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Immunology and Oncology Unit, Newcastle Mater Hospital, Newcastle, New South Wales, Australia

Requests for reprints: Peter Hersey, Immunology and Oncology Unit, Mater Hospital, Room 443, David Maddison Clinical Sciences Building, Corner King & Watt Streets, Newcastle, New South Wales 2300, Australia. Phone: 61-2-49-236828; Fax: 61-2-49-236184. E-mail: Peter.Hersey{at}newcastle.edu.au

	Abstract

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Protein kinase C (PKC) activation is believed to protect against apoptosis induced by death receptors. We have found however that the effect of activation of PKC on tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis of melanoma differs between cell lines. Pretreatment with phorbol 12-myristate 13-acetate (PMA) led to inhibition of apoptosis in the majority of the melanoma cell lines, but those with relatively low PKC expression were sensitized to TRAIL-induced apoptosis. Introduction of PKC into PKC-low cell lines reversed sensitization of the cells to TRAIL-induced apoptosis by PMA. In contrast, a dominant-negative form of PKC caused an increase in sensitivity. The changes in sensitivity to TRAIL-induced apoptosis were reflected in similar changes in conformation of Bax and its relocation from the cytosol to mitochondria. Similarly, there were concordant increases or decreases in mitochondrial release of second mitochondria-derived activator of caspase/DIABLO, activation of caspase-3, and processing of its substrates. Activation of PKC seemed to mediate its effects upstream of mitochondria but downstream of caspase-8 and Bid in that pretreatment with PMA did not cause significant changes in the expression levels of TRAIL death receptors, alterations in the levels of caspase-8 activation, or cleavage of Bid. PKC activated the antiapoptotic extracellular signal-regulated kinase 1/2 pathway, but inhibitors of this pathway only partially reversed the protective effect of PKC against TRAIL-induced apoptosis. These results provide further insights into the variable responses of melanoma to TRAIL-induced apoptosis and may help define responsive phenotypes to treatment of melanoma with TRAIL.

Key Words: Melanoma • TRAIL • Apoptosis • Protein kinase C

	Introduction

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Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptotic signaling is initiated by ligand-induced aggregation of death domains that reside on the cytoplasmic sides of the death receptors. This in turn orchestrates the assembly of adapter components, such as Fas-associated death domain that activate initiator caspases, caspase-8 and caspase-10, leading eventually to activation of effector caspases, such as caspase-3 (1, 2). We have shown in past studies that TRAIL-induced apoptosis of melanoma is largely mediated by the mitochondrial apoptotic pathway and that mitochondrial release of second mitochondria-derived activator of caspase (Smac) plays a critical role by binding to and inhibiting inhibitor of apoptosis protein family members (3, 4). The basis for the variation in Smac release remains largely unknown. It was suggested that the Bcl-2 protein family plays a pivotal role in regulating mitochondrion-mediated apoptosis by interaction between antiapoptotic members, such as Bcl-2, Bcl-x_L, and Mcl-1, and proapoptotic members such as Bax, Bad, and Bid (5). Among the latter, Bax seems essential for TRAIL-induced release of Smac from mitochondria (6, 7).

Protein kinase C (PKC) is a family of phospholipid-dependent serine/threonine kinases comprising at least 11 isoforms that play fundamental roles in signal transduction pathways that regulate cellular proliferation, differentiation, and apoptosis (8, 9). Activation of PKC by phorbol esters has been shown to have variable effects on apoptosis (10–16). In particular, activation of PKC seemed proapoptotic (10–12), whereas activation of PKC and PKC was antiapoptotic (13–16). Recent studies have implicated the PKC pathway in the protection of cells from apoptosis induced by death receptor ligation (13, 17–22). Activation of PKC has been reported to abrogate Fas-induced apoptosis through inhibition of death-inducing signaling complex formation by blocking Fas-associated death domain recruitment and thus caspase-8 activation (18–20). A similar mechanism has also been implicated in protection of HeLa cells from TRAIL-induced apoptosis (22). Moreover, inhibition of TRAIL-induced apoptosis by PKC activation was also suggested to occur at the level of proteolytic cleavage of caspase-8 or downstream of caspase-8-mediated Bid cleavage (20, 21).

In the present study, we have examined the potential interaction between PKC-mediated signal transduction and TRAIL-induced apoptotic signaling pathway in melanoma cell lines. We report that activation of PKC differentially regulates sensitivity of melanoma cells to TRAIL-induced apoptosis by modulating Bax activation, and this seems to be associated with the relative expression levels of PKC. Deficiency in PKC expression contributed to sensitization of melanoma to TRAIL-induced apoptosis.

	Materials and Methods

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Cell Lines
Human melanoma cell lines Me4405, Me1007, IgR3, Mel-FH, Mel-RM, Mel-CV, Mel-AT, and MM200 have been described previously (4). The cell lines were cultured in DMEM containing 5% FCS (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia).

Antibodies, Recombinant Proteins, and Other Reagents
Recombinant human TRAIL was supplied by Immunex (Seattle, WA). The preparation was supplied as a leucine zipper fusion protein. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma (Castle Hill, New South Wales, Australia). The PKC inhibitor, bisindolylmaleimide I (GF109203X), was from Calbiochem (Kilsyth, Victoria, Australia). The general caspase inhibitor Z-Val-Ala-Asp (OMe)-CH₂F and the caspase-3-specific inhibitor Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH₂F were from Calbiochem (La Jolla, CA). The rabbit monoclonal antibody (mAb) against active caspase-3 and mouse mAbs against caspase-8 and poly(ADP-ribose) polymerase were from PharMingen (North Ryde, New South Wales, Australia). Mouse mAbs against Bcl-2, Bcl-x_L, Mcl-1, Bax, and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), rabbit polyclonal antibodies against PKC, PKC, PKC, and inhibitor of caspase-activated DNase, and phosphorylated PKC, PKC, and PKC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody against Bid was from Cell Signaling (Beverly, MA). The rabbit polyclonal antibody against Smac was a kind gift from Dr. Xiao Dong Wang (Howard Hughes Medical Institute, Dallas, TX). The rabbit polyclonal antibody against ERK1/2 and the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 were purchased from Promega Corp. (Madison, WI). The rabbit polyclonal antibody against Bax (NT) was purchased from Upstate Biotechnology (Waltham, MA). The mAb against cytochrome c oxidase IV was purchased from Molecular Probes (Eugene, UR). Isotype control antibodies used were the ID4.5 (mouse IgG2a) mAb against Salmonella typhi supplied by Dr. L. Ashman (Institute for Medical and Veterinary Science, Adelaide, South Australia, Australia), the 107.3 mouse IgG1 mAb purchased from PharMingen (San Diego, CA), and rabbit IgG from Sigma.

Plasmid Vector and Transfection
The expression construct of pEF Bcl-2 was a kind gift from Dr. David Vaux (Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia), which was transfected into melanoma, cells and the resulting transfectants were maintained as described previously (3, 4).

PKC Adenovirus Vectors
Recombinant adenoviruses containing cDNA coding for PKC (Ax-PKC), a dominant-negative mutant of PKC (Ax-DN-PKC), and ß-galactosidase (Ax-lacZ) were kind gifts from Dr. H. Shinohara (Tokyo Medical and Dental University, Tokyo, Japan) and were used to infect melanoma cells as described previously (13).

Flow Cytometry
Immunostaining on intact and permeabilized cells was carried out as described previously (4). Analysis was carried out using a Becton Dickinson (Mountain View, CA) FACScan flow cytometer.

Apoptosis
Melanoma cells were seeded onto 24-well plates (Becton Dickinson, Lane Cove, New South Wales, Australia) overnight. Cells with or without pretreatment with PMA (100 ng/mL) for 30 minutes or GF109203X for 1 hour were then treated with TRAIL (200 ng/mL) for another 24 hours. Apoptotic cells were determined by the propidium iodide (PI) method as described elsewhere (4).

Western Blot Analysis
Methods used were as described previously (4, 23). Western blot analysis of ß-actin levels was included to show that equivalent amounts of protein were loaded in each lane. The data shown are representative of two individual experiments.

Preparation of Mitochondrial and Cytosolic Fractions
Methods used for subcellular fraction were similar to the methods described elsewhere (4, 23).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC Differentially Regulates Sensitivity of Melanoma Cells to TRAIL-Induced Apoptosis
Figure 1A shows that pretreatment with PMA inhibited TRAIL-induced apoptosis to varying degrees in five of eight melanoma cell lines (Mel-RM, Mel-CV, Mel-FH, MM200, and IgR3) ranging from 15% inhibition in IgR3 to 60% inhibition in Mel-RM and Mel-CV. In contrast, PMA increased TRAIL-induced apoptosis of Me4405 and Mel-AT by 25% and 13%. Inhibition of PKC with GF109203X resulted in opposing effects to those observed with PMA (Fig. 1B). Me1007, a caspase-8- and Bid-deficient line (2), was resistant to TRAIL-induced apoptosis even in the presence of PMA or GF109203X (Fig. 1A and B). Treatment with PMA or GF109203X alone was not cytotoxic to melanoma cells (Fig. 1A and B).

View larger version (40K): [in this window] [in a new window]   Figure 1. PKC differentially regulates sensitivity of melanoma cells to TRAIL-induced apoptosis. A and B, melanoma cells with or without pretreatment with PMA (100 ng/mL) for 30 min (A) or GF109203X (20 µmol/L) for 1 h (B) were treated with TRAIL (200 ng/mL) for another 24 h before measurement of apoptosis by the PI method using flow cytometry. Columns, mean of three individual experiments; bars, SE. C, Mel-RM and Me4405 cells with or without pretreatment with PMA (100 ng/mL) for 30 min or GF109203X (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 3 h before the processed form of caspase-3 was measured using a mAb that specifically recognizes the proteolytically processed form of caspase-3 in permeabilized cells using flow cytometry. Representative of three individual experiments. D, whole cell lysates from Mel-RM and Me4405 cells treated as in C were subjected to Western blot analysis of poly(ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated DNase (ICADL).

Sensitization of Melanoma Cells to TRAIL-Induced Apoptosis by PMA Is Associated with Deficient PKC Expression
Among the PKC isoforms that have been implicated in regulation of apoptosis, PKC is generally believed to be proapoptotic, whereas PKC and PKC are antiapoptotic (10–16). We therefore examined whether the differential effect of PKC on TRAIL-induced apoptosis was due to varying levels of PKC, PKC, and PKC expression in the cell lines. As shown in Fig. 2A, PKC was expressed at high levels in Mel-FH, MM200, IgR3, Mel-RM, and Mel-CV, moderate levels in Mel-AT and Me4405, and low levels in Me1007. PKC was expressed at moderate to high levels in all but MM200 and Me1007 cells. PKC was expressed at moderate to high levels in Me1007, MM200, Mel-RM, Mel-CV, Mel-FH, and IgR3 but was at very low levels in Mel-AT and Me4405 cells. It was of note that the latter seemed to be the only two cell lines that were sensitized to TRAIL by PMA (Fig. 1A).

View larger version (44K): [in this window] [in a new window]   Figure 2. Deficient PKC expression is associated with sensitivity of melanoma cells to TRAIL-induced apoptosis. A, expression of PKC, PKC, and PKC in melanoma cells. Whole cell lysates from melanoma cells were subjected to Western blot analysis. B, TRAIL induced rapid increases in phosphorylation of PKC and PKC. Whole cell lysates from Mel-RM and Me4405 cells with or without treatment with TRAIL (200 ng/mL) for the indicated times were subjected to Western blot analysis. C, the caspase-3 specific inhibitor Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH2F (z-DEVD-fmk) inhibited TRAIL-induced proteolytic cleavage of PKC and PKC. Mel-RM cells with or without pretreatment with Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH2F (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 3 h. Whole cell lysates were subjected to Western blot analysis. D, PKC was expressed in Me4405 cells infected with adenovirus carrying Ax-PKC but not those infected with adenovirus carrying Ax-lacZ. Whole cell lysates were subjected to Western blot analysis. E, Ax-PKC was phosphorylated by PMA and TRAIL. Whole cell lysates from Mel4405 carrying Ax-PKC with or without treatment with PMA (100 ng/mL), TRAIL (200 ng/mL), or the combination of both were subjected to Western blot analysis. F, Ax-PKC reverses PMA-mediated sensitization of Me4405 cells to TRAIL-induced apoptosis. Me4405 cells carrying Ax-PKC or Ax-lacZ with or without pretreatment with PMA (100 ng/mL) for 30 min were treated with TRAIL (200 ng/mL) for another 24 h before measurement of apoptosis by the PI method using flow cytometry. Columns, mean of three individual experiments; bars, SE. G, Ax-DN-PKC inhibits PMA- and TRAIL-mediated phosphorylation of PKC. Whole cell lysates from Mel-RM carrying Ax-DN-PKC or Ax-lacZ with or without treatment with PMA (100 ng/mL) or TRAIL (200 ng/mL) for 30 min were subjected to Western blot analysis. H, Ax-DN-PKC reverses PMA-mediated protection of Mel-RM cells from TRAIL-induced apoptosis. Mel-RM cells carrying Ax-DN-PKC or Ax-lacZ with or without pretreatment with PMA (100 ng/mL) for 30 min were treated with TRAIL (200 ng/mL) for another 24 h before measurement of apoptosis by the PI method using flow cytometry. Columns, mean of three individual experiments; bars, SE.

Figure 2B also shows that a 40-kDa band was detected in both Mel-RM and Me4405 in Western blot analyses of PKC 1 hour after exposure to TRAIL. This band persists until 16 hours after TRAIL treatment. Similarly, a weak 45-kDa band was detected in Western blot analyses of PKC in Mel-RM with a corresponding decrease in the expression levels of the native form of PKC. These are consistent with proteolytic cleavage of PKC and PKC by activated caspase-3 as described before (24–26). TRAIL-induced caspase-3 activation in melanoma cells is detectable as soon as 30 minutes after exposure to TRAIL and peaks at 3 hours (4). We therefore treated Mel-RM cells with the caspase-3 specific inhibitor Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH₂F for 1 hour before adding TRAIL for another 3 hours. Figure 2C shows that inhibition of caspase-3 markedly blocked the appearance of TRAIL-induced smaller forms of PKC and PKC. No indication of proteolytic cleavage of PKC was found after treatment with TRAIL.

To confirm a role of PKC in regulation of sensitivity of melanoma cells to TRAIL-induced apoptosis, we introduced PKC into PKC-deficient Me4405 cells by using an adenovirus expression system (13). Figure 2D and E shows that PKC was expressed at high levels in cells infected with adenovirus carrying the cDNA for PKC (Ax-PKC) and was readily phosphorylated by treatment with PMA or TRAIL. Pretreatment with PMA did not cause any increase in TRAIL-induced apoptosis in Me4405 cells expressing Ax-PKC (Fig. 3F).

View larger version (26K): [in this window] [in a new window]   Figure 3. PKC regulates sensitivity of melanoma cells to TRAIL-induced apoptosis downstream of Bid cleavage but upstream of mitochondrial apoptotic events. A, whole cell lysates from Mel-RM and Me4405 cells with or without treatment with TRAIL (200 ng/mL) in the presence or absence of PMA (100 ng/mL) or GF109203 (GF; 20 µmol/L) were subjected to Western blot analysis. B, regulation of mitochondrial release of Smac/DIABLO by PKC activation. Mel-RM and Me4405 with or without pretreatment with PMA (100 ng/mL) for 30 min or GF109203X (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 3 h. Mitochondrial (Mito.) and cytosolic (Cyto.) fractions were subjected to Western blot analysis. Western blot analysis of cytochrome c oxidase IV (COX IV) levels was included to show relative purity of the mitochondrial fractions. C, Bcl-2 was overexpressed in Mel-RM cells transfected with the cDNA encoding Bcl-2. Whole cell lysates were subjected to Western blot analysis. D, overexpression of Bcl-2 inhibited apoptosis induced by TRAIL regardless of the activation status of PKC. Mel-RM cells transfected with cDNA for Bcl-2 or vector alone with or without pretreatment with PMA (100 ng/mL) for 30 min or GF109203X (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 24 h. The percentage of apoptotic cells was quantitated by the PI method using flow cytometry. Columns, mean of three individual experiments; bars, SE.

PKC Regulates Sensitivity of Melanoma Cells to TRAIL-Induced Apoptosis Downstream of Bid Cleavage but Upstream of Mitochondrial Apoptotic Events
Figure 3A shows that pretreatment with either PMA or GF109203X did not cause any significant alteration in the processing of pro-caspase-8 or Bid. Consistent with this is that treatment with PMA or GF109203X for 3, 6, or 16 hours did not cause any change in the expression levels of TRAIL-R1 or TRAIL-R2 on the cell surface (data not shown).

As shown in Fig. 3B, TRAIL induced rapid release of Smac from mitochondria into the cytosol, which was markedly blocked by PMA but was enhanced by GF109203X pretreatment in Mel-RM cells. In contrast, TRAIL-induced release of Smac in Me4405 was potentiated by pretreatment with PMA but was reduced by pretreatment with GF109203X.

The role of mitochondria in PKC-mediated regulation of sensitivity to TRAIL-induced apoptosis was further confirmed by transfecting cDNA encoding Bcl-2 into Mel-RM cells (Fig. 3C). Figure 3D shows that TRAIL-induced apoptosis was completely inhibited in Bcl-2 transfectants irrespective of the presence of PMA or GF109203X.

PKC Modulates TRAIL-Induced Conformational Changes of Bax and Its Translocation from the Cytosol to Mitochondria
Translocation of Bax from the cytosol to mitochondria plays a key role in TRAIL-induced mitochondrial changes (27, 28). This involves a conformational change in Bax that exposes its NH₂ terminus that is otherwise not accessible for binding by Bax-NH₂-terminal epitope-specific antibodies in intact cells (28, 29). We studied if PKC affects TRAIL-induced conformational change of Bax by using an antibody directed against the NH₂-terminal region of Bax in flow cytometry (28, 29). As shown in Fig. 4A, pretreatment with PMA resulted in a marked decrease in the conformational change of Bax induced by TRAIL in Mel-RM cells but led to a moderate increase in Me4405 cells. In contrast, pretreatment with GF109203X promoted the conformational change of Bax in Mel-RM cells but slightly inhibited it in Me4405 cells.

View larger version (28K): [in this window] [in a new window]   Figure 4. Regulation of TRAIL-induced conformational changes of Bax and its translocation to mitochondria by PKC. A, pretreatment with PMA or GF109203X resulted in an increase or decrease in the conformational changes of Bax. Mel-RM and Me4405 cells with or without pretreatment with PMA (100 ng/mL) for 30 min or GF109203X (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 3 h. Conformational changes of Bax were measured in flow cytometry analyses using a Bax-NH2-terminal epitope-specific antibody in permeabilized cells. Representative of three individual experiments. B, pretreatment with PMA or GF109203X resulted in an increase or decrease in translocation of Bax from the cytosol to mitochondria. Mel-RM and Me4405 with or without pretreatment with PMA (100 ng/mL) for 30 min or GF109203X (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 3 h. Mitochondrial and cytosolic fractions were subjected to Western blot analysis. Western blot analysis of cytochrome c oxidase IV levels was included to show relative purity of the mitochondrial fractions.

Role of PKC-Mediated Activation of ERK1/2 in Protection of Melanoma from TRAIL-Induced Apoptosis
We have reported previously that Mel-RM and, to a lesser extent, Me4405 were sensitized to TRAIL-induced apoptosis by pretreatment with the MAPK kinase–specific inhibitor U0126 (23). We examined whether this MAPK pathway may be involved in PKC-mediated inhibition of TRAIL-induced apoptosis. In the presence of U0126, TRAIL-induced apoptosis was increased in both cell lines (Fig. 5A). Pretreatment with U0126 before adding PMA and TRAIL partially reversed the protective effect of PMA on TRAIL-induced apoptosis in Mel-RM cells but had no influence on the sensitizing effect of PMA on Me4405. The percentage of inhibition afforded by PMA decreased from 41.5% to 30.9% in the presence of U0126. Figure 5B shows that pretreatment with GF109203X markedly blocked both TRAIL- and PMA-induced phosphorylation of ERK1/2 in both Mel-RM and Me4405 cells.

View larger version (48K): [in this window] [in a new window]   Figure 5. Activation of ERK1/2 is partially responsible for protection of melanoma cells by PKC from TRAIL-induced apoptosis. A, inhibition of ERK1/2 reverses the protective effect of PKC on Mel-RM cells. Mel-RM and Me4405 cells with or without pretreatment with U0126 (20 µmol/L) were sequentially treated with PMA (100 ng/mL) and TRAIL (200 ng/mL). The percentage of apoptotic cells was then quantitated by the PI method using flow cytometry. Columns, mean of three individual experiments; bars, SE. B, inhibition of PKC blocked TRAIL-induced ERK1/2 activation. Mel-RM and Me4405 cells with or without pretreatment with PMA (100 ng/mL) for 30 min or GF109203X (20 µmol/L) for 1 h were treated with TRAIL (200 ng/mL) for another 1 h. Whole cell lysates were subjected to Western blot analysis.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Among the PKC isoforms, PKC is generally believed to be proapoptotic (10–12), whereas PKC and PKC are antiapoptotic (13–16). In view of this, we studied the relationship of the expression of these isoforms in melanoma cell lines to their sensitivity to TRAIL-induced apoptosis. The results indicate that the expression levels of PKC especially may play an important role in determining sensitivity of melanoma to apoptosis induced by TRAIL. This was supported by studies using adenovirus vector expression systems to express PKC in the PKC-deficient Me4405 cells, which reversed the potentiating effect of PMA on TRAIL-induced apoptosis. In contrast, expression of a dominant-negative PKC in PKC-expressing Mel-RM cells reversed the protective effect of PMA on TRAIL-induced apoptosis. These results are similar to those suggesting that PKC may protect against TRAIL-induced apoptosis of glioma cells (13). Our present results also support a proapoptotic role of PKC in melanoma in that cells with lower levels of PKC that were sensitized to TRAIL-induced apoptosis by PMA (Me4405 and Mel-AT) had relatively high levels of PKC. PKC was reported to be involved in protection against apoptosis in several types of cells (15, 16), but we did not find any relationship between the expression levels of PKC and sensitivity of melanoma cells to TRAIL-induced apoptosis. Others have shown that manipulation of PKC levels had little effect on apoptosis in response to the chemotherapeutic drug etoposide (30). The role that other isoforms of PKC may have in regulation of the sensitivity of melanoma cells to TRAIL is not known. Although the present results do not exclude a possible role for other PKC isoforms, such as PKC and PKC (31–33), they do however suggest that the relative contents of PKC may play an important part in determining sensitivity of melanoma cells to apoptosis induced by TRAIL.

We showed that both PKC and PKC are constitutively phosphorylated in melanoma cells and that TRAIL induces a rapid increase in their phosphorylation. These studies suggest that activation of PKC by TRAIL may provide positive or negative regulation of sensitivity of cells to TRAIL-induced apoptosis. This was supported by studies showing that a PKC inhibitor had opposing effects to PMA on sensitivity to TRAIL-induced apoptosis. Activation of PKC by TRAIL was reported to be protective in pancreatic adenocarcinoma cells (34). The present results, however, suggest that TRAIL-mediated activation of PKC protects melanoma cells from TRAIL-induced apoptosis in cells that express relatively high levels of PKC but promotes apoptosis in those that express relatively low levels of PKC.

The site in the apoptotic pathway at which PKC acts to affect TRAIL-induced apoptosis remains disputed (19–22). Whereas some studies showed that inhibition of TRAIL-induced apoptosis by activation of PKC occurred at the level of proteolytic cleavage of pro-caspase-8 (24), others showed that activation of PKC inhibited TRAIL-induced mitochondrial apoptotic events downstream of Bid cleavage (19, 21). More recently, it was shown that activation of PKC interfered with Fas-associated death domain recruitment to the death-inducing signaling complex (22). Our present results showed that the activation of PKC regulates TRAIL-induced apoptosis of melanoma by modulating Bax activation: (a) pretreatment with PMA or GF109203X did not cause significant changes in the expression levels of TRAIL death receptors, alterations in activation of caspase-8, and cleavage of Bid; (b) pretreatment with PMA resulted in either increased or decreased mitochondrial release of Smac/DIABLO, activation of caspase-3, and processing of its substrates; (c) overexpression of Bcl-2 markedly inhibited TRAIL-induced apoptosis in the presence of PMA or GF109203X; and (d) pretreatment with PMA or GF109203X led to increased or decreased levels in the conformational changes of Bax and its translocation from the cytosol to mitochondria.

A possible explanation for modulation of Bax activation by PKC is that PKC may regulate the expression of Bcl-2 family members. However, there was no change in the expression levels of Bcl-2, Bcl-x_L, Mcl-1, Bax, or Bak up to 12 hours after treatment with PMA or GF109203X when activation of Bax had already occurred (data not shown). It was reported recently that PKC interacts with Bax and promotes survival of human prostate cancer cells (35). This was tested in the present studies in melanoma cells by coimmunoprecipitation with a mAb against Bax or an antibody against PKC with or without exposure of cells to TRAIL, but no such association could be identified (data not shown). Given the complexity of multiple PKC isoforms that may act on Bax by different mechanisms, further studies using PKC isoform "knockouts" may assist in elucidating the role they may play in modulating Bax activation induced by TRAIL.

Our data also revealed that TRAIL induced proteolytic cleavage of PKC and PKC, which seemed to be caspase-3 dependent in that a caspase-3-specific inhibitor markedly blocked the appearance of the smaller forms of PKC and PKC. Treatment with either PMA or GF109203X did not induce appreciable levels of caspase-3 activation (data not shown) but was able to regulate TRAIL-induced caspase-3 activation by modulating mitochondrial apoptotic events. The latter is inhibitable by overexpression of Bcl-2 in melanoma cells (23, 36). We therefore believe that, even in the presence of PMA or GF109203X, Bcl-2 is able to inhibit cleavage of PKC and PKC and exerts its inhibitory effect on TRAIL-induced apoptosis by blocking TRAIL-induced release of mitochondrial apoptotic factors and subsequent activation of caspase-3.

PKC activates the MAPK ERK1/2 pathway by stimulating the Raf-Ras-MAPK kinase pathway in different cell types (26, 27). In this study, we showed that the protective effect of activation of PKC is at least in part associated with activation of ERK1/2 induced by TRAIL (23). This is because inhibition of ERK1/2 by the MAPK kinase–specific inhibitor partially reversed the protective effect on melanoma cells against TRAIL afforded by PMA. In addition, activation of ERK1/2 seems to be downstream of PKC in that inhibition of PKC markedly blocked TRAIL-induced activation of ERK1/2. However, the current studies also clearly show that an ERK1/2-independent mechanism(s) exists in PKC-mediated protection of melanoma against TRAIL-induced apoptosis. Previous studies have also reported that activation of PKC inhibited apoptosis by both MAPK-dependent and MAPK-independent pathways (19, 21). PKC-mediated activation of nuclear factor-B is known to play a role in protection of many types of cells from death receptor-induced apoptosis (34).

We believe that these results provide further important insights into the variable responses of melanoma cells to TRAIL-induced apoptosis. Measurement of relative contents of PKC isoforms may help define sensitive and resistant melanoma phenotypes to treatment based on TRAIL-induced apoptosis, and studies on this aspect are continuing. Should specific inhibitors of PKC become available, these in combination with TRAIL would be a promising regimen in treatment of melanoma and worthy of further exploration.

	Acknowledgments

 
We thank Drs. N. Oyaizu and H. Shinohara for the adenovirus constructs.

	Footnotes

 
Grant support: State Cancer Council, New South Wales, Australia.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/10/04; revised 1/21/05; accepted 2/ 7/05.

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