RESUMO
p51A, or TAp63gamma, a translation product of gene p51, or p63, was identified as a homolog of p53 in its primary structure and transactivating function. p53 plays a decision-making role in inducing either cell cycle arrest or apoptosis in response to DNA damage, and thereby preserves genome integrity of living cells. To compare the biological activities between p51A and p53, cell lines with low-level, constitutive expression of each protein were obtained by cDNA transfection of mouse erythroleukemic cells. Production of p51A with an apparent molecular mass of 57-kilodalton (kD) accompanied induction of p21waf1 and appearance of hemoglobin-producing cells. After DNA-damaging treatment either with ultraviolet light (UV) irradiation or with actinomycin D, the p51A protein accumulated in time courses corresponding to those of wild-type p53, and caused an increase in the hemoglobin-positive cell count. In contrast, p53-accumulated cells underwent apoptosis without exhibiting the feature of erythroid differentiation. The mode of p21waf1 and Bax-alpha upregulations varied between p51A- and p53-expressing cells and between the types of DNA damage. These results suggest the possibility that p51A induces differentiation under genotoxic circumstances. There may be cellular factors that control p51A protein stability and transactivating ability.
Assuntos
Ciclinas/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Genes p53/fisiologia , Fosfoproteínas , Proteínas Proto-Oncogênicas c-bcl-2 , Transativadores , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/genética , Dactinomicina/farmacologia , Genes Supressores de Tumor , Hemoglobinas/metabolismo , Leucemia Eritroblástica Aguda , Camundongos , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Proteínas Supressoras de Tumor , Raios Ultravioleta , Regulação para Cima , Proteína X Associada a bcl-2RESUMO
The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid differentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes differentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6 cells, EPO-induced differentiation was inhibited. Conversely, a dominant negative ras mutant (ras17N) induced differentiation even in the absence of EPO, suggesting that the basal Ras activity is essential for the maintenance of the undifferentiated phenotype and proliferative potential in this cell line. Rapid inactivation of ERK was observed after expression of ras17N. Slow but significant inactivation of ERK was also observed during EPO-induced differentiation. Furthermore, overexpression of a constitutively active mutant of ERK-activating kinase (MAPKK) was found to suppress erythroid differentiation, while pharmacological inhibition of MAPKK induced differentiation. These findings suggest that down-regulation of Ras/ERK signaling pathway may be an essential event in EPO-induced erythroid differentiation in this system.