RESUMEN
P21(Waf1) cyclin-dependent kinase inhibitor blocks cell cycle transition from G1 phase into DNA replication after DNA damage. The main targets of p21(Waf1) are Cyc 1E--Cdk2 and Cyc 1A--Cdk2 complexes, PCNA (proliferating cell nuclear antigen), a subunit of DNA polymerase delta, and E2F-1 transcription factor. The universal mechanism of cell cycle arrest in normal cells is determined as p21(Waf1) interaction with positive regulators of G1 phase. As a rule, DNA integrity control mechanisms are destroyed in the process of oncogenic transformation, which results in proliferation of genetically defective cells. The purpose of our study was to investigate molecular mechanisms of cell cycle regulation in transformants that are able (E1A + E1B-19kDa) or unable (E1A(+) + cHa-ras) to be arrested at G1/S checkpoint. We have shown that p21(Waf1) is able to form complexes with cyclins and Cdks, PCNA and E2F-1 transcryption factor, although it interacts with E1A oncoproducts in both transformants. The presence of E1A bound p21(Waf1) in cyclin-kinase complexes seems to be the cause of activating phosphorilation of Cdk2 at Thr-160 in cyclin A/E--Cdk2 complexes in both control and X-ray irradiated cells. Thus, the absence of G1/S arrest following irradiation in E1A + cHa-ras transformants and its presence in E1A(+) + E1B-19kDa transformants is not connected with differences in interaction of p21(waf1) with the main regulators of G1-to-S transition, but is realized through other not yet identified ways.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Oncogenes , Proteínas Proto-Oncogénicas/metabolismo , Proteínas E1A de Adenovirus , Animales , Ciclo Celular , Línea Celular Transformada/citología , Línea Celular Transformada/efectos de la radiación , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Factor de Transcripción E2F1/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Fase G1/efectos de la radiación , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas/genética , Ratas , Fase S/efectos de la radiación , Treonina , Rayos XRESUMEN
E1A + c-Ha-ras-transformants overexpressing bcl-2 oncogene are able to be arrested at the G1/S boundary of the cell cycle after DNA damage and upon serum starvation, this cell cycle blockage being accompanied by a decrease in the activity of cyclin E--Cdk2 complexes. Roscovitine-induced inhibition of cyclin-dependent kinases (Cdks) activity does not result in the G1/S arrest of E1A + c-Ha-ras + bcl-2-transformants. Roscovitine treatment causes an accumulation of G2/M cells, mainly at the expense of mitotic cells. However, the expression of Bcl-2 oncoproducts does not re-establish the regulation of mitotic events broken by introduction of E1A and c-Ha-ras oncogenes in normal cells, as revealed by the treatment of E1A + c-Ha-ras + bcl-2-transformants with nocodazole inducing mitotic arrest in normal cells. In spite of the elevated expression of antiapoptotic bcl-2 gene in transformants, nocodazole treatment results in mass apoptotic death preceded by polyploidy. Roscovitine also induces apoptosis with no polyploid cell accumulation being observed. Inhibition of Cdks activity with Roscovitine, as well as violation of microtubule depolymerization with nocodazole result in the apoptotic death in the tested cell lines sensitive (E1A + c-Ha-ras) and resistant (E1A + c-Ha-ras + bcl-2) to damaging agents. Thus, the application of Roscovitine, a specific inhibitor of Cdks, suggests that the decrease in Cdks activity in E1A + c-Ha-ras + bcl-2-transformants is not likely to be responsible for G1/S cell cycle arrest realization after damaging influences. Moreover, an antiproliferative effect of Bcl-2 in E1A + c-Ha-ras-transformants is restricted by restoration of cell cycle events at G1/S and G2/M boundaries, and does not concern the program of mitotic events regulation.
Asunto(s)
Genes bcl-2/fisiología , Mitosis/fisiología , Proteínas E1A de Adenovirus/genética , Animales , Antineoplásicos/farmacología , Apoptosis , División Celular , Línea Celular Transformada , Medios de Cultivo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fase G1 , Fase G2 , Genes ras , Mitosis/efectos de los fármacos , Nocodazol/farmacología , Poliploidía , Purinas/farmacología , Ratas , Ratas Wistar , RoscovitinaRESUMEN
Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.
Asunto(s)
Línea Celular Transformada/efectos de la radiación , Transformación Celular Neoplásica/efectos de la radiación , Daño del ADN , Fase G1 , Fase S , Proteínas E1A de Adenovirus/biosíntesis , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/biosíntesis , Proteínas E1B de Adenovirus/genética , Animales , Quinasas CDC2-CDC28/análisis , Línea Celular Transformada/citología , Ciclina A/análisis , Ciclina B/análisis , Quinasa 2 Dependiente de la Ciclina , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Fosforilación , RatasRESUMEN
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
The capability of REF cells transformed by EA + E1B-19 kDa and EA + cHa-ras oncogenes to realize the G1/S cell cycle arrest upon serum starvation was studied. The amount of cyclin-kinase inhibitor protein p27/Kip was shown to increase in both normal and transformed cells. However, the p27/Kip-bound cyclin-kinase complexes of transformed cells were found to be active, implying the functional inactivation of p27/Kip inhibitor. Nevertheless, in contrast to E1A + cHa-ras transformants, E1A + E1B-19 kDa transformants undergo the G1 cell cycle arrest. The G1 cell cycle block correlates with the decrease in cyclinE-Cdk2 activity. Since cyclinE-Cdk2 complexes need Thr-160 phosphorylation of Cdk2 by CAK-kinase for full activity, we have analysed the Cdk-7 associated activity upon serum starvation using gst-Cdk2 as a substrate. Serum starvation did not affect CAK activity either in E1A + cHa-ras or in E1A + E1B-19 kDa transformants. Thus, selective suppression of cyclineE-Cdk2 activity in E1A + E1B-19 kDa transformants upon serum starvation does not arise from the action of cyclin-kinase inhibitors, or from change in CAK activity.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Ciclo Celular/genética , Fibroblastos/patología , Genes ras/genética , Transducción de Señal/genética , Animales , Proteínas de Ciclo Celular/fisiología , Línea Celular Transformada , Transformación Celular Neoplásica , Transformación Celular Viral , Medio de Cultivo Libre de Suero , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/fisiología , Embrión de Mamíferos/citología , Fibroblastos/fisiología , Fase G1/genética , Ratas , Fase S/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
We studied the capability of E1A + cHa-ras and E1A + E1B19kDa transformants to undergo the G1/S arrest of the cell cycle following depletion of serum growth factors. It has been shown that serum starvation induced the G1/S arrest both in normal rat embryo fibroblasts (REF) and in E1A + E1B19kDa transformants, whereas E1A + cHa-ras transformed cells lost this feature. To analyse the mechanisms underlying these differences, we studied the expression of p27/KIP, its intracellular distribution and association with E1A oncoproducts. The content of the p27/KIP inhibitor of cyclin-dependent kinases was found to change a little upon transformation by two complementary oncogene pairs. However, serum starvation for 24 h led to a significant increase in the content of p27/KIP in E1A + E1B19kDa transformants, while E1A + cHa-ras cells accumulated p27/KIP less markedly. According to the immunofluorescence study, the p27/KIP inhibitor is located in the nucleus of both normal and transformed cells. Moreover, serum starvation did not lead to its inhibition due to redistribution to the cytoplasm in both cell lines. Also, we were unable to detect association of p27/KIP with E1A oncoproducts in immunoprecipitated complexes. The obtained data indicate that, in contrast to E1A + cHa-ras transformants, in E1A + E1B19kDa cells the p27/KIP inhibitor is functional and it is capable of inducing the G1/S block after serum starvation.