RESUMO
Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.
Assuntos
Senescência Celular , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Dano ao DNA , Proteínas Interatuantes com Canais de Kv , Senescência Celular/efeitos da radiação , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Radiação Ionizante , Reparo do DNA , Regulação da Expressão Gênica/efeitos da radiação , Proteínas RepressorasRESUMO
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 coordinated and faithful DNA damage response is of central importance for maintaining genomic integrity and survival. Here, we show that exposure of human cells to benzo(a)pyrene 9,10-diol-7,8-epoxide (BPDE), the active metabolite of benzo(a)pyrene (B(a)P), which represents a most important carcinogen formed during food preparation at high temperature, smoking and by incomplete combustion processes, causes a prompt and sustained upregulation of the DNA repair genes DDB2, XPC, XPF, XPG and POLH. Induction of these repair factors on RNA and protein level enhanced the removal of BPDE adducts from DNA and protected cells against subsequent BPDE exposure. However, through the induction of POLH the mutation frequency in the surviving cells was enhanced. Activation of these adaptive DNA repair genes was also observed upon B(a)P treatment of MCF7 cells and in buccal cells of human volunteers after cigarette smoking. Our data provide a rational basis for an adaptive response to polycyclic aromatic hydrocarbons, which occurs however at the expense of mutations that may drive cancer formation.