RESUMEN
In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.
Asunto(s)
Ciclinas/metabolismo , Proteínas Proto-Oncogénicas , beta Caroteno/farmacología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Fibroblastos , Técnica del Anticuerpo Fluorescente , Fase G1/efectos de los fármacos , Humanos , Liposomas/metabolismo , Fosforilación , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.