Myeloma cells selected for resistance to CD95-mediated apoptosis are not cross-resistant to cytotoxic drugs: Evidence for independent mechanisms of caspase activation

We have previously shown that selection for resistance to the anthracenes, doxorubicin or mitoxantrone, results in coselection for resistance to CD95- mediated apoptosis (Landowski et al: Blood 89:1854, 1997). In the present s tudy, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemother apeutic drugs. To address this question, we used two isogenic models of CD9 5-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for resistance to CD95-mediated apoptosis; and K562 cells expressing ectop ic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8 226/S, to agonistic anti-CD95 antibody resulted in a cell line devoid of CD 95 receptor surface expression and completely resistant to CD95-mediated ap optosis. Multiple clonal populations derived from the CD95-resistant cell l ine showed no difference in sensitivity to doxorubicin, mitoxantrone, Ara-C , or etoposide, demonstrating that cross-resistance between Fas-mediated ap optosis and drug-induced apoptosis occurs only when cytotoxic drugs are use d as the selecting agent. Using the inverse approach, we transfected the CD 95-negative cell line, K562, with a CD95 expression vector. Clones expressi ng variable levels of cell-surface CD95 were isolated by limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to ch emotherapeutic drugs. We show that CD95 surface expression confers sensitiv ity to CD95-mediated apoptosis; however, it does not alter response to chem otherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was identical in the CD95-sensitive and CD95-resistant cell lines in both isogenic cell systems. In addition, prior treatment with the CD95 rec eptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in 8226/S cells, but had no effect on doxorubicin cytotoxicity. These results show th at CD95 and chemotherapeutic drugs use common apoptotic effecters, but the point of convergence in these two pathways is downstream of CD95 receptor/l igand interaction. (C) 1999 by The American Society of Hematology.