RESUMO
The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. Paradoxically, while it can function as a tumor suppressor and inducer of apoptosis, it is overexpressed in a variety of human cancers. To resolve this enigma, we established cell-lines that overexpress PKR in non-transformed and in v-mos transformed CHO cells. Overexpression of PKR suppressed the proliferation of CHO cells by inducing a transient G0/G1 arrest, followed by a delayed G2/M arrest, which attenuated cell cycle progression. These effects were accompanied by early induction of p21/WAF-1 and delayed downregulation of CDC2 and cyclin B1. Induction of proapoptotic activity of the ectopic PKR paralleled the onset of G2/M arrest in CHO cells. However, while transiently inducing p21/WAF-1, PKR did not impose G2/M arrest or apoptosis in v-mos-transformed cells, nor was CDC2 or cyclin B1 down-regulated in those cells. These findings link the proapoptotic activity of PKR to the arrest of cell cycle at the G2/M phase. Consequently, the apoptotic activity of PKR could be counter-acted by an oncogene-like v-mos that overrides the G2/M arrest induced by PKR.
Assuntos
Apoptose , Proteína Quinase CDC2/metabolismo , Transformação Celular Neoplásica/metabolismo , Ciclina B/metabolismo , Ecdisterona/análogos & derivados , Proteínas Oncogênicas v-mos/farmacologia , eIF-2 Quinase/fisiologia , Animais , Células CHO , Ciclo Celular , Divisão Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica/patologia , Cricetinae , Ciclina B1 , Regulação para Baixo , Ecdisterona/farmacologia , Citometria de Fluxo , Cinética , Proteínas Oncogênicas v-mos/genética , Transfecção , Transformação Genética , eIF-2 Quinase/genéticaRESUMO
PC12 pheochromocytoma cells possess four known MEK activators: A-, B-, c-Raf-1 and MEKK. In order to examine whether differentiation factors or growth factors have a Raf isozyme preference for activation of the mitogenic cytoplasmic Raf-MEK-MAPK protein kinase cascade, the activation kinetics of these enzymes in response to epidermal growth factor (EGF) and nerve growth factor (NGF) were compared. An initial activation of all three Raf kinases was noticed, but only A- and B-Raf showed sustained activation by NGF, which was not seen after EGF treatment. Furthermore, expression of oncogenic versions of all three Raf kinases as well, as a potentially Raf-independent MEK activator, v-Mos, leads to activation of MAPK and to differentiation of PC12 cells. These data suggest a differential regulation of Raf kinases and that probably no alternative Raf substrates are involved in differentiation processes of PC12 cells.