Calphostin C triggers calcium-dependent apoptosis in human acute lymphoblastic leukemia cells

Recent studies have demonstrated that the naturally occurring perylenequino ne antibiotic calphostin C is a potent inhibitor of protein kinase C and ca n induce apoptosis in some tumor cell lines by an as yet unknown mechanism, Here we demonstrate that calphostin C induces dose-dependent apoptosis in DT40 chicken lymphoma B-cells, and targeted disruption of lyn, syk, btk, PL C gamma 2, or IP3R genes does not prevent or attenuate its cytotoxicity. In our study, calphostin C also induced rapid apoptosis in human acute lympho blastic leukemia (ALL) cell lines ALL-1 (BCR-ABL(+) pre-pre-B ALL), RS4;11 (MLL-AF4(+) pro-B ALL), NALM-6 (pre-B ALL), DAUDI (Burkitt's/B-cell ALL), M OLT-3 (T-ALL), and JURKAT (T-ALL), whereas other potent PKC inhibitors did not. In biochemical studies, calphostin C was discovered to induce rapid ca lcium mobilization from intracellular stores of ALL cell lines, and its cyt otoxicity against ALL cell lines was well correlated with the magnitude of this calcium signal. Calphostin C-induced apoptosis,vas markedly suppressed by BAPTA/AM, a cell-permeable Ca2+ chelator as well as NiCl2, an inhibitor of Ca2+/Mg2+-dependent endonucleases. Inhibition of the Ca2+/calmodulin-de pendent phosphatase calcineurin dth perfluoreperazine dimadeate (a calmodul in antagonist) or cyclosporin A (a specific inhibitor of calcineurin) also reduced the magnitude of calphostin C-induced apoptosis in ALL cell lines. Calphostin C was capable of inducing calcium mobilization and apoptosis in freshly obtained primary leukemic cells from children with ALL. Taken toget her, our results provide unprecedented evidence that calphostin C triggers a Ca2+-dependent apoptotic signal in human ALL cells.