Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox

Objective. Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (R R) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleoti de synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitu mor therapy. Materials and Methods. HL-60 and K562 leukemia cells were treated with incr easing doses of amidox and didox, Thereafter, the mode of cytotoxic drug ac tion was determined by Hoechst 33258/propidium iodide (HO/PI) double staini ng, annexin binding, DNA fragmentation, and caspase activation. This was co rrelated to the decrease in dNTP levels, Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatid ylserine were used to quantify apoptosis, Results. Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 muM amidox or 250 muM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100 % of the HL-60 cells died by apoptosis without an increase in necrosis, K56 2 cells were found to be resistant to amidox but not to didox, In HL-60 cel ls, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but n ot amidox-induced apoptosis, can be correlated with the decrease in dNTP le vels. Conclusions. The results suggests that amidox induces several apoptosis mec hanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox i n K562 cells, and because amidox hardly induces apoptosis in this cell line , no caspase cleavage is observed. Didox triggers distinct apoptosis pathwa ys in HL-60 and K562 cells. (C) 2001 International Society for Experimental Hematology, Published by Elsevier Science Inc.