The Pro-Apoptotic Drug Camptothecin Stimulates Phospholipase D Activity and Diacylglycerol Production in the Nucleus of HL-60 Human Promyelocytic Leukemia Cells¹ Free

Cell death by apoptosis is a fundamental process of mammalian organisms, in which it plays a major role in development, immune system maturation, tissue homeostasis, and aging. A growing body of evidence indicates that deregulation of apoptosis is most likely to have important implications for neoplastic transformation (1, 2). Moreover, the possibility of modifying sensitivity to apoptosis through its regulatory pathways has clear implications for the treatment of cancer (3). There is increasing evidence that diverse death signals activate different pathways that converge toward a conserved execution machinery, which depends on one or more members of a family of aspartic-acid-directed cysteine proteases called caspases (4). During the apoptotic process, dramatic morphological and biochemical changes take place in the nucleus (5, 6, 7, 8). Nuclear protein phosphorylation is likely to play an important role in the regulation of apoptosis (8). There are a few reports concerning the phosphorylation of nuclear proteins during apoptosis; in dexamethasone-treated thymocyte cultures, phosphorylation and cleavage of M_r 240,000 NuMA protein are very early events, detectable after a 30-min exposure to the hormone and preceding internucleosomal DNA cleavage (9). At present, there is no indication of the protein kinase that may phosphorylate NuMA during apoptosis. On the other hand, in thymocytes exposed to the pro-apoptotic fungal toxin, gliotoxin, a protein kinase A-dependent serine-phosphorylation of histone H3 has been reported (10). Very recently, the hyperphosphorylation of replication protein A middle subunit has been described in apoptotic Jurkat cells, and it seems mediated by both DNA- and cyclin-dependent protein kinases (11).

Another protein kinase that may be involved in apoptosis is PKC.³ The very first report linking nuclear PKC to apoptosis was published in 1994 by Trubiani et al. (12), who showed a redistribution of the kinase toward the nucleus in dexamethasone-treated thymocytes. In spontaneously apoptotic U937 cells, PKC-δ showed a reduced nuclear level of expression while PKC-α was increased in this cell compartment (13). A direct link between PKC and apoptosis was subsequently demonstrated in HL-60 cells induced to apoptosis by camptothecin (14). Indeed, it was shown that the α isoform of PKC phosphorylated lamin B 1 h after the addition of the drug, and this phenomenon preceded both proteolytic degradation of lamin B and DNA fragmentation. It is worth recalling that in HL-60 cells, PKC-β_II phosphorylates lamin B during the G₂-M phase transition (15), and this PKC isozyme is activated at the nucleus by DAG produced by a PI-PLC (16). In this article, we demonstrate that nuclei isolated from HL-60 cells exposed to camptothecin have more PKC-α protein than controls, and that the kinase is active. Moreover, the activation of nuclear PKC-α protein is preceded by a rise in nuclear DAG levels. Both the DAG rise and the activation of PKC-α at the nucleus were suppressed by propranolol (an inhibitor of PLD) but not by either ET-18-OCH₃ (a specific inhibitor of PI-PLC) or D609 (a purported inhibitor of PC-PLC). Interestingly, propranolol, but not the two other inhibitors, dramatically reduced the number of HL-60 cells entering apoptosis after exposure to camptothecin.

RPMI 1640, fetal bovine serum, 1,2-dioleyl-3-palmitoyl-glycerol, dioleylglycerol, CHAPS, phosphatidylserine, phosphatidic acid, normal goat serum, peroxidase-conjugated antirabbit, antigoat, and antimouse IgG, camptothecin, histone H1, leupeptin, aprotinin, PMSF, benzamidine, rabbit polyclonal antibody to PKC-α, DNase I, RNase A, and BSA were obtained from Sigma Chemical Co. (St. Louis, MO). Propidium iodide and phosphatidylethanol were from ICN Pharmaceuticals (Costa Mesa, CA). ET-18-OCH₃, propranolol, and D609 were from Calbiochem (La Jolla, CA). Enhanced chemiluminescence detection kit and NP40 were from Roche Molecular Biochemicals (Milan, Italy). [γ-³²P]ATP, phosphatidyl[methyl-[³H]]choline, [³H]PIP₂, and [³H] palmitic acid were from Amersham Pharmacia Biotech (Uppsala, Sweden). Protein A-Agarose and mouse monoclonal antibody to histone H1 (clone AE4) were from Upstate Biotechnology Incorporated (Lake Placid, NY). Goat polyclonal antibody to IGF-I receptor β chain (C-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). The Protein Assay kit (detergent-compatible) was from Bio-Rad (Hercules, CA). p81 paper and LK5D chromatography plates were obtained from Whatman International Ltd. (Maidstone, Kent, United Kingdom).

HL-60 human promyelocytic leukemia cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. For induction of apoptosis, cells were exposed for 3 h to 0.1 μg/ml DNA topoisomerase I inhibitor, camptothecin. Quantitative analysis of apoptosis was carried out by flow cytometry. Cells were fixed with 70% ethanol for 30 min at 4°C, washed in PBS, and left 10 min in PBS at 4°C. Propidium iodide was then added to each sample at a final concentration of 40 μg/ml. Flow analysis was performed by a FACStar Plus flow cytometer (Becton Dickinson, Palo Alto, CA) equipped with an argon ion laser tuned at 514 nm to excite propidium iodide. Propidium iodide red fluorescence was collected on a log scale and apoptotic cells were identified as a subdiploid peak.

This was accomplished essentially as reported by Fields et al. (17). All of the steps were executed at 4°C in buffers containing 0.1 mm Na₃VO₄, 20 mm NaF, 10 μm aprotinin, 10 μm benzamidine, and 1 mm PMSF. Cells were washed three times with PBS and hypotonically swelled in 50 mm of Tris-HCl (pH 7.4), 250 mm sucrose, and 5 mm MgSO₄, containing 1% (v/v) 2-mercaptoethanol for 10 min at 1 × 10⁷ cells/ml. Then, 10% (w/v) NP40 was added to a final concentration of 0.02% (w/v), and the cells were lysed with 50 strokes of a Dounce homogenizer using a B-type pestle. The lysate was layered over a cushion of 2.1 m sucrose, 50 mm of Tris-HCl (pH 7.4), 5 mm MgSO₄, and 1% 2-mercaptoethanol; and the nuclei were pelletted at 70,000 × g for 60 min in a Beckman SW28 rotor.

Purity of nuclear preparation was assessed by Western blotting analysis using either goat polyclonal antibody to IGF-I receptor β chain or monoclonal antibody to histone H1.

To obtain homogenates, cells were resuspended in 50 mm Tris-HCl, (pH 7.4), 1 mm EDTA, 1 mm EGTA, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.1 mm Na₃VO₄, 20 mm NaF, 10 μm aprotinin, 10 μm benzamidine, and 1 mm PMSF, and homogenized by 30 passages through a 25-gauge needle. Cytosolic extracts were prepared according to Shimizu et al. (14).

Nuclear extracts were prepared essentially as reported previously (18), with some modifications. Nuclei were resuspended in 5 mm Tris-HCl (pH 8.0), 1 mm EGTA, 1 mm EDTA, 0.1 mm Na₃VO₄, 20 mm NaF, 10 μm aprotinin, 10 μm benzamidine, 1 mm PMSF, and 0.3% Triton X-100, then ruptured by 50 passages through a 25-gauge hypodermic needle, and centrifuged at 5,000 × g to remove insoluble material. Nuclear extracts (1 ml, containing 500 μg of protein) was precleared by adding 5 μg of normal rabbit IgG and 10 μg of 50% Protein A-Agarose, followed by incubation for 1 h at 4°C and centrifugation at 12,000 × g for 10 min at 4°C. The samples were incubated for 4 h at 4°C under constant agitation with 5 μg of polyclonal antibody to PKC-α. 50% Protein A-Agarose (10 μg) was added and incubation proceeded for 1 h at 4°C under constant agitation, then centrifuged. The beads were washed once with lysis buffer and twice with kinase buffer [50 mm Tris-HCl (pH 7.4), 10 mm NaF, 1 mm Na₃VO₄, 0.5 mm EGTA, 0.5 mm EDTA, 2 mm MgCl₂, 5 μg/ml leupeptin, and 1 mm PMSF].

This was performed using the Bio-Rad Protein Assay (detergent compatible) according to the instructions of the manufacturer.

Proteins separated on SDS-PAGE (19) were transferred to nitrocellulose sheets. Sheets were saturated in PBS containing 5% normal goat serum and 4% BSA for 60 min at 37°C (blocking buffer), then incubated overnight at 4°C in blocking buffer containing the primary antibodies. After four washes in PBS containing 0.1% Tween 20, they were incubated for 30 min at room temperature with the appropriate peroxidase-conjugated secondary antibody, diluted 1:3,000 in PBS-Tween-20, and washed as above. Bands were visualized by the enhanced chemiluminescence method.

Immunocomplex beads were incubated in 50 μl of 20 mm Tris-HCl (pH 7.4), 10 mm MgCl₂, 10 μm ATP, 0.4 mg/ml histone H1, and 5 μCi of [γ-³²P]ATP in the presence of 1.2 mm CaCl₂, 40 μg/ml phosphatidylserine, 1.2 mm of CaCl₂, and 3.3 μm dioleylglycerol. Incubations were carried out at 30°C for 10 min. The reactions were terminated with 15 μl of acetic acid and spotted on to Whatman p81 paper, followed by washing with 0.75 mm H₃PO₄. Radioactivity was measured by Cerenkov counting.

The assay was performed according to Divecha et al. (20), using DAG kinase enzyme purified from rat brain. DAG was extracted from nuclei, whole cells, and cytosolic extracts dissolved in 20 μl of CHAPS (9.2 mg/ml) and sonicated at room temperature for 15 s. After the addition of 80 μl of reaction buffer [50 mm Tris-acetate (pH 7.4), 80 mm KCl, 10 mm Mg acetate, and 2 mm EGTA], the assay was started by the addition of 20 μl of DAG kinase enzyme followed by 80 μl of reaction buffer containing 5 μm ATP, and 1 μCi of [γ-³²P]ATP. Incubation was for 1 h at room temperature; then phosphatidic acid was extracted, chromatographed, and autoradiographed, and its radioactivity was counted in a liquid scintillation system (Betamatic IV, Kontron, Milan, Italy). Standard curves were obtained as reported by Divecha et al. (20), using 1,2-dioleyl-3-palmitoyl-glycerol as substrate.

This was accomplished in one of two ways:

(a) isolated nuclei were resuspended at 4°C in reaction buffer [50 mm Hepes-NaOH (pH 7.2), 2 mm EDTA, 0.5 mm EGTA, 5 mm DTT, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mm Na₃VO₄]. Nuclear protein (150 μg in 400 μl of buffer) was incubated with 100 μl of a Triton X-100 (6.25 mm), phosphatidyl[methyl-[³H]]choline (2.25 mm at 29 μCi/μmol) mixed micelle (3:1, Triton X-100:PC). The reaction mixture (a total reaction volume of 500 μl) was incubated at 37°C for 1 h, and the released water-soluble headgroups were separated by ion pairing with tetraphenylboron and quantified by liquid scintillation counting (21); or

(b) cells were labeled for 20 h in the presence of [³H]palmitic acid (5 μCi/ml). Nuclei were isolated and incubated (50 μg per assay in 200 μl final volume) for 30 min at 37°C in 25 mm Hepes-NaOH (pH 7.4), 100 mm KCl, 3 mm NaCl, 5 mm MgCl₂, 1 μm CaCl₂, 1 mm PMSF, 10 μm benzamidine and leupeptin, and 1.5% ethanol (22, 23). Total lipids were extracted (24), and the radioactivity that was incorporated was quantified at this time by scintillation counting. Phosphatidylethanol and phosphatidic acid were resolved from nuclear lipids by TLC according to standard methods (21, 22, 23, 24). Spots of interest were identified by comparison with authentic standards, scraped from the plates, and counted by scintillation counting.

Values were expressed as percentage of radioactivity in either phosphatidylethanol or phosphatidic acid with respect to total nuclear phospholipid.

The procedure outlined by Martelli et al. (18) was followed. Briefly, assays (100 μl) contained 100 mm 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.7), 150 mm NaCl, 0.06% sodium deoxycholate, 3 nmol [³H]PIP₂ (specific activity: 30,000 dpm nmol^-1), and 10 μg of nuclear protein. Incubation was for 30 min at 37°C. Hydrolysis was stopped by adding chloroform-methanol-HCl, and PIP₂ hydrolysis was quantified by liquid scintillation.

Data are the mean from three different experiments and are expressed as mean ± SD. The asterisk indicates significant differences (P < 0.01) in a Student’s paired t test. All of the other differences were found to be not significant with P > 0.01.

Because a very critical issue in the present study was the purity of nuclear preparations, we addressed it by Western blotting analysis. As shown in Fig. 1, our preparations of nuclei, when compared with whole cell homogenates, were highly enriched in histone H1 and devoid of the β chain of IGF-I receptor (which is expressed by these cells; see Ref. 25).

Then, we assayed PKC-α activity in isolated nuclei prepared from HL-60 cells treated for various times with the pro-apoptotic chemical camptothecin. Because various PKC isoforms have been reported to be present in HL-60 cell nuclei (26, 27), we prepared nuclear extracts and immunoprecipitated PKC-α using a specific polyclonal antibody. The immunoprecipitates were then tested for PKC-α activity using histone H1 as substrate. As shown in Fig. 2, nuclear PKC-α activity started to increase 30 min after the addition of camptothecin and peaked at 60 min, when an almost 4-fold increase was seen. After 150 min, the enzyme activity returned to basal levels.

We next wanted to determine whether the increase in nuclear PKC-α activity might be due to an increase in the protein amount present in isolated nuclei. As shown in Fig. 3,A, Western blotting analysis performed on isolated nuclei, revealed that the amount of PKC-α dramatically increased in parallel with the activity increase. In contrast, the levels of PKC-α protein detected in either whole HL-60 cell homogenates or cytosolic extracts remained constant (Fig. 3, B and C).

Nuclei were isolated from HL-60 cells after different times of camptothecin exposure and assayed for their DAG levels. Control nuclei contained about 37.2 ± 3.8 pmol of DAG per mg of protein, but after 15 min of treatment, there was an approximately 20% increase to 44.4 ± 4.5 pmol/mg of protein. At 30 min, the value rose to 122.6 ± 14.2 pmol/mg of protein, i.e., a nearly 3.5-fold increase (Fig. 4,A). Nuclear DAG levels peaked at 45 min after administration of the apoptotic stimulus and remained at almost the same level for up to 90 min. However, after 150 min, nuclear DAG mass returned to basal levels. Because HL-60 cell nuclei have been reported to contain at least two types of phospholipases that are capable of generating DAG (PI-PLC and PLD; see Refs. 28 and 29, respectively), we used inhibitors of these phospholipases to clarify the lipid source for nuclear DAG. As shown in Fig. 4,B, both the specific PI-PLC inhibitor, ET-18-OCH₃ (used at 100 μm), and the purported PC-PLC inhibitor D609 (30 μm) did not affect the rise in nuclear DAG. In contrast, propranolol (100 μm; a blocker of PLD-mediated DAG generation) dramatically reduced the nuclear DAG increase (Fig. 4,B). As an additional control, we measured the levels of DAG in whole HL-60 cell homogenates or cytosolic extracts. As presented in Fig. 4, C and D, no increase was seen at any time after camptothecin exposure.

We next measured PLD activity in isolated nuclei by two different methods. We first evaluated the levels of water-soluble headgroups released from phosphatidyl[methyl-[³H]]choline incubated in vitro with isolated nuclei. As shown in Fig. 5, the increase in nuclear PLD activity paralleled the increase in nuclear DAG presented in Fig. 4 A. Moreover, this nuclear PLD activity was sensitive to propranolol but not to ET-18-OCH₃.

Then, we measured in vitro nuclear PLD activity by an assay in which isolated nuclei, prepared from [³H]palmitic acid-labeled cells, are incubated in vitro in the presence of ethanol. Under these conditions, detection of PLD activity is based on the formation of phosphatidylethanol, a product that is generated from PLD by a transphosphatidylation reaction if ethanol is present (22, 23). As presented in Table 1, the percentage of radioactive phosphatidylethanol relative to other lipids measured in nuclei prepared from control cells was 0.41 ± 0.08. Forty-five min after exposure to camptothecin, the percentage had risen to 1.86 ± 0.24, i.e., a more than 4-fold increase. At 150 min, the percentage of phosphatidylethanol had returned to basal levels. Moreover, in the same Table, we also show that radiolabeled nuclear phosphatidic acid increased with a time-dependent accumulation similar to that seen for both phosphatidylethanol and DAG, which suggests a precursor-product of relationship, as observed by others previously (22).

As a further control, we measured PI-PLC activity in isolated HL-60 nuclei after treatment with camptothecin. The results are shown in Table 2. No enhancement of basal activity was measured at any time after incubation with the pro-apoptotic drug.

To test whether activation of PKC-α was dependent on the levels of DAG present in isolated nuclei, we measured the isozyme activity in nuclei prepared from cells that had been pretreated for 10 min with the inhibitors and then had been exposed to camptothecin. The results from these experiments are presented in Fig. 6. It is clear that whereas neither ET-18-OCH₃ nor D609 blocked nuclear PKC-α activation, propranolol did block it. Moreover, as shown in Fig. 7,A, Western blotting analysis revealed that, in nuclei obtained from cells that were pretreated with propranolol and exposed to camptothecin for 45 min, the amount of PKC-α protein was dramatically reduced in comparison with nuclei prepared from control cells (no pretreatment) or from cells pretreated for 10 min with either ET-18-OCH₃ or D609. In contrast, in nuclei obtained from cells exposed to the inhibitors but not to camptothecin, the nuclear PKC-α levels did not show changes (Fig. 7 B).

Finally, we evaluated whether or not the chemicals used to inhibit phosphalipases could block the induction of apoptosis caused by camptothecin. As shown in Table 3, propranolol, but not ET-18-OCH₃ nor D609, dramatically reduced the number of apoptotic HL-60 cells.

Proteolysis of lamin B has long been recognized as one of the most distinctive features of apoptotic nuclear changes (5, 30). Very recently, Shimizu et al. (14) demonstrated that in camptothecin-treated HL-60 cells a PKC-α-dependent lamin B phosphorylation preceded its proteolytic degradation as well as chromatin fragmentation. It is important to recall that in HL-60 cells lamin B phosphorylation also occurs at the G₂-M phase transition of the cell cycle. The protein kinase responsible for the phosphorylation of lamin B at the G₂-M phase transition was identified as the -β_II isozyme of PKC. In this case, however, phosphorylation causes a solubilization of lamin B followed by a breakdown of the nuclear envelope (31). Therefore, PKC-mediated lamin B phosphorylation during apoptosis may also affect nuclear and chromatin structure. Interestingly, Sun et al. (17) have reported that, in HL-60 cells during the G₂-M phase transition, there is an increase in the levels of nuclear DAG, which is responsible for the activation of PKC-β_II. Because of these results, we sought to determine whether in the nuclei of camptothecin-treated HL-60 cells, there also might be an increase in DAG mass that might explain the activation of PKC-α. Indeed, an increase in nuclear DAG linked to translocation and/or activation of PKC-α has been demonstrated to occur in other experimental models, mostly during cell proliferation (20, 32, 33).

We first demonstrated that in nuclei isolated from HL-60 cells treated with camptothecin, there is an increase in PKC-α activity. The activity enhancement started at 30 min after the initiation of treatment and returned to basal levels at approximately 150 min. This is in good agreement with the results of Shimizu et al. (14), which showed that in vivo phosphorylation of lamin B in camptothecin-exposed HL-60 cells was first detectable at 30 min after starting incubation and declined at 180 min. They also showed an induction of PKC-α activity in whole HL-60 cells that peaked around 60 min after the incubation of camptothecin and that was largely gone after 3 h. It may be that this increase in total-cell PKC-α activity reflects the enhancement of nuclear PKC-α activity that we have demonstrated to occur on a similar time scale. The same group (34) had previously reported that changes in soluble PKC-α activity in this experimental model were seen at later times and were due to a hyperphosphorylation of the kinase.

In contrast, the results of our Western blotting analysis showed that isolated HL-60 nuclei that were prepared from cells exposed to camptothecin retained more PKC-α protein. However, this increase is likely to be due to an insolubilization of PKC-α already resident in the nucleus because, by immunofluorescent staining, we did not see a translocation of cytoplasmic PKC-α to the nucleus after treatment with camptothecin,⁴ in contrast to the behavior of PKC-β_II in bryostatin-treated HL-60 cells (35).

This close association of PKC-α with the nucleus may be due to an increase in nuclear DAG levels. In fact, it has been previously hypothesized that PKC may be continuously cycling in and out of the nucleus and may become fixed in the nucleus by an increase of DAG, which can activate it (20). Using mass assays, we showed that an increase in the level of nuclear DAG preceded activation of PKC-α at the nucleus. Next, we tried to determine the source of nuclear DAG, because at least two different types of phospholipase activities have been shown to operate at the nuclear level and generate DAG, PI-PLC, and PLD (18, 28, 36, 37, 38). To this end, we used various inhibitors that have been reported to be specific for these enzymes (16, 33). The increase in nuclear DAG mass was not affected by either ET-18-OCH₃, a specific inhibitor of PI-PLC, or by D609, a purported inhibitor of PC-PLC. In this connection, it should be recalled that the existence of PC-PLC in mammalian cells is controversial (39), although some evidence points to the presence of such an enzyme in isolated nuclei (21, 40). In contrast, propranolol, a well- established blocker of PLD-mediated DAG generation, almost completely inhibited the DAG rise. It should be recalled that our nuclei were prepared according to a protocol that allows the conservation of the nuclear envelope (17). Thus, our results are suggestive of events that take place at the nuclear envelope level in agreement with the “NEST” hypothesis proposed by Raben et al. (41). However, there is also evidence that HL-60 nuclei—deprived of their envelope—still retain a PLD activity (29). Activation of PLD after treatment with camptothecin was demonstrated in isolated nuclei using two different enzymatic assays (42, 43, 44). Both of the assays demonstrated an approximately 4-fold increase in nuclear PLD activity. In contrast, we did not detect significant changes in nuclear PI-PLC activity. Furthermore, exposure of cells to propranolol, but not to either ET-18-OCH₃ or D609, prevented the rise in nuclear PKC-α activity and protein levels. An important role played by nuclear PLD in the control of apoptotic events elicited by camptothecin in HL-60 cells was also suggested by the fact that, of the three inhibitors we used, only propranolol was able to significantly reduce the number of apoptotic HL-60 cells measurable at various times after incubation with camptothecin.

Thus, a model could be envisioned in which nuclear PI-PLC and PKC-β_II are necessary for HL-60 cell proliferation and survival because PKC-β_II is essential for the transition from G₂- to M- phase of the cell cycle (45), and ET-18-OCH₃, which inhibits PI-PLC, induces apoptosis (16). In contrast, both nuclear PLD and PKC-α play a role in camptothecin-mediated apoptosis.

It may be that the differences in the composition of fatty acids between DAG generated by PI-PLC and those generated by PLD are important for attracting and/or activating different PKC isozymes at the nuclear level. Our results also demonstrate that after the camptothecin-treatment of HL-60, a rise occurred only in nuclear DAG, whereas DAG levels in other cell fractions did not change. However, we were unable to induce apoptosis in these cells by the use of membrane-permeant DAG analogues such as dioctanoyl-sn-glycerol (data not shown; see Ref. 46). Therefore, it seems that DAG is necessary but not sufficient to induce the apoptotic process in camptothecin-exposed HL-60 cells. We can speculate that lamin B, once it is phosphorylated by DAG-activated PKC-α, becomes more susceptible to proteases, which are then required—together with nucleases—for the completion of the execution phase of apoptosis. This is consistent with the observation that overexpression of mutated lamins A or B resistant to caspase cleavage delayed DNA fragmentation, a fact that could indicate the lamins are the only caspase substrates known to be directly involved in the execution phase of apoptosis (14).

It is still unclear how a nuclear PLD may become activated during apoptosis. Baldassare et al. (21) have demonstrated that a nuclear translocation of RhoA mediates the mitogen-induced activation of PLD involved in nuclear envelope signal transduction in IIC9 cells exposed to α-thrombin. In this connection, it should be emphasized that there are some reports hinting at an important role played by RhoA in the induction of apoptosis (for example, Ref. 47). Nevertheless, RhoA has also be demonstrated to counteract apoptosis, for example, by increasing Bcl-2 expression (48, 49). In any case, we think that involvement of RhoA in the activation of nuclear PLD activity during apoptosis should be further investigated.

An important role played by PKC-α during apoptosis has been suggested by Rusnak and Lazo (50) in DU-145 human androgen-independent prostatic carcinoma cells exposed to either etoposide or melphalan. However, other investigators came to opposite conclusions by studying COS cells (51). Because these differences may be due to the diverse cell types studied, future investigations entailing the use of transfected cDNAs for both PKC isoforms in sense or antisense orientation (52) should provide important clues as to the exact role(s) played by either PKC-α or PKC-β_II in HL-60 cells during cell proliferation and/or apoptosis.

In conclusion, we think that identification of a PLD-mediated pathway operating at the nuclear level during apoptosis may open new fields to the experimental use of specific inhibitors and/or activators that in the future may also become relevant clinically for the control of apoptotic events in human disease, including cancer.