Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases

Abstract

The human leukemia cell line, HL60 is very sensitive to various B can activate CPP32, 13 and apoptotic activation of proteaapoptotic stimuli and p53-null. The death-related cysteine prosome 14,15 and cathepsin D 16 have been reported. A nuclear teases of the caspases family play a central role in the scaffold protease has been postulated to activate apoptotic execution phase of apoptosis, and we recently reported the nuclease(s). 17 Our previous studies in human leukemia HL60 importance of serine protease activation in camptothecincells also indicated that apoptotic DNA fragmentation can be induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CEDinduced by serine proteases in a cell-free system and sup-3-related cysteine proteases) and serine proteases in cell death pressed by serine protease inhibitors. 18,19 induced by the topoisomerase I inhibitor, camptothecin, inThe goal of the present study was to investigate the effects HL60 cells and in a cell-free system. We found that CPP32 is of two cell-permeable protease inhibitors on camptothecin- also be used in a cell-free system. 18,32 In the present report, in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32we demonstrate that CPP32 is proteolytically activated in and serine proteases are activated in camptothecin-induced response to camptothecin-induced apoptosis in HL60 cells. apoptosis. Activation of CPP32 requires both caspase and serine proteaseKeywords: camptothecin; topoisomerase I; apoptosis; caspase;activation. Furthermore, we show that in a cell-free system, serine protease; HL60only serine protease inhibitors block DNA fragmentation and lamin B degradation. These results suggest that serine proteases can function both downstream and upstream of the Introduction CPP32 activation step in a cell death pathway.Apoptosis is well defined morphologically 1 and recent studies indicate that a number of evolutionary conserved genes reguMaterials and methods late a common cell death pathway that is conserved from worms to humans. 2,3 Asparate-specific cysteine proteases Drugs and chemicals (caspases) 4 play a central role in the execution phase of apoptosis. 5,6 The interleukin-1␤-converting enzyme (ICE) has Camptothecin was a generous gift from Drs ME Wall and MC been the first described homologue of the ced-3 death gene Wani (Research Triangle Park, NC, USA). It was freshly disproduct (CED-3) of Caenorhabditis elegans. 7 Other members solved in dimethyl sulfoxide at 10 mM and further diluted in of the caspase family including caspase 3 (CPP32/Yama/ water before each experiment. The caspase inhibitor, N-benApopain) 8,9 have been identified and recent evidence suggests zyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (Zthat several caspases can be activated sequentially in the same VAD-fmk), and the serine protease inhibitor, 3,4-dichloroisoapoptotic cell. 10 Poly(ADP-ribose)polymerase (PARP) 8,9 and coumarin (...

Human Apurinic/Apyrimidinic Endonuclease (Ape1) and Its N-terminal Truncated Form (AN34) Are Involved in DNA Fragmentation during Apoptosis

“…Thus, it is likely that AN34 is a cellular processing product of Ape1 after cleavage of Ape1 by a trypsin-like enzyme activated by caspase-3 during apoptosis. Several studies have reported that serine proteases may play a role in inducing chromatin DNA fragmentation during apoptosis (41)(42)(43). At the present time, the identity of the protease(s) involved in Ape1 processing and generation of AN34 remains unknown.…”

Sodium orthovanadate suppresses DNA damage-induced caspase activation and apoptosis by inactivating p53

“…Interestingly, the suppression of this apoptosis occurred exclusively among three wild-type p53 cell lines and not in two p53-null cell lines (Figure 4a and b). 1,[17][18][19][20] It is known that these three cell lines show p53-dependent apoptosis when stimulated by DNAdamaging agents. 1,17,18 In fact, these wild-type p53 cell lines showed the activation (accumulation) of p53 after the DNAdamaging treatments, and, as expected, the two p53-null cell lines did not (Figure 4d).…”

Caspase I-related protease inhibition retards the execution of okadaic acid- and camptothecin-induced apoptosis and PAI-2 cleavage, but not commitment to cell death in HL-60 cells

“…Camptothecin, a topoisomerase I inhibitor (reviewed in Rothenberg, 1997), was included in the study as an independent inducer of apoptosis (Del Bino et al, 1992;Shimizu and Pommier, 1997). We report that, although morphological and biochemical parameters of apoptosis, including PAI-2 cleavage, could be blocked by protease inhibition, the time course for commitment to cell death was not prolonged.…”