RESUMO
The retinoblastoma tumour suppressor protein RB is cleaved by caspases during apoptosis. Here we have mutated the caspase cleavage site in the carboxy terminus of the murine Rb protein in the mouse germ line to create the Rb-MI allele. After endotoxic shock, expression of Rb-MI inhibits apoptosis in the intestines, but not in the spleen, and promotes the survival of male mice. Fibroblasts expressing Rb-MI protein are protected from apoptosis induced by the tumour-necrosis factor-alpha type I receptor (TNFRI) but remain sensitive to cell death induced by DNA damage. Correspondingly, the release of cytochrome c and the activation of caspase-3 induced by TNFRI, but not by DNA damage, are defective in cells expressing Rb-MI. Our results highlight the importance of Rb cleavage in TNFRI-induced apoptosis.
Assuntos
Antígenos CD/metabolismo , Apoptose/genética , Caspases/metabolismo , Células Eucarióticas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/genética , Células 3T3 , Alelos , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Caspase 3 , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Células Eucarióticas/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Lipopolissacarídeos , Camundongos , Camundongos Knockout , Mutação/genética , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Retina/crescimento & desenvolvimento , Retina/metabolismo , Retina/efeitos da radiação , Choque Séptico/induzido quimicamente , Choque Séptico/genética , Choque Séptico/metabolismo , Transdução de Sinais/genética , Toxinas Biológicas , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1 to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1 proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/- and p21Cip1-/- 3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase-specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase-specific antitumor agents.
Assuntos
Proteínas de Ciclo Celular/metabolismo , AMP Cíclico/metabolismo , Replicação do DNA , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína do Retinoblastoma/metabolismo , Fase S , Animais , Apoptose , Northern Blotting , Ciclo Celular , Núcleo Celular/metabolismo , Proliferação de Células , Cromatina/metabolismo , Clonagem Molecular , Colforsina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21 , Relação Dose-Resposta a Droga , Citometria de Fluxo , Fase G1 , Humanos , Immunoblotting , Linfócitos/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Células NIH 3T3 , Fosforilação , Transdução de Sinais , Frações Subcelulares/metabolismo , Fatores de TempoRESUMO
The retinoblastoma protein (RB) suppresses cell proliferation and apoptosis. We have previously shown that RB degradation is required for tumor necrosis factor alpha (TNF-alpha) to induce apoptosis. We show here the identification of two apoptotic effectors, i.e., c-ABL tyrosine kinase and p73, which are activated by TNF-alpha following RB degradation. In cells expressing a degradation-resistant RB protein (RB-MI), TNF-alpha does not activate c-ABL. RB-MI also inhibits TNF-alpha-mediated activation of p73. Genetic deletion and pharmacological inhibition of c-ABL or p73 diminish the apoptotic response to TNF-alpha in human cell lines and mouse fibroblasts. Thymocytes isolated from Rb(MI/MI), Abl(-/-), or p73(-/-) mice are resistant to TNF-alpha-induced apoptosis compared to their wild-type counterparts. This is in contrast to p53(-/-) thymocytes, which exhibit a wild-type level of apoptosis in response to TNF-alpha. Thus, c-ABL and p73 contribute to apoptosis induced by TNF-alpha, in addition to their role in promoting DNA damage-associated cell death.