RESUMEN
Cell cycle progression is regulated through changes in the activity of cyclin-dependent kinases that are, in turn, regulated by the expression of their respective cyclin partners. In primary cells, cyclin E expression increases through the G(1) phase of the cell cycle and peaks near the G(1)/S boundary. The unscheduled expression of cyclin E in primary human fibroblasts leads to chromosomal instability that is greatly increased by loss of the p53 tumour suppressor. Intriguingly, ultraviolet light (UV), the most prevalent environmental carcinogen, is similarly known to induce chromosomal instability more dramatically in the absence of p53. Here we report that UV light transiently increased the expression of cyclin E in normal human fibroblasts. Strikingly, cyclin E levels remained elevated for an extended period of time in the absence of functional p53. UV-induced cyclin E expression was not restricted to the G(1)/S boundary but remained elevated throughout S phase and this correlated with a massive accumulation of p53-deficient fibroblasts in this phase of the cell cycle. Forced expression of cyclin E alone was insufficient to cause a similar S phase arrest but forced expression of cyclin E led to an increase in the proportion of UV-irradiated cells in S phase. The present work suggests that p53 affects S phase progression following UV exposure by preventing the sustained unscheduled expression of cyclin E and that this may limit the clastogenic and carcinogenic effects of UV light.
Asunto(s)
Ciclina E/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Proteína p53 Supresora de Tumor/fisiología , Rayos Ultravioleta , Ciclo Celular , Células Cultivadas , Ciclina E/biosíntesis , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Fase S , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
The antiproliferative effect of human bcl-2 gene transferred to E1A + c-Ha-ras-transformed rat embryo fibroblasts, which are characterized by the absence of cell cycle checkpoints after damage and by a high proapoptotic sensitivity was studied. Ionizing irradiation, adriamycin treatment, and serum starvation were shown to induce G1/S arrest in E1A + c-Ha-ras-transformants. Bcl-2 antiproliferative effect in E1A + c-Ha-ras-transformants was not associated with alterations in Cdk2, cyclin E and A contents. G1/S arrest following irradiation or serum starvation was accompanied by a decrease in kinase activity associated with cyclin E-cdk2, whereas G1/S arrest in tetraploid subpopulation after adriamycin treatment did not correlate with a decrease in cyclin E-associated kinase activity. Cyclin A-associated kinase activity did not decrease after any used treatment. Transfection of bcl-2 in E1A + c-Ha-ras-transformants resulted in elevated expression of cyclin-cdk complexes inhibitor p21/Waf-1, but not p27/Kip. Damaging agents caused p21/Waf-1 and p27/Kip accumulation, but bcl-2 overexpression did not restore functions of these inhibitors, since p21/Waf-1 and p27/Kip were unable to suppress cyclin-cdk complexes activity after damage. These results suggest that bcl-2 transfection in E1A + c-Ha-ras-transformants is likely to result in irradiation- or serum starvation-induced G1/S arrest accomplished by a selective decrease in cyclin E-associated kinase activity. Adriamycin-induced G1/S arrest seems to be realized via cyclin-cdk complexes activity-independent way involving antiproliferative targets downstream of cyclin E-cdk2 and cyclin A-cdk2 complexes.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Fase G1 , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/efectos de la radiación , Animales , Apoptosis , Línea Celular , Línea Celular Transformada , Medio de Cultivo Libre de Suero , Ciclina A/metabolismo , Ciclina E/metabolismo , Ciclina E/efectos de la radiación , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/efectos de la radiación , Doxorrubicina/farmacología , Embrión de Mamíferos/citología , Inhibidores Enzimáticos/metabolismo , Fibroblastos , Genes bcl-2/genética , Genes ras/genética , Humanos , Radiación Ionizante , Ratas , Fase S , TransfecciónRESUMEN
Introduction of the E1A early region of the human adenovirus type 5 impairs the ability of mammalian cells to arrest the cell cycle at G1/S after damage. Two-parameter fluorescent-activated cell sorting (FACS) with iododeoxyuridine revealed the radiation-induced G1/S arrest in rat embryo fibroblasts transformed with the complementing E1A + E1B-19 kDa oncogenes. This was due to selective inhibition of CycIE/Cdk2-associated kinase activity, while activities of type 2 kinase and of CyclA/Cdk2 complexes remained unchanged. The inhibitor of G1-phase cyclin kinases, p21/Waf1, was accumulated and interacted with target kinases both in normal and in transformed cells after irradiation. As shown by immunoprecipitation, p21/Waf1 formed complexes with the E1A on coproducts in the transformants, which possibly accounted for its functional inactivation. Kinase modification in cyclin-kinase complexes was assumed to play a key role in regulation of cyclin-dependent kinases in the transformants with inactivated p21/Waf1.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Quinasas CDC2-CDC28 , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Proteínas Proto-Oncogénicas , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/efectos de la radiación , Proteínas E1B de Adenovirus/efectos de la radiación , Animales , Células Cultivadas , Ciclina A/metabolismo , Ciclina A/efectos de la radiación , Ciclina E/metabolismo , Ciclina E/efectos de la radiación , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/efectos de la radiación , Ciclinas/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Rayos gamma , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Ratas , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/efectos de la radiaciónRESUMEN
In order to investigate the hypothesis that aberrant expression of cell-cycle regulatory proteins may represent early events in the process of carcinogenesis, levels of expression of the negative regulators p21(waf1/cip1) (p21), p27(kip1) (p27), and p16(ink4a) (p16) and/or the positive regulators cyclin D(1) and cyclin E were examined by western blot analysis in cells transformed in vitro by ionizing radiation. The levels of these proteins in 12 independently derived mouse 10T(1/2) cell clones transformed by 1.5 Gy of alpha radiation were compared with those in nine similarly derived nontransformed control clones. Constitutive levels of p21 were very low in all control clones, whereas p21 expression was significantly elevated in nine of 12 transformed clones. Two of the three transformed clones displaying low levels of p21 expressed increased levels of p53. p21 regulation was also altered in response to radiation in transformed clones as compared with controls, only minimal induction was observed 4 h following gamma irradiation. Western blot analysis indicated a constant expression of p27 protein but slightly decreased levels of p16 in these transformed clones. Cyclin D(1) was overexpressed in 11 of 12 transformed clones; in only two of these were the levels of cyclin E elevated. Overall, the results suggest that alterations in the expression of cell cycle regulatory proteins may represent important events in radiation-induced oncogenic transformation in vitro. Although the specific alterations vary among different transformed clones, overexpression and aberrant regulation of p21 appear to be the most frequent ones.