RESUMO
Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.
Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Senescência Celular/genética , DNA/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/metabolismo , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Células MCF-7 , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, mathematical models of the SOS network, translesion synthesis and mismatch repair are developed. Within the proposed models, the key pathways of these repair systems were simulated on the basis of modern experimental data related to their mechanisms. Our model approach shows a possible mechanistic explanation of the hypothesis that the bacterial mismatch repair system is responsible for attenuation of mutation frequency during ultraviolet-induced SOS response via removal of the nucleotides misincorporated by DNA polymerase V (the UmuD'2C complex).
Assuntos
Reparo de Erro de Pareamento de DNA/efeitos da radiação , Escherichia coli/metabolismo , Modelos Biológicos , Mutagênese/efeitos da radiação , Resposta SOS em Genética/efeitos da radiação , Raios Ultravioleta , Escherichia coli/genética , MutaçãoRESUMO
Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans. In C. elegans, involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Montagem e Desmontagem da Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Masculino , Raios UltravioletaRESUMO
As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.
Assuntos
Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Metilnitronitrosoguanidina/farmacologia , Adaptação Biológica , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular , Raios gama , Humanos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Mutação/efeitos dos fármacos , Timidina Quinase/metabolismoRESUMO
Low-dose hyper-radiosensitivity (HRS) is the phenomenon whereby cells exposed to radiation doses of less than approximately 0.5 Gy exhibit increased cell killing relative to that predicted from back-extrapolating high-dose survival data using a linear-quadratic model. While the exact mechanism remains to be elucidated, the involvement of several molecular repair pathways has been documented. These processes in turn are also associated with the response of cells to O6-methylguanine (O6MeG) lesions. We propose a model in which the level of low-dose cell killing is determined by the efficiency of both pre-replicative repair by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) and post-replicative repair by the DNA mismatch repair (MMR) system. We therefore hypothesized that the response of cells to low doses of radiation is dependent on the expression status of MGMT and MMR proteins. MMR (MSH2, MSH6, MLH1, PMS1, PMS2) and MGMT protein expression signatures were determined in a panel of normal (PWR1E, RWPE1) and malignant (22RV1, DU145, PC3) prostate cell lines and correlated with clonogenic survival and cell cycle analysis. PC3 and RWPE1 cells (HRS positive) were associated with MGMT and MMR proficiency, whereas HRS negative cell lines lacked expression of at least one (MGMT or MMR) protein. MGMT inactivation had no significant effect on cell survival. These results indicate a possible role for MMR-dependent processing of damage produced by low doses of radiation.
Assuntos
Reparo de Erro de Pareamento de DNA , Guanina/análogos & derivados , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Doses de Radiação , Tolerância a Radiação , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Guanina/metabolismo , Guanina/farmacologia , Humanos , Fenótipo , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , TemozolomidaRESUMO
One of the major health concerns on long-duration space missions will be radiation exposure to the astronauts. Outside the earth's magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation-sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56 Fe radiation on HSC function in Mlh1+/+ and Mlh1-/- marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. Stem Cells Translational Medicine 2018;7:513-520.
Assuntos
Reparo de Erro de Pareamento de DNA/efeitos da radiação , Raios gama , Células-Tronco Hematopoéticas/metabolismo , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Proliferação de Células/efeitos da radiação , Feminino , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Doses de RadiaçãoRESUMO
5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-20-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.
Assuntos
Dano ao DNA/genética , DNA Glicosilases/genética , Reparo do DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , 8-Hidroxi-2'-Desoxiguanosina , Extratos Celulares/genética , Extratos Celulares/farmacologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos da radiação , DNA Glicosilases/farmacologia , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Desoxiguanosina/análogos & derivados , Desoxiguanosina/genética , Desoxirribonuclease (Dímero de Pirimidina)/farmacologia , Células HeLa , Humanos , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Radiação Ionizante , Timina/análogos & derivadosRESUMO
Cdt1 is rapidly degraded by CRL4Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within â¼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for â¼30 min in NER-deficient XP-A cells, but was degraded within â¼60 min. The delayed degradation was also dependent on PCNA and CRL4Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Fase G1/efeitos da radiação , Proteólise/efeitos da radiação , Raios Ultravioleta , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Cinética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Xeroderma Pigmentoso/metabolismoRESUMO
To elucidate the role of the mismatch repair gene Mlh1 in genome instability during the fetal stage, spontaneous mutations were studied in Mlh1-deficient lacZ-transgenic mouse fetuses. Mutation levels were high at 9.5 days post coitum (dpc) and gradually increased during the embryonic stage, after which they remained unchanged. In addition, mutations that were found in brain, liver, spleen, small intestine and thymus showed similar levels and no statistically significant difference was found. The molecular nature of mutations at 12.5 dpc in fetuses of Mlh1+/+ and Mlh1-/- mice showed their own unique spectra, suggesting that deletion mutations were the main causes in the deficiency of the Mlh1 gene. Of note, fetuses of irradiated mice exhibited marked differences such as post-implantation loss and Mendelian distribution. Collectively, these results strongly suggest that high mutation ofMlh1-/--deficient fetuses has little effect on the fetuses during their early developmental stages, whereas Mlh1-/--deficient fetuses from X-ray irradiated mothers are clearly effected.
Assuntos
Desenvolvimento Embrionário/genética , Proteína 1 Homóloga a MutL/deficiência , Taxa de Mutação , Animais , Animais Recém-Nascidos , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Feto/embriologia , Feto/efeitos da radiação , Instabilidade Genômica/genética , Instabilidade Genômica/efeitos da radiação , Genótipo , Camundongos , Proteína 1 Homóloga a MutL/metabolismo , Raios X/efeitos adversosRESUMO
PURPOSE: To test the hypothesis that differences in DNA double-strand breaks (DSB) repair fidelity underlies differences in radiosensitivity. MATERIALS AND METHODS: A primary fibroblast culture (C42) derived from a pediatric cancer patient treated with reduced radiation doses consequent to a family history of radiosensitivity reminiscent of chromosomal fragility syndrome, was compared to a normal control (C29). DNA DSB rejoining and repair fidelity were studied by Southern blotting and hybridization to specific fragments: Alu repetitive sequence representing the overall DSB rejoining capacity in the genome and a 3.2 Mbp NotI restriction fragment on chromosome 21 for DSB repair fidelity. RESULTS: Although both assays showed statistically significant difference (p ≤ 0.05) between the two cell strains in residual misrepaired (un-or mis-rejoined) DSB (24 h after 30 or 80 Gy), the residual damage was lower in the Alu enriched genome assay compared to NotI assay (0.01-0.07 and 0.10-0.37, respectively). CONCLUSIONS: These results suggest that, in comparison to classic DSB repair experiment, an assay of measuring DNA DSB repair fidelity can provide better resolution and a more accurate estimate of misrepair of radiation-induced DNA damage, which underlies genomic instability and increased radiosensitivity.
Assuntos
Transtornos Cromossômicos/genética , Fragilidade Cromossômica/genética , Fragilidade Cromossômica/efeitos da radiação , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Tolerância a Radiação/genética , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/efeitos da radiação , Bioensaio/métodos , Pré-Escolar , Feminino , HumanosRESUMO
Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo de Erro de Pareamento de DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/fisiologia , Exodesoxirribonucleases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , DNA/genética , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/efeitos da radiação , Células HeLa , Humanos , Lasers/efeitos adversos , Camundongos , Proteína 1 Homóloga a MutL , Proteína 3 Homóloga a MutS , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transporte Proteico/genética , Transporte Proteico/efeitos da radiação , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiação , Fase S , Helicase da Síndrome de WernerRESUMO
Many studies have shown that DNA mismatch repair (MMR) has a role beyond that of repair in response to several types of DNA damage, including ultraviolet radiation (UV). We have demonstrated previously that the MMR-dependent component of UVB-induced apoptosis is integral to the suppression of UVB-induced tumorigenesis. Here we demonstrate that Msh6-dependent UVB-induced apoptotic pathway is both activated via the mitochondria and p53-independent. In addition, we have shown for the first time that caspase 2, an initiator caspase, localizes to the centrosomes in mitotic primary mouse embryonic fibroblasts, irrespective of MMR status and UVB treatment.
Assuntos
Apoptose , Caspase 2/metabolismo , Centrossomo/enzimologia , Reparo de Erro de Pareamento de DNA , Animais , Apoptose/efeitos da radiação , Caspase 9/metabolismo , Extratos Celulares , Células Cultivadas , Centrossomo/efeitos da radiação , Citocromos c/metabolismo , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos da radiação , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Microscopia Confocal , Mitocôndrias/enzimologia , Mitocôndrias/efeitos da radiação , Proteína 2 Homóloga a MutS/metabolismo , Transporte Proteico/efeitos da radiação , Receptores de Morte Celular/metabolismo , Transdução de Sinais/efeitos da radiação , Frações Subcelulares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Proteína X Associada a bcl-2/metabolismoRESUMO
Concurrent treatment with the methylating agent temozolomide during radiotherapy has yielded the first significant improvement in the survival of adult glioblastomas (GBM) in the last three decades. However, improved survival is observed in a minority of patients, most frequently those whose tumors display CpG methylation of the O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) promoter, and adult GBMs remain invariably fatal. Some, although not all, preclinical studies have shown that temozolomide can increase radiosensitivity in GBM cells that lack MGMT, the sole activity in human cells that removes O(6)-meG from DNA. Here, we systematically examined the temozolomide dose dependence of radiation killing in established GBM cell lines that differ in ability to remove O(6)-meG or tolerate its lethality. Our results show that minimally cytotoxic doses of temozolomide can produce dose-dependent radiosensitization in MGMT-deficient cells, MGMT-proficient cells, and MGMT-deficient cells that lack mismatch repair, a process that renders cells tolerant of the lethality of O(6)-meG. In cells that either possess or lack MGMT activity, radiosensitization requires exposure to temozolomide before but not after radiation and is accompanied by formation of double-strand breaks within 45 minutes of radiation. Moreover, suppressing alkyladenine-DNA glycosylase, the only activity in human cells that excises 3-methyladenine from DNA, reduces the temozolomide dose dependence of radiosensitization, indicating that radiosensitization is mediated by 3-methyladenine as well as by O(6)-meG. These results provide novel information on which to base further mechanistic study of radiosensitization by temozolomide in human GBM cells and to develop strategies to improve the outcome of concurrent temozolomide radiotherapy.
Assuntos
Neoplasias Encefálicas/radioterapia , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/análogos & derivados , Glioblastoma/radioterapia , Tolerância a Radiação/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Adulto , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Neoplasias Encefálicas/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/fisiologia , Dacarbazina/administração & dosagem , Dacarbazina/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Raios gama , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , Dose Máxima Tolerável , Temozolomida , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Poly(ADP-ribose) polymerases (PARPs) are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD(+) to a number of acceptor proteins. PARP-1, the best characterized member of the PARP family, which currently comprises 18 members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress. PARP is involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. Inhibitors of PARP-1 have been shown to enhance the cytotoxic effects of ionizing radiation and DNA-damaging chemotherapy agents, such as the methylating agents and topoisomerase I inhibitors. There are currently at least five PARP inhibitors in clinical trial development. Recent in vitro and in vivo evidence suggests that PARP inhibitors could be used not only as chemo/radiotherapy sensitizers, but also as single agents to selectively kill cancers defective in DNA repair, specifically cancers with mutations in the breast cancer-associated genes (BRCA1 and BRCA2). PARP becomes activated in response to oxidative DNA damage and depletes cellular energy pools, thus leading to cellular dysfunction in various tissues. The activation of PARP may also induce various cell death processes and promotes an inflammatory response associated with multiple organ failure. Inhibition of PARP activity is protective in a wide range of inflammatory and ischemia-reperfusion-associated diseases, including cardiovascular diseases, diabetes, rheumatoid arthritis, endotoxic shock, and stroke. The aim of this review is to overview the emerging data in the literature showing the role of PARP in the pathogenesis of cancer and inflammatory diseases and unravel the solid body of literature that supports the view that PARP is an important target for therapeutic intervention in critical illness.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Reparo de Erro de Pareamento de DNA/genética , Neovascularização Patológica/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/uso terapêutico , Radiossensibilizantes/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos da radiação , Ensaios Clínicos como Assunto , Terapia Combinada , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/imunologia , Radioterapia/efeitos adversosRESUMO
Mismatch repair (MMR) proteins contribute to genome stability by excising DNA mismatches introduced by DNA polymerase. Although MMR proteins are also known to influence cellular responses to DNA damage, how MMR proteins respond to DNA damage within the cell remains unknown. Here, we show that MMR proteins are recruited immediately to the sites of various types of DNA damage in human cells. MMR proteins are recruited to single-strand breaks in a poly(ADP-ribose)-dependent manner as well as to double-strand breaks. Using mutant cells, RNA interference and expression of fluorescence-tagged proteins, we show that accumulation of MutSbeta at the DNA damage site is solely dependent on the PCNA-binding domain of MSH3, and that of MutSalpha depends on a region near the PCNA-binding domain of MSH6. MSH2 is recruited to the DNA damage site through interactions with either MSH3 or MSH6, and is required for recruitment of MLH1 to the damage site. We found, furthermore, that MutSbeta is also recruited to UV-irradiated sites in nucleotide-excision-repair- and PCNA-dependent manners. Thus, MMR and its proteins function not only in replication but also in DNA repair.