Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation - Role of Bcl-2 protein family

Much evidence suggests that apoptosis plays a crucial role in cell populati on homeostasis that depends on the expression of various genes implicated i n the control of cell life and death. The sensitivity of human neuroblastom a cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments wi th retinoic acid (RA), 4 beta -phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decrease s PKC activity. Neuronal differentiation was evaluated by gamma -enolase, m icrotubule associated protein 2 (MAP2) and synaptophysin immunocytochemistr y. The sensitivity of the cells to thapsigargin-induced apoptosis was evalu ated by cell viability and nuclear fragmentation (Hoechst 33258) and compar ed with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein express ion of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than control s. Conversely, the cells treated with staurosporine were more susceptible t o apoptosis. In parallel with morphological modifications, the expression o f inhibitors and activators of apoptosis was directly dependent upon the di fferentiating agent used. Bcl-2 expression was strongly increased by PMA an d drastically decreased by staurosporine as was Bcl-x(L) expression. Bax an d Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 e xpression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrat e a relationship between the continuous activation of PKC, the expression o f Bcl-2 protein family and the resistance of differentiated SK-N-SH to apop tosis.