNA cross-links and DNA double-strand breaks (DSBs). Repair of these lesions are important in preventing apoptosis. An exception is O 6 -methylguanine-thymine lesions, which require mismatch repair for triggering apoptosis. Apoptosis induced by many chemical genotoxins is the consequence of blockage of DNA replication, which leads to collapse of replication forks and DSB formation. These DSBs are thought to be crucial downstream apoptosistriggering lesions. DSBs are detected by ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) proteins, which signal downstream to CHK1, CHK2 (checkpoint kinases) and p53. p53 induces transcriptional activation of pro-apoptotic factors such as FAS, PUMA and BAX. Many tumors harbor mutations in p53. There are p53 backup systems that involve CHK1 and/or CHK2-driven E2F1 activation and p73 upregulation, which in turn transcribes BAX, PUMA and NOXA. Another trigger of apoptosis upon DNA damage is the inhibition of RNA synthesis, which leads to a decline in the level of critical gene products such as MKP1 (mitogen-activated protein kinase phosphatase). This causes sustained activation of JNK (Jun kinase) and, finally, AP-1, which stimulates death-receptor activation. DNA damage-triggered signaling and execution of apoptosis is cell-type-and genotoxin-specific depending on the p53 (p63 and p73) status, death-receptor responsiveness, MAP-kinase activation and, most importantly, DNA repair capacity. Because most clinical anti-cancer drugs target DNA, increasing knowledge on DNA damage-triggered signaling leading to cell death is expected to provide new strategies for therapeutic interventions.
Journal of Cellular Biochemistry, 2008
It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS 3 cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N 0-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS 3 cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS 3 cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS 3 cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS 3 cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS 3 cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS 3 cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.
p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage
The EMBO journal, 2000
p53-mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem (ES) cells that express p53 with Gln and Ser in place of Leu25 and Trp26, respectively. p53(Gln25Ser26) was stable but did not accumulate after DNA damage; the expression of p21/Waf1 and PERP was not induced, and p53-dependent repression of MAP4 expression was abolished. Therefore, p53(Gln25Ser26) is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53(Gln25Ser26) was phosphorylated at Ser18 but was not acetylated at C-terminal sites, and its DNA binding activity did not increase, further supporting a role for p53 acetylation in the activation of sequence-specific DNA binding activity. Most importantly, p53(Gln25Ser26) mouse thymocytes and ES cells, like p53(-/-) ...
Cancer research, 1994
The present study assessed the role ofthep53 tumor suppressor gene in cell cycle arrest and apoptosis following treatment of Burldtt's lymphoma and lymphoblastoid cell lines with ‘y-rays, etoposide, nitrogen mustard, and cisplatin. Cell cycle arrest was measured by flow cytometry; p5.3 and @ protein levels were measured by Western blotting; cell sur vival was measured in 72-96-h growth inhibition assays and by trypan blue staining, and apoptotic DNA fragmentation was assessed by either agarose gel electrophoresis or a modified ifiter elution method. We found that y-rays and etoposide induced a strong G1 arrest in the wild-typeps3 lines while nitrogen mustard and cisplatin induced relatively little G1 arrest. All agents failed to induce G1 arrest in cells containing mutantp53 genes. The degree of C1 arrest observed with these agents correlated with the rate ofp53 and p2JW41fI/C4PI protein accumulation: ‘y-rays and etopo side induced rapid accumulation of both p5.3 and p21@'@―1; nitrogen mustard and cisplatin induced slow accumulation of p5.3 and no major accumulation of the @2jWafI/C1@1 protein. Despite differences in G1 arrest and kinetics ofp53 orp21@―@ protein accumulation, all agents tended to decrease survival to a greater extent in the wild-type p5.3 lines com pared to the mutant p5.3 lines. Cell death in the wild-type p53 lines was associated with intracellular DNA degradation into oligonucleosomal sized DNA fragments, indicative ofapoptosis.
Carcinogenesis, 1999
A network of interacting cellular components is known to mediate the regulatory role of tumor suppressor protein p53 in genomic stability. DNA repair machinery is considered to be one of these vital cellular components. To investigate the modulatory function of p53 on the repair of DNA damage and related effects, we have studied the responses of human p53-wild-type (p53-WT), p53-mutant (p53-Mut) and p53-nullizygous (p53-Null) cells following exposure to UV irradiation. Absence of wild-type p53 function coincided with an enhanced sensitivity to UV, as well as induction of apoptosis. However, the lack of wild-type p53 expression did not affect the response of its signal transducer protein, p21. Repair analysis of specific genomic sequences, at a single nucleotide resolution, revealed that the removal of cyclobutane pyrimidine dimers in a non-transcribed strand was significantly slower in p53-Mut and p53-Null cell lines compared with the normal p53-WT cells. However, the repair of the transcribed strand was comparable in the three cell lines. Thus, p53 is required for the efficient nucleotide excision repair (NER) of the global genomic DNA, but not for the transcription-coupled repair of the essential genes. The decreased global NER, due to the lost p53 function, seems to be responsible for the conjoined cytotoxicity and apoptosis of human cells subjected to DNA stress damage.
Oncogene, 2004
Unicellular organisms respond to the presence of DNA lesions by activating cell cycle checkpoint and repair mechanisms, while multicellular animals have acquired the further option of eliminating damaged cells by triggering apoptosis. Defects in DNA damage-induced apoptosis contribute to tumorigenesis and to the resistance of cancer cells to a variety of therapeutic agents. The intranuclear mechanisms that signal apoptosis after DNA damage overlap with those that initiate cell cycle arrest and DNA repair, and the early events in these pathways are highly conserved. In addition, multiple independent routes have recently been traced by which nuclear DNA damage can be signalled to the mitochondria, tipping the balance in favour of cell death rather than repair and survival. Here, we review current knowledge of nuclear DNA damage signalling, giving particular attention to interactions between these nuclear events and apoptotic processes in other intracellular compartments.
Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents
Proceedings of the National Academy of Sciences, 1999
All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O 6 -alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O 6methylguanine (O 6 MeG) and O 6 -chloroethylguanine (O 6 CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutS␣ branch of the DNA mismatch repair pathway (but not the MutS branch) is absolutely required for signaling the initiation of apoptosis in response to O 6 MeGs and is partially required for signaling apoptosis in response to O 6 CEGs. Further, O 6 MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutS␣-dependent. Despite this, MutS␣-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.
Functional connection between p53 and caspase-2 is essential for apoptosis induced by DNA damage
Oncogene, 2006
Recent findings have established caspase-2 as an important apical regulator in apoptotic pathways leading from DNA damage to release of mitochondrial cytochrome c and subsequent activation of effector caspases. Yet, the molecular map connecting the embarking stimuli of genotoxic stress with caspase-2 activation remains to be elucidated. Here, we address the question of potential caspase-2 regulators by examining 5-fluorouracil (5-FU)induced apoptosis in wild-type and p53-deficient human colon carcinoma cells. Apoptosis was observed only in p53 þ / þ cells and was preceded by caspase-2 activation. Hence, although no direct interaction between p53 and caspase-2 was observed in the cell system used, our data clearly demonstrate that a functional connection between these two proteins is essential for initiation of the 5-FUinduced apoptotic process. Proposed mediators of caspase-2 activation include PIDDosome complex proteins PIDD and RAIDD. Surprisingly, the presence of a complex encompassing at least RAIDD, PIDD and caspase-2 was verified in both p53 þ / þ and p53 À/À cells, also in the absence of 5-FU treatment. Thus, our results confirm the participation of PIDD and RAIDD in PIDDosome complex formation but question their role as sole mediators of caspase-2 activation. This assumption was further supported by siRNA transfections targeting PIDD or RAIDD. In conclusion, our findings support the hypothesis of p53 as an upstream regulator of caspase activity and provide data concerning caspase-2 processing mechanisms. As suppression of caspase-2 expression in 5-FU-treated cells also affects the level of the p53 protein, possibilities of a reciprocal interaction between these proteins are discussed.
Oncogene, 2004
Various tumor-therapeutic drugs and environmental carcinogens alkylate DNA inducing O 6 -methylguanine (O 6 MeG) that provokes cell death by apoptosis. In rodent fibroblasts, apoptosis triggered by O 6 MeG is executed via the mitochondrial damage pathway. Conversion of O 6 MeG into critical downstream lesions requires mismatch repair (MMR). This is thought to signal apoptosis upon binding to O 6 MeG lesions mispaired with thymine. Alternatively, O 6 MeG lesions might be processed by MMR giving rise to DNA double-strand breaks (DSBs) during replication that finally provoke apoptosis. To test this, we examined apoptosis triggered by O 6 MeG in human peripheral lymphocytes in which O 6 -methylguanine-DNA methyltransferase (MGMT) had been inactivated by O 6 -benzylguanine (O 6 BG) and which were not proliferating or proliferating upon CD3/CD28 stimulation. Treatment with N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG) or the anticancer drug temozolomide induced apoptosis only in proliferating, but not resting cells. With exceptional high alkylation doses (X15 lM of MNNG), apoptosis was also observed in resting lymphocytes, albeit at a lower level than in proliferating cells. This response was not affected by O 6 BG, suggesting that replication-independent apoptosis at high dose levels is caused by lesions other than O 6 MeG. O 6 MeG-triggered apoptosis in proliferating lymphocytes was preceded by a wave of DSBs, which coincided with p53 and Fas receptor upregulation, while Fas ligand, Bax and Bcl-2 expression was not altered. Treatment with anti-Fas neutralizing antibody attenuated MNNG-induced apoptosis in MGMT-depleted proliferating lymphocytes. The data suggest that O 6 MeG is converted by MMR and DNA replication into DSBs that trigger apoptosis by p53 stabilization and Fas/CD95/Apo-1 upregulation. This is supported by the finding that ionizing radiation, inducing DSBs on its own, provokes apoptosis in lymphocytes in a replication-independent way. The strict proliferation dependence of apoptosis triggered by O 6 MeG may explain the specific killing response of MGMT-deficient proliferating cells, including tumors, to O 6 MeG generating anticancer drugs and suggests that tumor proliferation rate, Fas responsiveness, MGMT and MMR status are important prognosis parameters.
Carcinogenesis, 2001
We have demonstrated previously that the toxicity of 5-hydroxymethyl-2Ј-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G 2 and that hmdUrd-treated cells underwent apotosis after arrest in late S/G 2 phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.