Protein Kinase C (cid:1) Plays a Critical Role in Mannosylerythritol Lipid-induced Differentiation of Melanoma B16 Cells*

Mannosylerythritol lipid (MEL), a novel extracellular glycolipid from yeast, was found to inhibit the proliferation of mouse melanoma B16 cells in a dose-dependent manner and to induce the apoptosis of B16 cells at concentrations higher than 10 (cid:2) M (Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Geltinger, C., Nakahara, T., Murata, T., and Yokoyama, K. K. (1999) Cancer Res. 59, 482–486). We show here that exposure of B16 cells to MEL (5 (cid:2) M ) for 2 days resulted in an increase of the levels of differentiation-associated markers of melanoma cells such as melanogenesis and tyrosinase activity, which were accompanied by morphological changes. The MEL-induced differentiation of B16 cells at this concentration was closely associated with arrest of the cell cycle at G 1 phase, but no significant population of apo- ptotic cells was identified. Expression of protein kinase C (cid:1) (PKC (cid:1)

Abnormal cellular differentiation is a well established characteristic of tumor cells. A promising approach to the treatment of cancer involves the induction of the terminal differentiation and growth arrest of cancer cells. Because the discovery that polar organic compounds can induce the specific differentiation of erythroleukemia cells (1), a variety of reagents, including polar organic compounds, short-chain fatty acids, and retinoids, have been shown to induce features of differentiation in many lines of cancer cells, including melanoma cells (2)(3)(4)(5)(6). Our ability to manipulate the phenotypes of neoplastic cells with these reagents provides us with ways to unravel the mechanisms that underlie cellular differentiation, as well as to develop potentially useful therapeutic reagents (7). Melanoma is well known as a chemotherapy-resistant cancer (8), and it has been suggested that melanoma might be a suitable target for therapy with differentiation-inducing agents (9).
Mannosylerythritol lipid (MEL), 1 a novel extracellular glycolipid produced by yeast, induces the granulocytic differentiation of HL-60 promyelocytic leukemia cells and alters the composition of cell surface glycosphingolipids (GSLs) (10). Gangliosides and GSLs, which are ubiquitous constituents of the plasma membrane of mammalian cells, play an important role in the modulation of cellular proliferation, oncogenesis and differentiation (11,12). Cellular differentiation and oncogenic transformation are accompanied by dramatic changes in both absolute and relative levels of GSLs (13). Yeast-derived glycolipids differ from those derived from mammalian cells in terms of specific substituents, but their backbones are similar. We postulated that MEL might affect other tumor cells, in addition to inducing the cellular differentiation of HL-60 cells.
The molecular mechanisms responsible for the induction of the differentiation of melanoma cells are poorly understood. However, there is evidence that protein kinase C (PKC) might be associated with such differentiation. PKCs form a multigene family of serine-and threonine-specific kinases. They appear to play roles in a wide variety of cellular processes that include the functions of some membrane receptors, and the proliferation and differentiation of cells (14 -16). PKC␣ is expressed ubiquitously, while PKC␤ is found only in some tissues, and PKC␥ appears to be restricted almost exclusively to brain tissue (14). Such distribution suggests possible functional differences among these enzymes. PKC is associated with the differentiation that is induced by retinoic acid (RA), by ␣melanocyte-stimulating hormone and by dehydroepiandrosterone in cell lines such as F9 teratocarcinoma cells and melanoma cells (17)(18)(19). For example, increased expression of PKC␣ plays a key role in the RA-triggered differentiation of melanoma cells (20,21), while PKC␤ appears to be critical for the regulation of melanogenesis in both human melanocytes and S91 mouse melanoma cells (17,22). Thus, the roles of the isoforms of PKC appear to depend on the cell type. Specific activators and inhibitors of PKC have proved useful in attempts to analyze the effects of PKC on various transformation processes. However, such compounds do not discriminate between isozymes. Antisense oligodeoxynucleotides (AS-ODNs) and ribozymes are more suitable for studies of the roles of individual isoforms.
In this study, we evaluated the physiological effects of MEL on B16 melanoma cells and found that MEL induced cell cycle arrest and the differentiation of the cells in culture. We showed that PKC␣ was involved in this induced differentiation by using an AS-ODN that was specific to mouse PKC␣, as well as by inducing the expression of a constitutively active form of PKC␣ in B16 cells.

EXPERIMENTAL PROCEDURES
Cell Culture and Materials-Mouse melanoma B16 4A5 cells (referred to hereafter as B16 cells) were obtained from the Riken Cell Bank (Tsukuba, Ibaraki, Japan) and maintained in DMEM supplemented with 10% fetal bovine serum or in serum-free DMEM-ITES (DMEM plus insulin, transferrin, ethanolamine, and selenite) medium, as described elsewhere (23), at 37°C in a humidified atmosphere of 5% CO 2 . MEL was prepared and purified essentially as described by Kitamoto et al. (24), and administered to cells at the indicated concentrations. A WST-1 cell counting kit (Dojin Laboratories, Kumamoto, Japan) was used to monitor the number of viable cells. RA, dimethyl sulfoxide (Me 2 SO) and dexamethasone were obtained from Sigma Chemical Co.
Measurements of Melanin Content and Tyrosinase Activity and the Flow Cytometric Analysis of the Cell Cycle and Apoptotic Cells-The methods used for measurements of melanin content and tyrosinase activity and the flow cytometric analysis of cultures of B16 cells have been described elsewhere (23,25). One unit of tyrosinase activity was defined as the activity that caused an increase in absorbance at 280 nm of 0.001/min. Melanin content was measured at an absorbance of 490 nm, and the concentration of melanin was calibrated with synthetic melanin (Sigma Chemical Co.).
Immunoblotting of PKC-Untreated and MEL-treated B16 cells were lysed with ice-cold RIPA buffer (1ϫ phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with phenylmethylsulfonyl fluoride (Sigma Chemical Co.; 25 g/ml), aprotinin (Sigma Chemical Co.; 30 l/ml) and sodium orthovanadate (Sigma Chemical Co.; 1 mM) at 4°C for 15 min. Cell lysates were collected and crude samples of protein were purified as described by Wessel et al. (26). Protein concentrations of samples were determined by the Bradford method using delipidated albumin (Sigma Chemical Co.) as the standard (27). For Western blotting analysis of PKCs, equal amounts of samples were subjected to electrophoresis on a 7.5% polyacrylamide gel in the presence of SDS (SDS-PAGE) and transferred to a nitrocellulose membrane. Immunoblotting was performed with poly-clonal antibodies specific to PKC␣, PKC␤1, PKC␤2, PKC␥, PKC␦, PKC⑀, PKC and PKC (Santa Cruz Biotechnology. Inc., Santa Cruz, CA) or polyclonal antibodies against PKC␣ (Promega Co., Madison, WI). The products of immunoreactions were detected with an enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech).
Immunoprecipitation and Assay of PKC Activity in Vitro-After collection of cell lysates in RIPA buffer, samples were precleared with Protein A/G Plus-agarose beads (Santa Cruz Biotechnology Inc.) at 4°C for 15 min. The beads were removed by centrifugation, and samples were then incubated with polyclonal antibodies specific for PKC␣ (Promega Co.) at 4°C for 1 h. After addition of Protein A/G Plus-agarose beads, mixtures were incubated at 4°C for an additional hour. The beads were then washed three times with RIPA buffer and suspended in kinase assay buffer for measurment of PKC activity in vitro, using a PKC assay kit (Upstate Biotechnology, Lake Placid, NY). In brief, 10 l of substrate mixture (500 M PKC substrate peptide) were mixed with 10 l of a solution of protein kinase A (PKA) inhibitor, 10 l of a solution of PKC lipid activator (phosphatidylserine and diglyceride) and 10 l of the prepared enzyme in a microcentrifuge tube. The reaction was started by the addition of 10 l of a solution of ATP that contained [␥-32 P]ATP (Amersham Pharmacia Biotech, specific activity 3000 Ci/ mmol). The mixture was agitated gently and incubated at 30°C for 10 min. The reaction was stopped by removal of 25 l of the reaction mixture and placing it in the center of a piece of P81 phosphocellulose paper (Whatman). After 30 s, the paper was immersed in 0.75% phosphoric acid. After washing, the paper was soaked in acetone for 2 min and then transferred to a 5-ml scintillation vial. A scintillation mixture was added and the radioactivity on the paper was measured in a scintillation counter.
Treatment of B16 Cells with Antisense Oligodeoxynucleotides Directed against PKC␣ mRNA-B16 cells were cultured under normal conditions until they reached a 70 -80% confluence. Then cells were rinsed with serum-free DMEM and incubated in a mixture of AS-ODN or S-ODN or control oligodeoxynucleotides (Tsukuba Research Laboratory, Toagosei Co., Tsukuba, Japan) and the LipofectAMINE TM reagent (Life Technologies, Inc.) in OPTI-MEMI reduced-serum medium reagent (Life Technologies, Inc.) for 4 -5 h. The AS-ODN and the Lipo-fectAMINE TM reagent were then removed, and cells were cultured in normal medium for the indicated times. Cells were used for immunoprecipitation, immunoblotting of PKC, and Northern blotting analysis.
Northern Blotting Analysis-Total RNA was isolated from target cells using the Trizol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Then 25 g of total RNA from each sample was fractionated on a 1% agarose gel that contained 2.2 M formaldehyde and bands of RNA were transferred to a Hybond-N TM filter (Amersham Pharmacia Biotech). For Northern blotting analysis, filters were incubated in Rapid-hyb buffer (Amersham Pharmacia Biotech) at 65°C for 30 min before hybridization with a 32 P-radiolabeled cDNA probes for mouse PKC isoforms or rat ␤-actin at 65°C for 18 h. Filters were washed with 0.2ϫ SSC plus 0.5% SDS for 10 min and then exposed to RX-film (Fuji Film; Tokyo, Japan) with an intensifying screen. Before rehybridization, filters were treated with a solution of boiling 0.5% SDS for 10 min.
Transfection of B16 cells with Plasmids-B16 cells were cultured under normal conditions to 70 -80% confluence. Then cells were cotransfected with pcDNA3 (Invitrogen BV, Groningen, The Netherlands), which harbored a gene for neomycin resistance, and the pEF-HisB expression vector (Invitrogen BV) that encoded a His tag-fused constitutively active form of PKC␣ (28) by calcium precipitation, as described elsewhere (29). Control cells were transfected with pcDNA3 only. After transfection, cells were plated on medium that contained 500 g/ml of G418 (Life Technologies, Inc.). Clones of resistant cells were selected and stable colonies were examined with monoclonal antibodies against the His tag (Qiagen GmbH, Hilden, Germany) for immunoprecipitation and polyclonal antibodies specific for PKC␣ for Western blotting. Selected cells were routinely grown in the presence of selective pressure from G418.

RESULTS
The MEL-induced Differentiation of B16 Cells-We investigated the effects of MEL on the differentiation induction of B16 cells by examining the effects of MEL on the production of melanin and tyrosinase activity, which are markers of the differentiation of melanoma cells (30,31). Fig. 1A shows the melanin content of B16 cells after culture for 2 days. The amount of melanin in untreated cells was 0.10 Ϯ 0.02 g/g of protein, while that in 5 M MEL-treated cells was 0.18 Ϯ 0.02 g/g of protein. RA, dimethyl sulfoxide (Me 2 SO), and dexamethasone were used as positive controls because each has been reported to induce the differentiation of melanoma cells (32)(33)(34). Treatment of cells with Me 2 SO and with dexamethasone resulted in the enhanced production of melanin. However, RA did not induce significantly enhanced melanogenesis. The tyrosinase activity of B16 cells that had been treated with 5 M MEL for 2 days was 6-fold greater than that of untreated cells (Fig. 1B). These results indicate that MEL enhanced tyrosinase activity, with resultant increased melanogenesis, and suggest that B16 cells undergo differentiation upon treatment with MEL. When added to the reaction mixture directly, MEL had no detectable effect on tyrosinase activity (data not shown).
The morphology of untreated B16 cells (Fig. 1C) was typical of that of mouse melanoma cells in culture. The cells resembled fibroblasts and were often clumped together. On the second day of cultivation, B16 cells exposed to 5 M MEL extended dendrites and arranged themselves alongside one another (Fig.  1D), suggesting that cellular differentiation had occurred also at a morphological level.
MEL Interrupts the Cell Cycle at the G 1 Phase-We next examined whether the MEL-induced differentiation of B16 cells might be accompanied by effects on the cell cycle. As shown in Fig. 2, A and B, flow cytometry revealed that populations of cells exposed to 5 M MEL for 2 days showed a significant decrease in the relative number of cells in the S phase as compared with untreated cells (from 33.6 to 11.1%) and a marked increase in the relative number of cells in the G 1 phase (from 57.0 to 80.8%), while there was little change in the cell population in the G 2 /M phase (from 6.2 to 5.9%). Thus, the effects of MEL on the differentiation-induction of B16 cells can be attributed, at least in part, to the induction of cell cycle arrest at the G 1 phase. Cultures of B16 cells exposed to 5 M MEL showed no significant increase in the relative number of apoptotic cells, as compared with untreated cells (see Fig. 2, C and D).
Enhanced Expression of PKC␣ in Response to MEL-As an intracellular calcium-dependent and phospholipid-dependent protein kinase, PKC has been implicated as a key messenger in the cellular signaling associated with the proliferation and differentiation of cells (14 -16). In an attempt to obtain insight into the molecular pathways that lead to cellular differentiation in response to MEL, we examined whether MEL might influence the expression of PKC. We cultured cells in the presence and in the absence of MEL for the indicated periods of time and harvested them. Then we performed Western blotting analysis to compare relative levels of expression of PKC␣. PKC␣ protein was almost undetectable in MEL-treated and in untreated B16 cells after cultivation for 3 h. After further cultivation, for up to 24 h, there was an increase in the expression of PKC␣ in both MEL-treated and untreated B16 cells and the increases were time-dependent. There was, moreover, no obvious difference between the levels of PKC␣ in the two types of culture. However, the expression of PKC␣ in untreated cells decreased markedly at 48 h, while MEL-treated cells continued to display high-level expression of PKC␣ (Fig. 3A). The level was ϳ2.3-fold higher than that in untreated cells, as determined by densitometric scanning. The enzymatic activities of PKC␣ during the incubation of cells with MEL were also measured and have a good correlation with the expression levels of PKC␣ protein (Fig. 3B). Neither PKC␤, PKC␥, nor PKC were detected in MEL-treated or in untreated cells and the levels of PKC␦, PKC⑀, and PKC were not significantly altered (see Fig.  5D). These results suggest that PKC␣-related pathways might possibly be involved in a MEL-triggered signal pathway in B16 cells.
Antisense Oligodeoxynucleotides Directed against PKC␣ mRNA Counteract the MEL-induced Effects on B16 Cells-To examine the possible relationship between the MEL-triggered differentiation of B16 cells and enhancement of the expression of PKC␣, we introduced phosphorothioate AS-ODNs targeted to the mouse gene for PKC␣ into B16 cells to suppress the expression of PKC␣. We used AS-ODNs with sequences that should be specific to the mRNA for PKC␣. As shown in Fig. 4, we selected four AS-ODNs directed toward the mRNA for PKC␣. To examine the effects of nonspecific suppression by AS-ODNs, we also used a random 23-mer ODN (N23) as a control. B16 cells were exposed to the various AS-ODNs (200 nM) during cultivation. In the case of the four AS-ODNs specific for PKC␣, no. 3642 was the most effective in reducing the level of PKC␣ protein, and it reduced the level to at least 10-fold lower than that in B16 cells treated with N23 (200 nM) and that in untreated cells (Fig. 5A). The AS-ODNs against other target sequences in the mRNA for PKC␣ did not cause such a significant decrease in the level of PKC␣ itself. We also examined the effects of sense (S)-ODNs corresponding to nt 960 -981 and nt 2636 -2659 of PKC␣ mRNA and found no decrease in the levels of PKC␣ (Fig. 5B). We next examined the effects of AS-ODN on the level of PKC␣ mRNA in B16 cells by Northern blotting. Two transcripts of the gene for PKC␣ were identified with sizes of 10.8 and 3.8 kilobase. The level of PKC␣ mRNA was dramatically reduced in B16 cells that had been treated with AS-ODN no. 3642 (Fig. 5C). Incubation of B16 cells with S-ODN (no. 3846, 200 nM) had no detectable effect on the level of mRNAs for PKC␣ (Fig. 5C). The introduction of AS-ODNs or S-ODNs did not change the levels of the expression of other PKCs (Fig.  5D). No expression of PKC␤1, PKC␤2, PKC␥, and PKC was detected in B16 cells. The relative levels of other PKC families such as PKC␦, PKC⑀, and PKC were not changed. Thus, an AS-ODN specific for a target site in PKC␣ transcripts suppressed the expression of PKC␣ in a sequence-specific manner.
We next performed an assay of PKC␣ activity in vitro. As shown in Fig. 5E, treatment with 5 M MEL for 2 days stimulated the phosphorylation activity of PKC␣, which reached a level that was 2.5-fold higher than the level in untreated control cells. This result was consistent with the MEL-induced enhancement of the expression of PKC␣ (Fig. 3). Exposure of B16 cells to AS-ODN no. 3642 resulted in a clear decrease in PKC activity, while S-ODN no. 3846 had only a minimal effect (Fig. 5E).
We then tested whether B16 cells that lacked PKC␣ could still respond to MEL. As shown in Fig. 5F, B16 cells treated with 5 M MEL for 2 days produced more than double the amount of melanin, as compared with the control cells. However, cells exposed to both MEL and AS-ODN no. 3642 exhibited no increase in melanogenesis. B16 cells treated with S-ODN resembled control B16 cells, in terms of the response to MEL (Fig. 5, F and G). These results indicated that PKC␣ plays a critical role in the MEL-induced differentiation of B16 melanoma cells. We noticed that the morphology of B16 cells incubated with AS-ODN or S-ODN changed slightly. We observed more round-shaped cells in both cases, as compared with the untreated B16 cells (Fig. 5G, panels a-c); however, the number of the melanin-stained cells (see black cells in panels d-f) was significantly reduced after treatment with AS-ODN as compared to S-ODN (panel C). Although we do not know the exact reason for more round-phenotyped cells appearing in the case of AS-ODN or S-ODN as compared with the control cells, this might be caused by the nonspecific effect of oligodeoxynucleotides, because AS-ODN, S-ODN, and N23 resulted in similar changes in the morphology of the cells. However, in terms of the production of melanin, both cases exhibited similar levels to untreated B16 cells (Fig. 5F).

The Effects of Expression of a Constitutively Active Form of PKC␣ Mimic the Effects of MEL on Melanogenesis in B16
Cells-We introduced the pEFHisB expression vector that included a His tag-fused and constitutively active form of PKC␣ (28), which had point mutations at amino acid residues 22 and 25, into B16 cells. We selected positive stable clones of B16 cells and evaluated two representative clones, A␣13-1 and A␣13-4, for the expression of the exogenous gene for PKC␣ (Fig. 6A).
B16 cells that expressed the constitutively active form of PKC␣ (A␣13-4) had a constitutively high level of melanin (Fig. 6B), which resembled the MEL-triggered melanogenesis in B16 cells. It has also been found that exogenous expression of the constitutively active form of PKC␣ has no detectable effect on the expression of PKC␤1, PKC␤2, PKC␥, and PKC (no expression) and the expression of PKC␦ and PKC⑀ was not significantly changed (Fig. 6C). The results indicate that PKC␣ is closely associated with the differentiation of B16 melanoma cells. Taken together with the results in Fig. 6, these data indicate that PKC␣ plays a key role in the MEL-induced differentiation of B16 cells. A␣13-4 cells also proliferated at a lower rate than the parent B16 cells (Fig. 6D). This result was consistent with the results in Fig. 6B, which showed that A␣13-4 cells had a greater potential for spontaneous differentiation than the parent cells. However, A␣13-4 cells were less susceptible to MEL in terms of the induction of melanogenesis than the parent B16 cells (data not shown). The reason for this phenomenon remains to be determined. It is possible that, once PKC␣ has reached a threshold level for commitment to cellular differentiation, further expression of PKC␣ has no additional effect on the induction of the differentiation of B16 cells.

DISCUSSION
In the present study, we examined the effects of MEL on B16 melanoma cells. MEL at 5 M inhibited the proliferation of B16 cells and stimulated both tyrosinase activity and melanogenesis, with accompanying arrest of the cell cycle at the G 1 phase and morphological alterations (Figs. 1 and 2). All of these results suggest that MEL induced arrest of the cell cycle and the differentiation of cells. To our knowledge, there have been no other reports of the induction of the differentiation of melanoma cells by an extracellular microbial glycolipid.
We examined the MEL-triggered signaling pathway in B16 cells, focusing on protein kinase C, which has been reported to play an important role in the regulation of cellular proliferation and differentiation (35)(36)(37). Western blotting analysis demonstrated that PKC␣, PKC␦, PKC⑀, and PKC were the isoforms of PKC expressed in B16 cells (Fig. 5, A, B, and D). We did not detect the expression of PKC␤1, PKC␤2, PKC␥, and PKC (Fig.  5D). Other groups have reported that only the mRNAs for PKC⑀ and PKC can be detected in B16 cells, with the level of each transcript being extremely low (38). Exposure of B16 cells for 48 h to 5 M MEL, which was the optimal concentration for induction of differentiation, resulted in enhanced expression of PKC␣, at a level that was ϳ2.3-fold higher than the level in untreated cells (Fig. 3A). In an attempt to understand the molecular mechanism of the MEL-induced differentiation of B16 cells, we introduced AS-ODNs specific for PKC␣ mRNA into B16 cells. Several AS-ODNs inhibited the expression of PKC␣ to a varying degree and counteracted MEL-induced melanogenesis (Figs. 4 and 5). Furthermore, the results of the expression of a constitutively active form of PKC␣ mimicked the results of the stimulation of B16 cells by MEL (Fig. 6). These results strongly suggest that PKC␣ plays a critical role in the MEL-induced differentiation of B16 cells.
Several studies have demonstrated that PKC plays an important role in the growth and progression of certain tumors. For example, in the case of B16-F1 melanoma cells, clones that overexpressed PKC␣ have prolonged doubling times and increased melanin production. These phenotypic characteristics are also observed during the RA-induced differentiation of melanoma cells (21). It has been also reported that PKC␣ is necessary and sufficient to increase progression through the upregulation of p21 Cip1 in glioma cells (39). By contrast, the exogenously expressed PKC␣ in MCF-7 breast cancer cells leads to a more aggressive neoplastic phenotype, and cells exhibit an enhanced rate of proliferation, anchorage-independent growth, and increased tumorigenicity in nude mice (40). Expression after transfection of PKC␤1 and PKC␥ in fibroblasts results in a phenotype typical of cellular transformation (41), whereas overexpression of PKC␤1 in colon cells results in growth inhibition (42). Therefore, modified levels of PKC, either elevated or decreased, appear to be closely linked to abnormal proliferation, with the specific effect depending on the type of tumors or isoform in question. Our data imply that increased expression of PKC␣ plays a critical role in the MELinduced differentiation of B16 cells.
It is unclear how MEL triggers the expression of PKC␣. MEL was reported previously to perturb the composition of cellsurface glycolipids, such as GM3 and lactosylceramide, in HL-60 promyelocytic leukemia cells (10). It was shown recently that GM3, a kind of GSL, is closely associated with c-Src and Rho in the GM3-enriched microdomain of the surface membrane of B16 melanoma cells. Such organizational units might be directly involved in signal transduction (43). Some glycolipids are known to undergo marked cancer-associated changes (44), and PKC has been reported to be involved in the regulation of glycolipid sulfotransferase activity in renal carcinoma cells (45). The possible link between PKC and cell surface glycolipids remains to be determined and might be the key to an understanding of how signals for differentiation are transmitted to the nucleus to activate target genes.
At a differentiation-inducing concentration (5 M), MEL had no significant apoptosis-inducing effect on B16 cells (Fig. 2D). We reported previously that, at concentrations above 10 M, MEL is a potent inducer of apoptosis in B16 melanoma cells in vitro (23). Therefore, the effects of MEL on B16 cells are concentrationdependent. Because G 1 arrest has been observed in MEL-induced differentiation and apoptosis (Fig. 2, A and B; Ref. 23), we speculate that after stimulation of G 1 arrest by MEL, B16 cells might respond to different doses of MEL via different, but related signal cascades. The detailed correlations and differences between pathways that lead to MEL-induced differentiation and apoptosis remain to be resolved.
In this study, we selected four AS-ODNs specific for mouse PKC␣ mRNA in an attempt to suppress the expression of PKC␣ during MEL-induced differentiation of B16 cells. AS-ODN no. 3642 was the most effective in perturbing the expression of PKC␣, while S-ODN no. 3846 had no detectable effect on the expression of PKC␣. Thus, the AS-ODNs appeared to act in a sequence-specific manner. AS-ODN no. 3642 at 200 nM significantly decreased expression of PKC␣ in B16 cells on day 2. However, at above 300 nM no. 3642 was toxic to the cells. This result supports our previous conclusion that MEL (above 10 M) induces apoptosis of B16 cells together with the suppressed expression of PKC activity (46).
MEL induces the differentiation of several different lines of carcinoma cells (10,46,47,48). The details of the molecular mechanisms of MEL-induced differentiation are now being investigated. The data presented here provide evidence that MEL, a yeast-derived glycolipid, triggers the differentiation of malignant B16 melanoma cells in culture and that PKC␣ plays a key role in the MEL-induced signaling pathway to cellular differentiation. These results might provide the groundwork for the use of microbial extracellular glycolipids as novel reagents for the treatment of melanoma.