RESUMO
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Caspase 8/metabolismo , Dano ao DNA , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Animais , Apoptose , Carcinoma Hepatocelular/patologia , Proliferação de Células , Senescência Celular , Doença Crônica , Cruzamentos Genéticos , Reparo do DNA , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Instabilidade Genômica , Hepatectomia , Hepatócitos/patologia , Histonas/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática , Masculino , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de RiscoRESUMO
Bacterial infection leads to consumption of short-lived innate immune effector cells, which then need to be replenished from hematopoietic stem and progenitor cells (HSPCs). HSPCs express pattern recognition receptors, such as Toll-like receptors (TLRs), and ligation of these receptors induces HSPC mobilization, cytokine production, and myeloid differentiation. The underlying mechanisms involved in pathogen signal transduction in HSCs and the resulting biological consequences remain poorly defined. Here, we show that in vivo lipopolysaccharide (LPS) application induces proliferation of dormant HSCs directly via TLR4 and that sustained LPS exposure impairs HSC self-renewal and competitive repopulation activity. This process is mediated via TLR4-TRIF-ROS-p38, but not MyD88 signaling, and can be inhibited pharmacologically without preventing emergency granulopoiesis. Live Salmonella Typhimurium infection similarly induces proliferative stress in HSCs, in part via TLR4-TRIF signals. Thus, while direct TLR4 activation in HSCs might be beneficial for controlling systemic infection, prolonged TLR4 signaling has detrimental effects and may contribute to inflammation-associated HSPC dysfunction.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Imunidade Inata , Salmonella typhimurium/fisiologia , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4-CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery-an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Replicação do DNA/genética , Instabilidade Genômica/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Ciclinas/genética , Ciclinas/metabolismo , Humanos , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.
Assuntos
Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Fase G1 , Células-Tronco Embrionárias Murinas/metabolismo , Rad51 Recombinase/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Blastocisto/metabolismo , Western Blotting , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Eletroforese em Gel de Campo Pulsado , Citometria de Fluxo , Histonas/metabolismo , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Mitose , Mórula/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína de Replicação A/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm(+/-)) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm(+/-) males did not significantly differ from that in wild-type BALB/c mice. Acute exposure to 1 Gy of gamma-rays did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm(+/-) and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analysed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.