RESUMEN
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.
Asunto(s)
Senescencia Celular , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Daño del ADN , Proteínas de Interacción con los Canales Kv , Senescencia Celular/efectos de la radiación , Senescencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Humanos , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas de Interacción con los Canales Kv/genética , Radiación Ionizante , Reparación del ADN , Regulación de la Expresión Génica/efectos de la radiación , Proteínas RepresorasRESUMEN
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.
Asunto(s)
Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Senescencia Celular/genética , ADN/genética , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F4/genética , Puntos de Control del Ciclo Celular , Línea Celular Transformada , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN/metabolismo , Daño del ADN , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Reparación de la Incompatibilidad de ADN/efectos de la radiación , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F4/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Rayos gamma , Humanos , Proteínas de Interacción con los Canales Kv/genética , Proteínas de Interacción con los Canales Kv/metabolismo , Células MCF-7 , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación/efectos de los fármacos , Reparación del ADN por Recombinación/efectos de la radiación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
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.
