Activation of PKCα Downstream from Caspases during Apoptosis Induced by 7-Hydroxystaurosporine or the Topoisomerase Inhibitors, Camptothecin and Etoposide, in Human Myeloid Leukemia HL60 Cells*

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCα, βI, βII, δ, and ζ activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCζ and inhibited kinase activity of PKCβI, βII, and δ. PKCα activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCα activity during apoptosis in HL60 cells. However, CPT did not affect PKCβI, βII and ζ, and activated PKCδ. PKCα activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCα autophosphorylation in vitro and increased phosphorylationin vivo. We also found that not only PKC δ but also PKC βI was proteolytically activated in HL60 cells during apoptosis. The PKCα activation and hyperphosphorylation were abrogated byN-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCδ and βI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCα and proteolytic activation for PKC δ and βI.

In the present study, we investigated PKC isoform activities during UCN-01-induced apoptosis in human myeloid leukemia HL60 cells. HL60 cells were chosen for these studies because they undergo rapid apoptosis to various agents including UCN-01. This apoptosis is p53-independent as HL60 cells are p53 null (22) and does not require protein synthesis (14,23). It is preceded by transient activation of cyclin B1/Cdc2 kinase (14,24). The changes of PKC activity were also examined in HL60 cells undergoing apoptosis after treatment with the topoisomerase inhibitors, CPT and etoposide (VP-16). We found that UCN-01 inhibited PKC␤I, ␤II, and ␦ activity in drug-treated cells as well as in vitro. For PKC␣, UCN-01 first inhibited the kinase activity and then activated PKC␣. CPT-induced apoptosis was associated with marked increase in PKC␣ and ␦ activities. The mechanisms of PKC activation and their relationship to caspases (ICE-like proteases) are investigated. Cell Culture-Human promyelocytic leukemia HL60 cells were grown at 37°C in the presence of 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD), 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin.
PKC Assays-Immunocomplex beads were obtained by incubating different anti-PKC antibodies with cytosolic extracts in 20 l of reaction buffer containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 10 M cold ATP, 0.4 mg/ml histone H1, 40 g/ml PS, 3.3 M dioleoylglycerol, 5 Ci of [␥-32 P]ATP in the absence or presence of 1.2 mM CaCl 2 depending on the PKC isoform activity measured. Incubations were carried out at 30°C for 10 min. Samples were loaded onto 12% SDS-PAGE gels (NOVEX, San Diego, CA) and electrophoresed at 120 V for 2 h. For quantification of kinase activity in immunoprecipitates, gels were dried, and the extent of histone H1 phosphorylation was measured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis of PKCs-Cytosolic samples were electrophoresed at 120 V on 12% SDS-PAGE gels and electrophoretically transferred to Immobilon membranes (Millipore, Bedford, MA) for 2 h at 30 V. The membranes were blocked overnight in phosphate-buffered saline-Tween 20 (PBS-T) containing 5% non-fat dried milk. Probing with selected anti-PKC antibodies (1 g/ml in PBS-T) for 1 h was followed by incubation with horseradish peroxidase-labeled anti-rabbit IgG (1:1000 dilution). After washing in PBS-T, membranes were developed using the enhanced chemiluminescence (ECL) detection system (NEN Life Science Products).
PKC␣ Phosphorylation in Cells-HL60 cells were spun and resuspended at 10 6 cells/ml in phosphate-free RPMI 1640 medium supplemented with 10% dialyzed fetal bovine serum once. After 1 h, 50 Ci/ml [ 32 P]orthophosphate was added to cell cultures for 1 h. Then cells were chased in isotope-free medium for 1 h prior to UCN-01 treatment. Cytosol extracts were boiled in electrophoresis sample buffer and analyzed by 10% SDS-PAGE gels. Gels were dried and autoradiographed at Ϫ70°C.
Detection of DNA Fragmentation and Reconstituted Cell-free System-DNA fragmentation related to apoptosis was measured by filter elution assay as described previously (8,25).

UCN-01-and CPT-induced Apoptosis Are Associated with
Increased PKC Activity in HL60 Cells-Our previous studies indicated that UCN-01 and CPT are potent inducers of apoptosis in HL60 cells (14,27). After 3 h of drug treatment, 60 -90% of the cells exhibit apoptosis with typical morphological changes and internucleosomal fragmentation. To investigate whether PKC was involved in apoptosis induced by these anticancer drugs, we treated HL60 cells with UCN-01 or CPT and extracted cytosol and membrane fractions at various times. The synthetic peptide, MBP4 -14, was used as a specific and sensitive substrate to assay PKC activity (26). We found that phosphorylation of MBP4 -14 first decreased during the first hour of UCN-01 treatment compared with control and then increased progressively above control values 3 h after the beginning of treatment. CPT increased the phosphorylation of MBP4 -14 already after 1 h of treatment (Fig. 1). These measurements were performed in cytosolic fractions. In membrane fractions, there was no significant difference between basal and stimulated levels of MBP4 -14 phosphorylation and no change of MBP4 -14 phosphorylation after exposure to UCN-01 or CPT for 3 h (not shown). These data indicate that PKC activity is induced by UCN-01 and CPT treatment in HL60 cells undergoing apoptosis.
UCN-01-and CPT-induced Apoptosis Are Associated with Up-regulation of PKC␣ Activity-The above data raised the question as to what kinds of PKC isoforms were activated by UCN-01 or CPT. MBP4 -14 is selective for PKC␣, ␤I, ␤II, and ␦ isoforms but has been reported to be a poor substrate for other isoforms (26) . Protein G-Sepharose beads were first washed with nucleus buffer and after with kinase buffer. Histone H1 kinase reactions were carried out at 30°C for 10 min and were terminated by adding SDS-PAGE sample buffer. Proteins were separated by 12% SDS-PAGE. Gels were dried and exposed in PhosphorImager cassettes.
know that UCN-01 inhibits PKC␣, ␤, ␥, ␦, and ⑀ and does not inhibit PKC in vitro. Since PKC␥ was undetectable in HL60 cells and PKC⑀ was expressed only very weakly using Western blotting (data not shown) (1), we used anti-PKC␣, ␤I, ␤II, , and ␦ antibodies and immunoprecipitation to determine the effects of UCN-01 and CPT on PKC isoform activities. Fig. 2 shows that UCN-01 inhibited all the PKC isoforms tested at 1 h of treatment, which is consistent with the known anti-PKC activity of UCN-01 in vitro (10). However, at 3 h, PKC␣ activity was markedly stimulated (5-fold) by UCN-01. CPT increased PKC␣ and ␦ activities but had no effects on PKC␤I and ␤II activities. Neither UCN-01 nor CPT affected PKC activity (data not shown). These data indicate that apoptosis induced by UCN-01 and CPT treatment is associated with marked PKC␣ activation.

Suppression of UCN-01-and CPT-induced Apoptosis by the Caspase Inhibitor, z-VAD-fmk, Is Associated with Lack of PKC␣
Activation-Caspases (ICE-like proteases) are key mediators of apoptosis (28 -30), and we previously showed that the caspase inhibitor z-VAD-fmk prevents UCN-01-induced apoptosis in HL60 cells (14). Fig. 3A, shows that z-VAD-fmk also prevents apoptotic DNA fragmentation induced by CPT in HL60 cells. However, z-VAD-fmk does not inhibit the DNA fragmentation induced by UCN-01-and CPT-activated cytosol in cell-free system (8,14,25) (Fig. 3B). Under these conditions, we examined whether z-VAD-fmk affected the PKC changes induced by UCN-01 or CPT in HL60 cells undergoing apoptosis. We found that z-VAD-fmk blocked the activation of PKC␣ induced either by UCN-01 or CPT. However, z-VAD-fmk did not suppress the CPT-induced activation of PKC␦ (Fig. 4A) and had no effects on  4. Effects of UCN-01, CPT, VP-16, and z-VAD-fmk on PKC␣ activity in HL60 cells. HL60 cells were exposed to either 10 M UCN-01 or 1 M CPT in the absence or presence of 50 M z-VAD-fmk for 3 h (panels A and B). HL60 cells were treated with 50 M VP-16 for the indicated times (panel C). Cytosol fractions from untreated or treated cells were immunoprecipitated with the indicated anti-PKC␤ antibodies (panel A) or anti-PKC␣ and ␦ antibodies (panels B and C). PKC activity was measured using histone H1 as substrate.
FIG. 5. In vitro effects of UCN-01, CPT, and z-VAD-fmk on PKC␣ activity. A, cytosols from untreated HL60 cells were immunoprecipitated with anti-PKC␣ antibody. UCN-01 or CPT was added to the immunoprecipitates. Then histone H1 kinase activity was measured. B, cytosol was prepared from untreated cells or from cells treated with CPT or UCN-01 for 3 h. Cytosols were then incubated with 50 M z-VAD-fmk for 30 min at 30°C. Samples were immunoprecipitated with anti-PKC␣ antibody. Histone H1 kinase activity was measured. DMSO, dimethyl sulfoxide. the inhibitions of PKC␤I, ␤II, and ␦ activities induced by UCN-01 (Fig. 4). We next tested whether PKC␣ was activated in response to the topoisomerase II inhibitor, VP-16. We found that VP-16 also activated PKC␣ in HL60 cells undergoing apoptosis (8) (Fig. 4C). These data suggested that apoptosis induced by different pathways (topoisomerase inhibition for CPT or VP-16 or protein kinase inhibition for UCN-01) is associated with PKC␣ activation in HL60 cells. The observation that z-VAD-fmk blocked PKC␣ activation suggests that PKC␣ is downstream from caspases during apoptosis in HL60 cells.
Apoptotic PKC␣ Activation Is Not Due to a Direct Effect of UCN-01 or CPT, and Suppression of PKC␣ Activation Is Not Due to a Direct Effect of z-VAD-fmk on PKC␣ Activity-To further investigate the mechanisms by which UCN-01 or CPT regulate PKC␣ activity, we first tested whether UCN-01 or CPT directly affected PKC␣ activity using in vitro kinase assays. PKC␣ immunoprecipitates obtained from control cytosol were tested for kinase activity with UCN-01 or CPT. Fig. 5A shows that UCN-01 did not activate but rather inhibited PKC␣ activity directly. CPT had no direct effect on PKC␣ activity (Fig. 5A). z-VAD-fmk was also tested in this system and did not exhibit a direct effect on PKC␣ activation induced by UCN-01 or CPT (Fig. 5B). These results indicated that the PKC changes observed in cytosols from cells treated with UCN-01 or CPT in the absence or presence of z-VAD-fmk were indirect.
We next examined whether the activation of PKC␣ by UCN-01 or CPT and the effects of z-VAD-fmk might be due to alterations of the PKC␣ protein. Western blot analyses were carried out on cytosols from untreated, UCN-01-or CPTtreated HL60 cells using anti-PKC␣ antibody. PKC␣ protein levels remained unchanged after drug-treatments (Fig. 6A). Thus, PKC␣ activation by UCN-01 and CPT was not due to changes in PKC␣ protein levels. We also measured the other PKC isoforms. Protein levels of PKC␤I, ␤II, and ␦ did not change significantly (Fig. 6, B-D). Interestingly, UCN-01 and CPT induced PKC␤I and ␦ cleavages when HL60 cells underwent apoptosis, and z-VAD-fmk blocked these proteolytic effects. Proteolytic activation of PKC␦ by caspases is consistent with results obtained in U-937 cells (21). However, our data suggest that PKC␤I is also cleaved by caspases during apoptosis in HL60 cells.
Since neither proteolytic activation nor change in protein levels appear to account for PKC␣ activation, we next tested the effects of UCN-01 and CPT on the PKC␣ autophosphorylation. It is indeed known that autophosphorylation is one of the important modes of regulation of PKC activity (31). Autophosphorylation assays for PKC␣ were carried out essentially as described for histone H1 phosphorylation except that histone H1 was omitted from the reactions. Fig. 7A shows that treatment of HL60 cells for 3 h with either UCN-01 or CPT increased PKC␣ autophosphorylation (Fig. 7A) and that z-VADfmk blocked this increased autophosphorylation (Fig. 7B). Finally, we performed experiments to test in vivo phosphorylation of PKC␣ in HL60 cells treated with UCN-01. Fig. 8 shows that UCN-01 increased PKC␣ phosphorylation after 1 h of treatment and that z-VAD-fmk inhibited the UCN-01-induced PKC␣ phosphorylation. These results suggest that caspase activation during apoptosis in HL60 cells is upstream from PKC␣ autophosphorylation and activation.

DISCUSSION
The results of Fig. 1 show that although UCN-01 is a PKC inhibitor and CPT is a topoisomerase I inhibitor, both of them induced activation of PKC. Using immunoprecipitation assay for PKC activities, we found that UCN-01 inhibited PKC␤I, ␤II, and ␦ activities in whole cells as well as in vitro but that PKC␣ was activated in HL60 cells undergoing apoptosis. Apoptosis induced by CPT, a DNA topoisomerase I inhibitor was also associated with activation of PKC␣ as well as PKC␦ activation.
Caspases play a central role in the apoptosis pathways (28 -30). z-VAD-fmk, a cell-permeable caspase inhibitor with broad specificity, blocks apoptosis induced by various stimuli including treatment by UCN-01 or CPT. Our results demonstrate  8. UCN-01-induced apoptosis is associated with PKC␣ phosphorylation in vivo. HL60 cells were incubated in phosphatefree RPMI 1640 medium supplemented with 10% dialyzed fetal bovine serum for 1 h. Fifty Ci/ml [ 32 P]orthophosphate was then added for 1 h. Cells were washed once with phosphate-free medium for 1 h and then 10 M UCN-01 was added. Cytosols were prepared at the indicated times and were immunoprecipitated with anti-PKC␣ antibody. The beads were boiled in sample buffer and analyzed by SDS-PAGE gel electrophoresis. The gel was dried and exposed to Kodak Rx film at Ϫ70°C. that z-VAD-fmk selectively inhibited the activation of PKC␣ induced by UCN-01 and CPT. On the other hand, z-VAD-fmk did not affect the inhibition of PKC␤I, ␤II, and ␦ activities by UCN-01 and the activation of PKC␦ by CPT. At the same time, we found that z-VAD-fmk blocked the proteolytic cleavages of PKC␤I and PKC␦, suggesting that PKC␦ cleavage is not the only mechanism for PKC␦ activation. For PKC␣, we found no evidence that PKC␣ activation is associated with proteolytic cleavage.
Our data (Fig. 5) rule out that UCN-01, CPT, or z-VAD-fmk directly affected PKC␣. PKC␣ activation is not due to changes in PKC␣ protein levels since control and cells treated with UCN-01 or CPT in the absence and presence of z-VAD-fmk had approximately equal amounts of PKC␣ protein as detected by Western blot. Phosphorylation has emerged as an important mode of regulation for PKC (3,32). For example, mutation of Thr to Ala in PKC␣ (Thr-497) results in an inactive kinase (33), and PKC autophosphorylation has been shown to regulate PKC activity by trans-phosphorylation at the activation loop followed by autophosphorylation (31,32). Our results show that UCN-01 and CPT activated autophosphorylation of PKC␣ and that z-VAD-fmk reversed such effects. Using [ 32 P]orthophosphate metabolic labeling, we also found increased phosphorylation of PKC␣ in HL60 cells undergoing apoptosis and blockade of these effects by z-VAD-fmk. These findings suggest that alterations of PKC␣ phosphorylation might be important for modulating PKC␣ activity and might be regulated by caspases.
The mechanisms (pathways) by which UCN-01 and CPT induce phosphorylation and activation of PKC␣ are yet unknown. We previously showed that both CPT and UCN-01 induce transient and unscheduled cyclin B1/Cdc2 kinase activation before the onset of apoptosis in HL60 cells treated with CPT or UCN-01 (14,24). CPT and DNA damaging agents have also been shown to activate other protein kinases including DNA-dependent protein kinase (34), jun kinase (35,36), and c-Abl (36). Further investigation will be required to establish the connections between activation of various protein kinases and caspases during apoptosis and to establish the functional significance of PKC␣ activation in the apoptotic response of HL60 cells.