Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVDaminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100-and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(N ␣ -benzyloxycarbonylglutamyl-N ⑀ -biotinyllysyl)-aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one-and twodimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.Recent studies (reviewed in Refs. 1-5) indicate that the cytotoxicity of virtually all chemotherapeutic agents is accompanied by apoptosis in susceptible cell lines. Likewise, experiments in animals (6 -9) and studies of circulating blasts from leukemia patients (10) have provided evidence that chemotherapy is accompanied by apoptosis in vivo. Moreover, it has been suggested that resistance to the cytotoxic effects of chemotherapeutic agents can result from resistance to chemotherapyinduced apoptosis (8,11,12). These observations highlight the potential importance of understanding the factors that control apoptosis.

Abstract. Previous studies have shown that in neuronal cells the developmental phenomenon of programmed cell death is an active process, requiring synthesis of both RNA and protein. This presumably reflects a requirement for novel gene products to effect cell death. It is shown here that the death of nerve growth factor-deprived neuronal PC12 cells occurs at the same rate as that of rat sympathetic neurons and, like rat sympathetic neurons, involves new transcription and translation. In nerve growth factor--deprived neuronal PC12 cells, a decline in metabolic activity, assessed by uptake of [3H]2-deoxyglucose, precedes the decline in cell number, assessed by counts of trypan blue-excluding cells. Both declines are prevented by actinomycin D and anisomycin. In contrast, the death of normeuronal (chromaffin-like) PC12 cells is not inhibited by transcription or translation inhibitors and thus does not require new protein synthesis. DNA fragmentation by internucleosomal cleavage does not appear to be a consistent or significant aspect of cell death in sympathetic neurons, neuronal PC12 cells, or nonneuronal PC12 cells, notwithstanding that the putative nuclease inhibitor aurintricarboxylic acid protects sympathetic neurons, as well as neuronal and nonneuronal PC12 cells, from death induced by trophic factor removal. Both phenotypic classes of PC12 cells respond to aurintricarboxylic acid with similar dose-response characteristics. Our results indicate that programmed cell death in neuronal PC12 cells, but not in nonneuronal PC12 cells, resembles programmed cell death in sympathetic neurons in significant mechanistic aspects: time course, role of new protein synthesis, and lack of a significant degree of DNA fragmentation.TAINMENT of proper cell number in each region of the organism is an essential aspect of development and morphogenesis. To accomplish this, not only is the rate of celt proliferation tightly regulated, but there is also a program of cell death which results in the elimination of specific subsets of cells (28,43,46). Programmed cell death (PCD) t provides a rapid and efficient method for achieving proper tissue mass and cell number as well as for eliminating supernumerary or dysfunctional cells. During neuronal development, PCD operates to ensure precise quantitative matching of presynaptic and postsynaptic pools of cells (28). Determination of neuronal survival appears to occur via an intercellular competition for a limited supply of target-derived neurotrophic support (28). Thus, an augmented supply of nerve growth factor (NGF) reduces ha vivo death of sympathetic and sensory neurons while NGF deprivation, in vivo or in vitro, causes them to die (21). PCI2 cells, grown in serum-containing medium, do not require NGF for their survival although treatment with NGF induces neuronal

The activation of multiple interleukin-1b converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an a nity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identi®ed six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these ®ndings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

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We describe a timetable of events during programmed cell death (PCD) in neuronal PC12 cells, specifically, Ras signaling, immediate-early gene (IEG) expression, DNA fragmentation and commitment to PCD. Commitment occurs over a period from 10-20 hr after NGF withdrawal. Ras signaling declines rapidly after NGF removal, reaching minimal levels within 2-4 hr, well before the onset of commitment. DNA fragmentation, detected by TUNEL reaction, begins about 24 hr after NGF withdrawal, well after all cells are committed, but coincident with the onset of cell dissolution previously determined by trypan blue exclusion (Mesner et al., 1992). Among the IEGs studied here, c-jun and TIS21 are expressed within 6 hr after NGF withdrawal. Expression of c-fos, egr-1, and TIS11 does not begin until 20 hr after NGF withdrawal. IEG expression generally ends by 24 hr after NGF withdrawal. The IEGs TIS7 and nur77 are not expressed during PCD, yielding a pattern distinct from that following other stimuli. An identical pattern of IEG expression occurs in non-neuronal PC12 cells deprived of serum, although expression begins at 10-14 hr after serum withdrawal. A similar IEG expression pattern was observed in Rat-1 fibroblasts, with various genes expressed 6-18 hr after serum withdrawal. In none of these cell types did expression of the stress-related gene Hsp70 change following trophic factor withdrawal. The distinctive pattern of IEG expression described here should facilitate identification of intracellular regulatory signals active during PCD.