Alteration of the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR cells: Influence of drug transport abnormalities

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin-ceramide pathway . We have now investigated sphingomyelin (SM) hydrolysis, ceramide generati on, and apoptosis in vincristine-selected multidrug resistant (MDR) HL-60 c ells (HL-60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydr olysis, and ceramide generation) and apoptosis in both parental and MDR cel ls, when used at isotoxic doses (i.e., I and 100 mu M for HL-60 and HL-60/V inc, respectively), However, in MDR cells treated with either 10 mu M DNR o r 1 mu M DNR in association with the P-glycoprotein (P-gp) blocker verapami l (treatment conditions which yield an intracellular DNR concentration simi lar to that achieved with 1 mu M in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR-induced SM cycle in MDR cells is not directly related to P-gp. We have also investigated the influence of intracellular drug loc alization on the DNR-induced SM-cycle in HL-60/Vinc cells. In these cells, DNR at 10 mu M is mainly localized in cytoplasmic vesicles, white the drug is diffusely distributed when used at 100 mu M, A diffuse distribution patt ern was also observed when MDR cells were treated with 1 mu M DNR in associ ation with the cyclosporine derivative PSC-833, but not with verapamil. In parallel, PSC-833, but not verapamil, restored the induction of the SM cycl e and the apoptotic potential of DNR, and markedly increased drug cytotoxic ity in MDR cells. Our results suggest that altered intracellular drug trans port plays an important role in limiting ceramide generation and cell death in MDR cells. (C) 1999 Wiley-Liss, Inc.