RESUMO
Particular protein kinase inhibitors block a senescence-like phenomenon in SVts8 cells induced by a shift up in temperature. We characterized cellular proteins with affinity chromatography using one such inhibitor as a ligand. Two proteins of 56 and 100 kDa were found to be dephosphorylated specifically, probably due to induction of a protein phosphatase activity(s).
Assuntos
Envelhecimento/fisiologia , Antígenos Transformantes de Poliomavirus/biossíntese , Proteínas/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Células Cultivadas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos , Temperatura Alta , Humanos , Ligantes , Peso Molecular , Fosforilação , Inibidores de Proteínas Quinases , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
Immortal human fibroblasts, SVts8 cells, which express a heat-labile SV40 large T antigen, induces a senescence-like phenomenon in response to upward shift in temperature. Cells with arrested division show strong induction of senescence-associated beta-galactosidase. We examined how p53 and pRB are involved in this phenomenon since they are major targets of the T antigen. Transfection of cells with plasmids encoding the wild-type T antigen or human papilloma virus type 16 E6/E7 proteins completely abolished the arrest in cell division, a plasmid encoding the E6 protein suppressed it markedly, while a plasmid encoding E7 had no effect. Plasmids encoding dominant-negative p53 mutants also suppressed the arrest in cell division to various degrees. Upon temperature shift, p21 mRNA was upregulated 10-fold in SVts8 cells, but only slightly in clones expressing the wild-type T antigen or dominant-negative p53 mutants. These data demonstrate that p53 plays a major role in this senescence-like phenomenon.