Apaf-1 overexpression partially overcomes apoptotic resistance in a cisplatin-selected HeLa cell line

Inhibition of caspase-3-mediated apoptosis has been hypothesized to be asso ciated with chemoresistance. Investigations of apoptosis revealed that cyto solic cytochrome c is associated with a complex of apoptotic protease activ ating factor-1 (Apaf-1), an adapter molecule, and caspase-9 to activate cas pase-3. However, whether these apoptotic molecules are involved in acquired cisplatin resistance is not understood. The present work shows reduced act ivation of caspase-3 and apoptosis in a cisplatin-selected HeLa cell line. Ac-DEVD-CHO, a caspase-3 inhibitor, inhibited cisplatin-induced apoptosis a bout 60-70% in both cell lines. Ac-LEHD-CHO, a caspase-9 inhibitor or Ac-IE TD-CHO, a caspase-8 inhibitor, inhibited cisplatin-induced caspase-3 activa tion and apoptosis similarly in both cell lines. In addition, cisplatin ind uced the activation of caspase-9, the upstream activator of caspase-3, in a dose-dependent manner, and the activation of caspase-9 was less induced in resistant cells. The accumulation of cytosolic cytochrome c, an activator of caspase-9, and the induction of the mitochondrial membrane-associated vo ltage-dependent anion channel were also reduced in cisplatin-resistant cell s. However, the concentration of Bcl-2 family proteins in cisplatin-resista nt cells was normal. The concentration of Apaf-1 was unaltered in both cell lines. Increasing the cellular concentration of Apaf-1 through the transie nt expression of the gene increased the induction of apoptosis in resistant cells, associated with enhanced activation of caspase-9, caspase-3 and DNA fragmentation factor. Regression analysis reveals that the modification fa ctor, the ratio of the slope in the linear range of the dose-response curve with Apaf-1 to the slope without Apaf-1, is 1.5 and 4.75 in the HeLa and c isplatin-resistant HeLa cells, respectively. These results indicate that ap optosis and caspases are less induced in cisplatin-selected HeLa cells. The y also suggest that ectopic overexpression of Apaf-1 may partially reverse the acquired cisplatin resistance. (C) 2001 Published by Elsevier Science B .V. on behalf of the Federation of European Biochemical Societies.