Increased Expression of Protein Kinase C, Plays a Key Role in Retinoic Acid-induced Melanoma Differentiation*

cells induced by retinoic acid (RA) is preceded by a large increase in protein kinase C, (PKC,) mRNA and pro- tein. To determine the role of PKC, in the differentiation program, we stably transfected B16-F1 cells with a plasmid containing the full length PKC, cDNA driven by an SV40 promoter. Two out of thirty-two colonies screened were determined to overexpress PKC by 2-&fold according to Western blot analysis and PKC enzyme activity. When compared to control cells (wild-type cells and cells transfected only with the neomycin resistance gene), PKC, overexpressing clones dis- played longer doubling times, diminished anchorage-independent growth, and increased melanin production. RA treatment of control cells mimicked these phenotypic characteristics. When injected subcutaneously into syngeneic mice, PKC, overexpressing clones produced smaller tumors and had longer latencies than control cells. These findings, combined with the fact that phorbol esters down-regulate PKC and antagonize RA action suggest that PKC, plays a key role in the RA-induced melanoma differentiation. with a Centricon-lO'" microconcentrator (Amicon). Protein concen- trations of samples were determined by the Bradford method (17). Samples were diluted to equal protein concentrations and assayed with a commercially available PKC assay system (Amersham) in the presence and absence of 12-0-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine. The system utilizes synthetic, PKC-specific, substrate peptides which become phosphorylated with the radiolabeled phosphate group from [-y-"'P]ATP. At the end of the reaction, the radiolabeled peptide was separated from unincorporated 'v2P by the use of an affinity paper for the peptide. The degree of phosphorylation was determined by liquid scintillation counting. Enzyme activity was calculated from counts/min taking into account the specific activity of the radioisotope and reaction time. Southern Analysis of PKC, Restriction Fragments-Genomic DNA from wild type cells and clones H and K was digested with 10 units of HindIIIlpg of DNA at 37 "C overnight. 15 pg of digested DNA was electrophoretically separated on a 1% agarose gel, alkali-denatured, and transferred to Hybond-N" membrane (Amersham). The full length purified PKC,, cDNA insert obtained from the transfection plasmid was used as a probe. 100 ng of insert was labeled by the random oligonucleotide primer method (Amersham). 100 p1 of probe (4.5 X lo5 cpmlpl) was added to the membrane. The blot was hybrid- ized for 48 h followed by a 5-min wash in 2 X SSC at room tempe-rature, two 30-min washes in 2 X SSC with 1% SDS (w/v) at 65 "C, and a final 10-min wash of 0.1 X SSC at room temperature. The blot was then exposed to film (Kodak X-AR) for 24 h with two intensifying screens at -80 "C.

PKC, levels occurs relatively early during the differentiation program, the question arises as to whether this enzyme mediates some of the phenotypic changes induced by RA ( i e . decreased monolayer growth rate, elimination of anchorageindependent growth, and increased melanin production). This question was addressed by establishing and characterizing stable transfectants of B16-F1 cells that overexpress PKC, in the absence of exogenous RA.

MATERIALS AND METHODS
Stable Transfection of B16-FI Cells-We co-transfected early passage B16-Fl cells with a PKC, cDNA expression vector (YK504) (14) driven by an SR,,promoter (15) and the pSV40-neo plasmid encoding the gene for neomycin resistance at a 1:10 ratio (pSV40neo:YK504). Transfection was accomplished by the calcium phosphate/glycerol shock procedure (16). Cells transfected only with pSV40-neo (Neo) served as negative controls throughout most experiments. Neomycin-resistant clones were selected in Dulbecco's modified Eagle's medium (DMEM with 10% newborn calf serum) containing 1 mg/rnl of the neomycin derivative G418 (Gibco).
Western Blot Analysis for PKC,-Cells were seeded at 2 X 105/100mm dish. After attachment (6 h), cells were refed with DMEM containing 10% bovine calf serum with or without 10 p~ RA. RA was always handled in subdued light. Forty eight hours after addition of RA, cells were washed twice with PBS and harvested in 250 pl of lysis buffer (10 mM Tris (pH 7.5), 1 mM EDTA, 1% glycerol, 1 pg/ml leupeptin, 1 pg/ml pepstatin, 50 pg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). Harvested cells were lysed on ice by three consecutive 10-s sonications with a Tekmarm sonic disruptor at power setting 60. Protein concentrations were determined by the BCA@ (Pierce) protein assay. Crude samples were diluted to equal protein concentrations and electrophoretically separated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel. Proteins were electrically transferred to Hybond-C Extra'" nitrocellulose membrane (Amersham). The membrane was incubated overnight in blocking solution (5% nonfat dry milk in Tris-buffered saline (TBS), pH 8.0, containing 0.2% Tween). A 1:25 dilution of monoclonal anti-PKC, antibody (Seikagaku) was added for 2 h. This solution was removed, and the blot was washed several times in blocking solution followed by a 1-h incubation with a 1:3000 dilution of rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham), washed several times in blocking solution and one final time in blocking solution lacking the milk. Reactive bands were visualized by the enhanced chemiluminescence method (Amersham). All washes and incubations were performed at room temperature.
Enzyme Activities of Partially Purified PKC-Culture conditions were the same as those described for Western blot. Cells were lysed on ice with 20 mM Tris, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 5% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and 10 pg/ml aprotinin. Complete cell disruption was further ensured by three consecutive 10-s sonications with a TekmaP sonic disruptor at power setting 60. The total cell lysate was centrifuged at 12,000 X g for 15 min. The supernatant was loaded onto a DEAEcellulose anion exchange column (Cellex-D"', Bio-Rad) previously equilibrated with column buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol). The column was washed with 15 volumes of column buffer. The PKC fraction was eluted with 2 volumes of column buffer containing 100 mM NaCl and concentrated with a Centricon-lO'" microconcentrator (Amicon). Protein concentrations of samples were determined by the Bradford method (17). Samples were diluted to equal protein concentrations and assayed with a commercially available PKC assay system (Amersham) in the presence and absence of 12-0-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine. The system utilizes synthetic, PKCspecific, substrate peptides which become phosphorylated with the radiolabeled phosphate group from [-y-"'P]ATP. At the end of the reaction, the radiolabeled peptide was separated from unincorporated 'v2P by the use of an affinity paper for the peptide. The degree of phosphorylation was determined by liquid scintillation counting. Enzyme activity was calculated from counts/min taking into account the specific activity of the radioisotope and reaction time.
Southern Analysis of PKC, Restriction Fragments-Genomic DNA from wild type cells and clones H and K was digested with 10 units of HindIIIlpg of DNA a t 37 "C overnight. 15 pg of digested DNA was electrophoretically separated on a 1% agarose gel, alkali-denatured, and transferred to Hybond-N" membrane (Amersham). The full length purified PKC,, cDNA insert obtained from the transfection plasmid was used as a probe. 100 ng of insert was labeled by the random oligonucleotide primer method (Amersham). 100 p1 of probe (4.5 X lo5 cpmlpl) was added to the membrane. The blot was hybridized for 48 h followed by a 5-min wash in 2 X SSC a t room temperature, two 30-min washes in 2 X SSC with 1% SDS (w/v) a t 65 "C, and a final 10-min wash of 0.1 X SSC at room temperature. The blot was then exposed to film (Kodak X-AR) for 24 h with two intensifying screens a t -80 "C.
Monolayer Growth Curves and Doubling Times-Cells were seeded at a density of 1 X lo4 viable cells/lOO-mrn tissue culture dish (Falcon) in 8 ml of DMEM containing 10% bovine calf serum. Viability was determined by trypan blue dye exclusion. All cultures had more than 95% viable cells. Six hours after seeding, WT-RA received a 1:lOOO dilution of a 10 mM RA stock in dimethyl sulfoxide (Me2SO) to a final concentration of 10 p~ in DMEM. RA was handled in subdued light at all times. Media were changed a t 48 h. Cells were incubated at 37 "C in 7% CO1, 93% air. At 12-h intervals, cells were harvested by trypsinization, and viable cells were counted with a hemacytometer. (Means were determined to be significantly different from wild type by ANOVA followed by Newman-Keuls multiple comparisons.) Colony Formation in Soft Agarose-A 3-ml bottom layer of freshly prepared 1% agarose/DMEM/lO% serum containing the appropriate RA concentrations was dispensed into 60-mm dishes and allowed to solidify. The top layer consisted of 5000 viable cells suspended in 3 ml of 0.3% agarose/DMEM/lO% serum containing the respective RA concentrations. Following solidification of the top layer, a 2-ml DMEM liquid overlay, containing the appropriate RA concentrations, was added to each dish. The liquid phase was changed every 48 h. RA was always handled in subdued light. Viable colonies were counted 10 days after seeding. Only colonies greater than or equal to 300 pm in diameter were counted.
Relatiue Melanin Content of Media and Cells-Cells were seeded so that after 4 days their densities would be equal (approximately 1.7 X 10' cells/lOO-mm dish). After attachment, cells were refed with or without 10 p~ RA in DMEM. After 48 h, cells were refed with their respective RA concentrations. Exactly 10 ml of medium was added to each dish. On the 4th day, 1.5-ml aliquots of spent medium were taken and centrifuged a t 12,000 X g for 10 min. The relative melanin contents of supernatants were determined by measuring the A492 ",,,.
From each dish, 5 X 10' cells were collected by trypsinization followed by a 2000 X g centrifugation. The cell pellets were dissolved in 1 ml of 1 N KOH at 80 "C for 1 h. This hydrolysate was centrifuged at 12,000 X g for 10 min. The relative melanin concentrations of the supernatants were determined by measuring the A a t 492 nm.
Tumorigenicity in Syngeneic Mice-Thirty-two male, 4-6-weekold, C57BL/6 mice (Jackson Laboratories) were randomly allocated into four groups, each containing 8 animals. Each mouse received 6 X lo5 viable cells suspended in 0.1 ml of phosphate-buffered saline injected subcutaneously into the right flank. Viability and cell number were determined by trypan blue dye exclusion and counts on a hemacytometer. Each group of mice was injected with a different cell type; clones H, K, Neo, and WT respectively. Following injection, mice were palpated daily to determine tumor latency. We considered latency to be the number of days after injection on which the tumor could first be palpated. T o prevent unnecessary morbidity and suffering, we killed all mice on the 14th day after injection. Mice were killed by nitrogen gas inhalation. Tumors were excised and wet tumor weights were determined with a Mettler'" analytical balance. Statistical significance was determined by the nonparametric Kruskal-Wallis test followed by Dunnets multiple comparisons (comparing experimental groups to a control group) (18).

RESULTS
We transfected early passage B16-F1 cells with a PKC, cDNA expression vector as described under "Materials and Methods." Thirty-two G418-resistant clones were screened, yielding two consistently positive clones, designated as H and K, respectively. These clones were determined to overexpress PKC, based on both Western blot analysis ( Fig. 1) and enzyme activity (Fig. 2). The relative quantities of PKC,, were determined by scanning the Western blot autoradiograms with a computerized densitometer. These analyses showed that clones H and K contained approximately 3.3 and 2.3 times more PKC,, respectively, than wild type (WT) cells. Western blots also showed that RA treatment induced a further increase in PKC, levels in both overexpressing clones as well as the previously described PKC, induction in control cells (WT and cells transfected only with the neomycin resistance gene (Neo)). Both positive clones were further screened for increased PKC enzyme activity using a commercially available kit (Amersham) (Fig. 2). We found that clones H and K showed increased specific PKC activities 3.3 and 2.0 times greater, respectively, than those of WT or Neo cells (Fig. 2). As expected, RA treatment increased PKC activities in all cells. There was also a significant difference in PKC activities between clones H and K ( p < 0.025, Newman-Keuls).
T o confirm that H and K were indeed distinct clones that had stably incorporated the foreign PKC, gene, a Southern blot restriction analysis was performed (Fig. 3). The different Hind-I11 restriction patterns observed between DNA obtained from wild type cells and that from clones H and K illustrate that both clones have incorporated foreign PKC, DNA into their genomes. Furthermore, since clones H and K have different restriction patterns from each other, these data demonstrate that H and K are indeed separate clones.
In monolayer growth rate experiments, we found that cells from clones H and K had slower growth rates than untreated WT and Neo cells (Fig. 4). As we expected, RA treatment depressed the monolayer growth of WT and Neo cells. In comparing the growth of the two overexpressing clones, we found that by 48 h clone H had fewer cells per dish than clone K ( p < 0.05, Newman-Keuls). This finding correlated with the PKC levels and activities in these clones ( Fig. 1 and 2). When clones H and K were treated with RA, their respective  growth rates did not significantly decrease (data not shown). At the light microscopy level, we were unable to detect obvious morphological differences between PKC, overexpressing clones and control cells. The ability of PKC, overexpressing clones to form viable colonies in soft agarose was significantly lower than that of seeded into each dish. Dishes were refed with their respective media every 48 h. Ten days after seeding, colonies equal to or greater than 300 pm in diameter were counted. Wild type ( W T ) and transfected ( H , K, and Neo) B16 cells. The data are presented as the means 2 S.D. of triplicate plates. * mean is significantly different from WT, p < 0.001; Newman-Keuls. untreated WT and Neo cells (Fig. 5). Cells from clone H formed significantly fewer colonies than those from clone K ( p < 0.001, Newman-Keuls). As with the monolayer growth rate data, the difference in soft agarose growth between clones H and K correlated with their respective PKC levels. As expected, RA treatment of W T cells decreased their ability to form viable colonies in a concentration-dependent fashion. The colony-forming abilities of clones H and K were also reduced in a dose-dependent fashion when treated with RA (data not shown).
As an estimate of the degree of differentiation, we analyzed the cells for melanin production (intracellular as well as secreted melanin (Fig. 6). Cells from clones H and K contained and secreted more melanin than untreated control cells. RA  treatment of W T cells resulted in melanin levels comparable t o those from clones H and K. In light of the large reduction in soft agarose colony formation by the PKC, overexpressing clones, we decided to study their tumorigenicity. When syngeneic C57BL/6 mice were injected subcutaneously with the different cell types, we observed statistically significant differences in tumor latencies ( p < 0.01) and tumor wet weights ( p < 0.01) between the two PKC, overexpressing clones and their WT and Neo counterparts (Table I). Tumors, originating from control cells, weighed more, had shorter latencies, and commonly traversed the peritoneal membrane, massively involving the peritoneal cavity. Peritoneal involvement was much less common in tumors arising from clones H and K (data not shown).
We also excised and checked the lungs of each animal for the presence of melanotic foci which would suggest metastasis. None of the 32 mice showed evidence of metastasis to the lungs. This finding was expected, however, since the B16-F1 is a cell line of relatively low metastatic potential, and 14 days of tumor growth are a very short period for spontaneous metastasis to occur.

DISCUSSION
In this report, we show that B16 cells transfected with and overexpressing PKC, exhibit phenotypes very similar to those of wild type cells treated with RA. Both of the PKC, overexpressing clones showed slower monolayer growth rates, dimin-ished capabilities to form viable colonies in soft agarose, and increased melanin contents as well as secretion. When injected into syngeneic mice, both PKC, overexpressing clones produced smaller tumors and had longer latency times than control cells.
It is important to note that the level of PKC, overexpression achieved in both clones was lower than that induced by RA treatment of WT or Neo cells. The reason for this finding could be explained in that PKC, is apparently associated with diminished growth (Fig. 4). Therefore, we might have been selecting cells that overexpress an apparently negative growth regulator. If a cell should overexpress very large amounts of PKC,, it might grow so slowly that no colony would be formed. Therefore, we believe that inducing higher levels of PKC, in this system would require transfection with PKC, under the control of an inducible promoter.
Other laboratories have transfected various cells with other PKC isozymes (19)(20)(21). In two of these reports, the investigators transfected nonmalignant fibroblasts and obtained clones which overexpressed PKC, and PKCo,, respectively, at very high levels. In both cases, the PKC overexpressors assumed some characteristics of transformed cells (19,21). When malignant HT29 (human colon carcinoma) cells were transfected with PKCB1, the overexpressing cells (11-to 15fold) showed evidence of diminished malignancy (20). Also, when PKCB protein was introduced into erythroleukemia cells, they differentiated at a faster rate (22). In this report, we show evidence that B16 melanoma cells transfected with and overexpressing the PKC, isozyme at levels 2-4-fold higher than wild type cells, acquire a more differentiated phenotype. An important difference between the HT29 study (20) and ours is that we did not have to treat our overexpressing clones with phorbol esters in order to induce a phenotypic change. This might be explained by the presence of factors in the cell culture medium (i.e. calf serum or autocrine factors) which could stimulate a constant diacylglycerol production, thus activating the enzyme in the absence of additional cofactors such as phorbol esters. This would suggest that in B16 melanoma cells the limiting factor is the amount of PKC,, enzyme and not the activators of the enzyme.
Our transfectants produced lower levels of PKC, than those in other systems, yet the phenotype of B16 melanoma cells is apparently quite sensitive to small changes in the level of this enzyme. Furthermore, the data seem to suggest that some phenotypic traits, i.e. melanin production and monolayer growth, require lower levels of PKC, in order to saturate downstream regulators than other traits, such as colony formation in soft agarose.
The sole fact that the PKC, overexpressing clones yielded smaller tumors than their control counterparts does not necessarily indicate that their growth rates, following latency, were significantly slower. Longer latency periods by themselves could have resulted in such findings. On the other hand, one could speculate that the selective pressures within the animal might have resulted in the growth of cells that have lost PKC, overexpression. Due to the rapid tumor growth and its short duration, we were unable to accurately calculate tumor growth rates.
Although the disparate actions of PKC overexpression on the phenotypes of the host cell still seem an enigma, similar findings apply to the ras oncogene. Some cells, when transfected with v-ras, adopted a transformed phenotype (23-25), while others are induced to differentiate (26). Thus, it becomes apparent that the cellular milieu in which certain regulatory proteins function dictate the biological response.
RA has been repeatedly shown to induce differentiation in several different types of cancer cells. The mechanism by which RA induces differentiation is still largely unknown.
Our observations, together with the findings that prolonged phorbol ester treatment of B16 cells depletes the cells of PKC,, and counteracts the effects of RA treatment (10, 27), provide strong evidence that PKC, plays a key role in RAinduced B16 cell differentiation.