In Vitro Phenotypic Alteration of Human Melanoma Cells Induced by Differentiating Agents: Heterogeneous Effects on Cellular Growth and Morphology,Enzymatic Activity,and Antigenic Expression

Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G₁ phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and -γ-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B863 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of -γ-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells.     

Growth inhibition of murine melanoma by butyric acid and dimethylsulfoxide

Treatment of B16-F10 melanoma cells with dimethylsulfoxide (DMSO) or butyric acid (BA) inhibits cell growth and delays tumor appearance in syngeneic mice. Both agents induce morphological changes in these cells. Treatment of melanoma cells with DMSO results in a marked increase in tyrosinase activity and melanin content. BA, on the other hand, does not increase melanin content and decreases tyrosinase activity. The data show that there are marked differences in the effect of DMSO and BA on melanin biosynthesis, whereas both agents inhibit cell growth and cause a delay in tumor appearance. These findings indicate that decreased proliferation of melanoma cells and induction of melanin biosynthesis are not necessarily associated phenomena.     

Involvement of phospholipase D1 in melanogenesis of mouse B16 melanoma cells

In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.     

Effects of dimethyl sulfoxide (DMSO) on microfilament organization, cellular adhesion, and growth of cultured mouse B16 melanoma cells

Cell shape is involved in a variety of cellular activities including proliferation, adhesion, migration, and transformation. Agents known to promote differentiation, such as retinoic acid, butyrate, and dibutyryl cyclic AMP, induce marked alterations in cell shape which are often accompanied by changes in cell functions. In this paper we study the effects of the differentiating polar solvent dimethyl sulfoxide (DMSO) on cytoskeleton, adhesion, and growth properties of cultured mouse B16 melanoma cells. DMSO induced a progressive reorganization of the cytoskeleton which was fully developed in 4 days of continuous exposure to the agent. DMSO-treated cells developed thick and regularly oriented microfilament bundles of the stress fiber type ending at vinculin-rich areas of focal contact between the ventral membrane and the substratum (interference reflection microscopydark adhesion plaques). Such a rearrangement of the cytoskeleton resulted in increased adhesion to the substratum and inhibition of cell growth in comparison to control untreated cells. Cells which became highly flattened and tightly adherent after exposure to DMSO for 4 days progressively reverted their phenotype to that of control untreated cells within 3 days of DMSO withdrawal. Namely, they lost stress fibers and adhesion plaques, became rounded and less adherent, and increased their growth rate. These results indicate that DMSO can change the transformed appearance of B16 mouse melanoma cells to a phenotype which is typical of a variety of nontransformed cells in culture.     

Comparative effects of butyrate and N6, 2'-O-dibutyryladenosine-3':5'-cyclic monophosphate on growth, differentiation and gene expression in U937 human monoblastoid cells.

Administration of 1mM sodium butyrate or N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) inhibits the growth activity of U937 human monoblastoid cells by blocking them at the G1 or at the G1 + G2 phases of the cell cycle, respectively. Both agents induce the differentiation of U937 cells, as proved by the increased expression of the maturation-associated CD11b antigen and by the increased capacity to reduce nitroblue tetrazolium. RNA blot assays indicate that butyrate and dbcAMP decrease the expression of ornithine decarboxylase and c-myc genes, and stimulate the expression of the vimentin gene. However, while dbcAMP induces c-fos mRNA accumulation, butyrate did not affect the expression of this proto-oncogene.     

Retinoic acid blockade of imidazole-induced tyrosinase expression in B16 melanoma cultures: similar effects of the active retinoid and triiodothyronine

The effect of retinoic acid on the induction of tyrosinase (EC 1:14.18.1) by imidazole was determined in cultured B16/C3 melanoma cells. Retinoic acid could block the induction of enzyme activity within 3 hours of addition to the culture medium at a physiological concentration (10nM). The blockade was similar to that of 3,3',5-L-triiodothyronine (T3) already reported. mRNA hybridizable to a tyrosinase DNA probe was induced by imidazole while retinoic acid and T3 blocked that increase. These observations suggest that retinoic acid can mimic the action of T3 in B16 melanoma cells in culture.     

Chemical inducers of differentiation,dimethylsulfoxide, butyric acid,and dimethylthiourea,induce selective ultrastructural patterns in B16 melanoma cells

The morphology and ultrastructure of B16 melanoma cells was examined after treatment of the cells with the chemical inducers of differentiation dimethylsulfoxide (DMSO), butyric acid, and dimethylthiourea (DMTU). The treated B16 melanoma cells seemed to be enlarged and more flattened, and to possess dendrite-like structures as revealed by scanning electron microscopy. The main ultrastructural features, depicted by transmission electron microscopy in DMSO-treated B16 cells were: a marked increase in melanin granules, migration of the melanin granules to the dendrites, and appearance of melanosome aggregates. Butyric acid did not induce melanin biosynthesis; however, it stimulated rough endoplasmic reticulum (RER) formation all over the cytoplasm. The DMTU-treated cells also showed a well developed RER accompanied by early stages of melanosomes and melanin granules. The increase in the endoplasmic reticulum was also reflected by enhancement of NADPH cytochrome c reductase activity, an enzymatic marker of the endoplasmic reticulum. The mitochondria in the DMTU-treated cells were swollen with disrupted cristae. The results indicate that DMSO, butyric acid, and DMTU induce different ultrastructural patterns in B16 melanoma cells. These findings correlate with the biochemical alterations induced in melanoma cells by these agents.     

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies

Effect of six organic solvents—methanol, ethanol, propanol, dimethyl sulphoxide (DMSO), N,N-dimethyl formamide (DMF), and glycerol on the conformation and interaction of catalase and anticatalase antibodies were studied with the aim of identifying the solvents in which antigen–antibody interactions are strong. The antigen binding activity of the antibodies in the various organic solvents increased in the following order: ethanol < methanol < no organic solvent < propanol < DMSO < DMF < glycerol. The structure of both the antibody and the antigen molecule was affected significantly in 40% concentration of the organic solvents used in this study. Catalase activity was inhibited in DMSO. However, the enzyme was activated in DMF upto about 50% of its concentration.     

Cytomorphological changes in human tumor cells exposed to high-polar compounds

The effects of some high-polar compounds on the cytomorphological characteristics of cultured cells of synovial and osteogenic human sarcomas (Sa-2 and Sa-4) were studied. Incubation of Sa-2 cells with 1 or 2% dimethylsulfoxide (DMSO) or 100 mM dimethylformamide (DMF) during 4-6 days induced the cellular, structural and functional changes in Sa-2 cells which were considered as manifestations of pseudoepithelial differentiation. DMSO did not influence the activity of Sa-2 cell enzymes, while DMF suppressed their activity, mainly that of acid and alkaline phosphatases. DMSO and DMF induced in Sa-4 cultured the appearance of the cells which were similar cytomorphologically to normal osteoblasts. DMSO and DMF significantly depressed the activity of acid phosphatase in Sa-4 cells and transformed the positive reaction of alkaline phosphatase to the negative one. DMSO and DMF prolonged the time of the doubling of Sa-2 and Sa-4 cells. The effects of DMSO and DMF were reversible. Methylformamide (200 mM) and dimethyl acetamide (0,1 and 10 mM) did not induce analogous changes in human sarcoma cells.     

Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Differential effects of sodium butyrate and dimethylsulfoxide on gamma-glutamyl transpeptidase and alkaline phosphatase activities in MCF-7 breast cancer cells

Sodium butyrate and dimethylsulfoxide (DMSO), two known chemical inducers of cell differentiation, were examined on MCF-7 breast cancer cells. Both agents reduce the proliferative capacity of MCF-7 cells, as reflected by inhibition of colony formation in semisolid agar. Sodium butyrate is shown to enhance markedly the activity of two plasma membrane-bound enzymes, alkaline phosphatase and gamma-glutamyl transpeptidase. DMSO does not enhance the activity of these enzymes, but rather induces a small decrease in gamma-glutamyl transpeptidase activity. The present results show that although both agents inhibit cell proliferation, they have a distinct effect on phenotypic expression.     

Differentiation of TERA-2 human embryonal carcinoma cells into neurons and HCMV permissive cells

Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis-diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2'deoxyribose (BUdR), 5-iodouracil 2'deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%-80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA.     

Acute effects of two melanocytolytic agents, hydroquinone and beta-mercaptoethanolamine, upon tyrosinase activity and cyclic nucleotide levels in murine melanomas

The acute in vitro actions of two potent melanocytolytic agents, hydroquinone (HQ) and beta-mercaptoethanolamine (MEA), were determined in the B-16, Cloudman S-91 and Harding-Passey (HP) murine melanomas grown in vivo. Drug treated melanoma dice (5--480 min) were analyzed for tyrosinase activity and cyclic nucleotide levels (cAMP, cGMP). HQ and MEA effects on tyrosinase activity are complex and vary with tumor type, duration of treatment and agent tested. MEA or HQ inhibited B-16 tyrosinase activity. With combined drug therapy, low concentrations of MEA plus HQ stimulate B-16 tyrosinase activity while high concentrations of the drugs have little effect on enzymatic activity. MEA depresses tyrosinase activity while HQ elevates enzymatic activity in the S-19 melanoma. Both high and low concentrations of the combined drugs (MEA plus HQ) elicit the same response, stimulation at 10 min followed by continued depression of tyrosinase activity for the remainder of the 4 h study period. MEA initially stimulates HP tyrosinase activity followed by depression of enzymic activity. In contrast, HQ initially depresses HP tyrosinase activity followed by stimulation of enzyme activity. In combination the drugs inhibit HP tyrosinase activity. The effects of MEA and/or HQ on murine melanoma cyclic nucleotide levels are equally complex. MEA or HQ elevate cAMP and cGMP levels in all three tumors with the exception of S-91 cGMP levels which are not altered. In combination the drugs increase cyclic nucleotide levels in each of the three tumor types but at different times. No correlation is present between cyclic nucleotide levels and tyrosinase activity. Thus, the action of increased cyclic nucleotide levels in melanogenesis can not be separated from the direct actions of MEA and HQ upon melanogenesis. The divergent effects of MEA and/or HQ on tyrosinase activity and cyclic nucleotide levels in these melanomas are not correlated with the known in vivo melanocytolytic activity of these drugs. Thus, these parameters appear to be inadequate indicators of melanoma cell viability in chemotherapeutic screening of drugs effective in destroying malignant melanoma.     

Inhibition of D1, D2, and a cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a product of lipid peroxidation, is an highly reactive aldehyde that, at concentration similar to those found in normal cells, blocks proliferation and induces a granulocytic-like differentiation in HL-60 cells. These effects are accompained by a marked increase in the proportion G0/G1 cells. The mechanisms of HNE action were investigated by analyzing the expression of the cyclins and cyclin-dependent protein kinases (CDKs), controlling the cell cycle progression. Data obtained by exposing cells to dimethyl sulfoxide (DMSO) were used for comparison. 4-Hydroxynonenal downregulated both mRNA and protein contents of cyclins D1, D2, and A until 24 h from the treatments, whereas DMSO inhibited cyclin D1 and D2 expression until the end of experiment (2 days) and induces an increase of cyclin A until 1 day. Cyclins B and E, and protein kinase CDK2 and CDK4 expressions were not affected by HNE, whereas DMSO induced an increase of cyclin E, B, and CDK2 from 8 h to 1 day. These data are in agreement with previous results indicating a different time-course of accumulation in G0/G1 phases of cells treated with HNE and DMSO and suggest that the HNE inhibitory effect on proliferation and cell cycle progression may depend by the downregulation of D1, D2, and A cyclin expression.     

The effects of histamine H2 receptor antagonists on melanogenesis and cellular proliferation in melanoma cells in culture

B16-C3 murine melanoma, A375P human melanotic melanoma, and C32 human amelanotic melanoma cells were incubated in the presence of (0-4 mM) H2-antagonists, ranitidine and cimetidine. Cell proliferation, tyrosinase activity and melanin content were monitored. H2-antagonists stimulated tyrosinase activity and melanin accumulation in B16-C3 cells in a dose- and time-dependent manner. Stimulation of enzyme activity and pigment production was accompanied by inhibition of cellular proliferation in B16-C3 cells. The inhibitory concentration of cimetidine was approximately 2-fold higher than that of ranitidine. H2-antagonists failed to stimulate melanogenesis in A375P or C32 cells, but inhibited cellular proliferation in both cell lines. These results are the first demonstration of H2-antagonist induced phenotypic changes in malignant melanoma cells in vitro, and represent a novel mechanism for the previously described in vivo antitumor effects of these agents.     

Effect of thymidine analogs on tyrosinase activity and mRNA accumulation in mouse melanoma cells

The thymidine analog 5-bromodeoxyuridine (BrdU) has been found to inhibit terminal differentiation of a variety of cell types without significantly affecting the growth of these cells. We have compared the effect of BrdU with two other thymidine analogs, 5-iododeoxyuridine and 5-fluorodeoxyuridine, on the growth, tyrosinase activity, and tyrosinase-mRNA accumulation in BL-6 mouse melanoma cells. We show that all three analogs inhibit growth and tyrosinase activity, but only BrdU significantly inhibits the accumulation of tyrosinase mRNA. We consider these results in the light of current understanding of BrdU action.     

Cellular sialic acid level and phenotypic expression in B16 melanoma cells: comparison of spontaneous variations and bromodeoxyuridine- and theophylline-induced changes

The relation of the total cellular content of sialic acid to phenotypic expression of B16 mouse melanoma cells was examined by using phenotype-modifying reagents and more than 10 cloned cell lines with spontaneous phenotypic variations. The sialic acid content changed in a growth phase-dependent manner with a peak in the early log phase of growth. This peak completely disappeared when cells were treated with 5-bromodeoxyuridine (BrdU), suggesting its relation to quasi-normal phenotypes of the treated cells. BrdU treatment also reduced the cellular sialic acid content itself and resulted in the suppression of the activity of tyrosinase, the key enzyme for melanogenesis, and a considerable increase in cell-to-substratum adhesiveness. Treatment with theophylline, in contrast, markedly elevated the sialic acid content, which was accompanied by dramatic increments in tyrosinase activity and pigmentation as well as a slight increase in adhesiveness. The results show a correlation of sialic acid level with tyrosinase expression but not with cell adhesion. From comparison of spontaneous phenotypic variations, the correlation of sialic acid level with tyrosinase activity was confirmed, while there was only a slight correlation with adhesiveness. It is thus suggested that sialylation/desialylation, being reflected as variations in cellular sialic acid content, is implicated in melanoma cell differentiation in terms of tyrosinase expression.