Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes

Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.     

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.     

Inhibition of apoptosis by the actin-regulatory protein gelsolin

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.     

Bcl-2 relieves the trans-repressive function of the glucocorticoid receptor and inhibits the activation of CPP32-like cysteine proteases

Glucocorticoid induces apoptosis in immature lymphocytes which is inhibitable by Bcl-2. Although glucocorticoid-mediated signal transduction is well understood, the mechanism of the induction of apoptosis by the activated glucocorticoid receptor as well as the inhibition of apoptosis by Bcl-2 remains enigmatic. Here we report that overexpressed Bcl-2 relieves the glucocorticoid receptor-mediated repressive function on the AP-1 activity and completely inhibits the activation of CPP32-like cysteine proteases. In contrast, glucocorticoid receptor-mediated transactivation was not affected by Bcl-2. This suggests that glucocorticoid may induce apoptosis by repressing transactivation by AP-1 which is relieved by Bcl-2. Furthermore, we report evidence that, in contrast with CPP32-like proteases, ICE-like proteases are not involved in this apoptotic pathway.     

Purification and characterization of an interleukin-1beta-converting enzyme family protease that activates cysteine protease P32 (CPP32)

CPP32, a member of the interleukin-1beta-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2alpha, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events.     

Retinoic acid treated P19 embryonal carcinoma cells differentiate into oligodendrocytes capable of myelination

Retinoic acid treatment of P19 embryonal carcinoma cells induces their differentiation into cultures containing neurons and astrocytes. We present two lines of experimentation indicating that oligodendrocytes also develop from retinoic acid-treated P19 cells. We isolated an immortal cell line from retinoic acid-treated P19 cell cultures whose proliferation is dependent upon epidermal growth factor. Upon removal of the growth factor these cells differentiate into both astrocytes and oligodendrocytes as determined by immunostaining with antibodies to the astrocyte marker glial fibrillar acidic protein and the oligodendrocyte markers, myelin associated glycoprotein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase. This cell line appears to be a bi-potential glial precursor. We also found that oligodendrocytes developed directly from P19 cells when retinoic acid-treated cells were transplanted into the brains of neonatal rat pups. Cells that developed into oligodendrocytes migrated into fiber bundles up to several millimeters from the site of the graft. These P19-derived oligodendrocytes appeared to myelinate axons from host neurons. Thus, retinoic acid-treated P19 cells differentiate into neurons, astrocytes and oligodendrocytes, the three cell types that normally develop from embryonic neuroectoderm, indicating that these cell cultures differentiate in a fashion closely resembling that of embryonic neuroectoderm.     

Identification of an interleukin-1 beta converting enzyme-like activity that increases upon treatment of P19 cells with retinoic acid as the proteasome

We examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation. The interleukin-1 beta converting enzyme (ICE)-like Ac-YVAD-MCA hydrolytic activity was increased about 6-fold by treatment with retinoic acid. This activity was inhibited by N-ethylmaleimide and Ac-YVAD-H but not by E-64, EDTA, PMSF, or amastatin. The ICE-like activity in P19 cells eluted as a single peak just after the void volume on gel filtration. No ICE-like activity was observed at a molecular mass of 30-50 kDa. Enzymatic purification, Western blot analysis, and an immunoabsorption study demonstrated that the ICE-like activity in P19 cells is caused by the proteasome, and is stimulated during retinoic acid-induced differentiation. The proteasome purified from mouse liver also cleaved Ac-YVAD-MCA. These results strongly suggest that the proteasome is a major ICE-like proteinase in P19 cells and may be involved in the neural differentiation and the apoptotic pathway.     

Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the 'response to injury' hypothesis

BACKGROUND: Oxidized LDL (oxLDL) is believed to play a key role as a triggering molecule that causes injury to the endothelium as an early event in atherogenesis. However, the mechanisms by which oxLDL injures endothelial cells are entirely unknown. We speculate that oxLDL may activate a cellular suicide pathway that leads to apoptosis. METHODS AND RESULTS: Human umbilical venous endothelial cells (HUVEC) were incubated with increasing doses of native or oxLDL for 18 hours. Apoptosis of HUVEC was measured with an ELISA specific for histone-associated DNA fragments and confirmed with DNA laddering. Native LDL had no effect, but incubation with oxLDL dose-dependently induced apoptosis of HUVEC. Induction of apoptosis by oxLDL was associated with increased CPP32-like protease activity, which is the major enzyme that initiates the proteolytical cascade leading to cell death. Specific inhibition of CPP32 activity completely abrogated oxLDL-induced apoptosis. The antioxidants N-acetylcysteine and the combination of vitamins C and E prevented oxLDL-induced apoptosis, abrogated the enhancement of CPP32-like protease activity, and inhibited the proteolytic cleavage of CPP32 into its active subunit p17. CONCLUSIONS: oxLDL activates the suicide pathway leading to apoptosis of endothelial cells by enhancing CPP32-like protease activity. The oxLDL-mediated activation of CPP32 appears to involve the elaboration of reactive oxygen species. Activation of the cell death effector CPP32 by oxLDL may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis.     

Ceramide induces apoptosis via CPP32 activation

Although both ceramide and interleukin-1beta converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell-permeable ceramide induced cleavage and activation of CPP32, a Ced-3/ICE-like protease, but not ICE. Ceramide-induced apoptosis of Jurkat cells was blocked by the CPP32-specific tetrapeptide inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both anti-Fas- and ceramide-induced apoptosis. These results indicate that CPP32 activation is required for ceramide-induced apoptosis, and suggest sphingomyelin-ceramide pathway functions upstream of CPP32.     

Not for resuscitation: two decades of challenge for nursing ethics and practice

The interleukin-1 beta converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH2F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 microM) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH2F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH2F), and CPP32-like proteases (Z-DEVD-CH2F), failed to prevent apoptotic death. An 35S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH2F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH2F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.     

Distinct interleukin-1beta-converting enzyme family proteases mediate cisplatin- and staurosporine-induced apoptosis of mouse proximal tubule cells

Interleukin-1beta converting enzyme (ICE) family proteases (caspases) are known to be implicated as important effectors of apoptotic pathways. The purpose of this study was to elucidate the role of ICE family proteases in apoptosis of mouse cells derived from the terminal proximal tubule (S3) treated with cisplatin, an anti-tumor drug, or staurosporine, a protein kinase C inhibitor. For this purpose, we measured the activities of ICE family proteases and examined the effects of tetrapeptide and viral ICE family protease inhibitors on the activities of ICE family proteases in and the degree of apoptosis of S3 cells treated with cisplatin and staurosporine. RT-PCR analysis revealed that S3 cells as well as mouse kidney express mRNA for ICE and CPP32, an ICE family protease. Results of enzymatic analysis, determination the degree of DNA fragmentation and cytotoxicity test suggest that CPP32 mediates cisplatin-induced apoptosis of S3 cells, whereas ICE family proteases other than CPP32 mediate staurosporine-induced apoptosis of S3 cells. In conclusion, distinct ICE family proteases mediate apoptosis of mouse proximal tubule cells depending on the stimuli to which the cells are exposed.     

Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.     

Intracellular K+ suppresses the activation of apoptosis in lymphocytes

Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K+ regulates the apoptotic process by controlling the activity of death enzymes. We show that K+, at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. Direct measurement of K+ by inductively coupled plasma/mass spectrometry and flow cytometry indicates a major decrease in intracellular K+ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K+. Disruption of the natural K+ electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K+ concentration is a necessary, early event in programmed cell death.     

Characterization of Toscana virus-defective interfering particles generated in vivo

Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.     

Histamine does not potentiate cyclic AMP-mediated amylase secretion in the guinea-pig pancreatic acinar cells

Some chemotherapeutic agents, as well as TNF and Fas, induce apoptotic cell death in tumor cells, but the cellular components involved in the process have not yet been identified. Interleukin 1 beta converting enzyme (ICE) is a mammalian homolog of CED-3, a protein required for programmed cell death in nematode Caenorhabditis elegans. We found that a selective inhibitor of ICE/ced 3 family proteases, benzyloxycarbonyl Asp CH2OC(O) 2 6,-dichlorobenzene (Z-Asp-CH2-DCB). completely blocked the apoptotic cell death of human leukemia cells caused by etoposide, camptothecin, 1-beta-D-arabinofuranosyl cytosine (Ara-C) and adriamycin. Moreover, in antitumor agent-treated U937 cells, an ICE-like (CPP32-like) protease was strongly activated. These results indicate that ICE/ ced 3 family proteases are involved in antitumor agent-induced apoptosis. Activation of ICE family proteases plays a key role in apoptosis. However, the subsequent mechanisms resulting in apoptosis are largely unknown. We identified actin as a substrate of ICE family proteases. Cleavage of actin and other substrate proteins by ICE family proteases could be critical in the ongoing process of antitumor agent-induced apoptosis in tumor cells.     

Pharmacological characterization of a simple behavioral response mediated selectively by central adenosine A1 receptors, using in vivo and in vitro techniques

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.     

Functional activation of Nedd2/ICH-1 (caspase-2) is an early process in apoptosis

The ICE/CED-3 family of proteases (caspases) play a central role in the execution phase of apoptosis. These proteases are synthesised as precursor molecules that require processing at specific aspartate residues to produce the two subunits that comprise the active enzyme. The activation of some of these proteases has been shown to occur during apoptosis. Here we show that Nedd2/ICH-1 (caspase-2) is activated during apoptosis induced by a variety of apoptotic stimuli. This activation occurs very early upon treatment of cells with apoptotic agents and appears to precede the activation of CPP32 (caspase-3). The activation of Nedd2 was not seen in cells that are resistant to apoptosis. These observations suggest that Nedd2 is an early effector in the pathway leading to cell death. Our observations also lend weight to the hypothesis that a group of caspases containing long prodomains are the first to be activated in response to apoptotic signals and that they lie upstream of a second class of caspases such as CPP32 containing short or no prodomains.     

Cooperative interception of neuronal apoptosis by BCL-2 and BAG-1 expression: prevention of caspase activation and reduced production of reactive oxygen species

Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N-acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert-butyl-alpha-phenylnitrone, and the antioxidant, N-acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.