Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

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.     

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.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.     

Evidence for a calcium/calmodulin involvement in density-dependent melanogenesis in murine B16 melanoma cells.

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to alpha MSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. alpha MSH-stimulated melanin production was extremely density-dependent but alpha MSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as alpha MSH.     

Evidence for a Calcium/Calmodulin Involvement in Density-Dependent Melanogenesis in Murine B16 Melanoma Cells

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to αMSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. αMSH-stimulated melanin production was extremely density-dependent but αMSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as αMSH.     

Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells. Evidence for a differential induction of two distinct isoenzymes.

Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.     

Regulation of mammalian melanogenesis by tyrosinase inhibition

Melanocyte stimulating hormone (MSH) specifically induces differentiation of mammalian melanocytes. To further define the biochemical events elicited by this stimulus, we have cloned murine melanoma cells which are either highly responsive or nonresponsive to MSH, and have examined their ultrastructural appearance, their melanogenic activities, and also their expression of tyrosinase. We have found that the basal levels of melanogenic activity in pigmented and nonpigmented cells correlate with expression of surface MSH receptors rather than with production of tyrosinase. Nonpigmented cells produce a potent, highly stable inhibitor of melanogenesis; this inhibitor acts directly on tyrosinase to dramatically and abruptly suppress melanin production. This posttranslational control of tyrosinase activity may represent a critical regulatory point in mammalian pigmentation.     

Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors.

Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 microM-2 mM) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.     

Alpha tocopheryl succinate inhibits melanocyte-stimulating hormone (MSH)-sensitive adenylate cyclase activity in melanoma cells

D-alpha tocopheryl succinate (vitamin E succinate), which is known to induce differentiation and growth inhibition in murine B-16 melanoma cells, reduced basal and melanocyte-stimulating hormone (MSH)-stimulated adenylate cyclase (AC) activity in vitro. Vitamin E succinate treatment also reduced sodium fluoride- and forskoline-stimulated AC activity of melanoma cells in vitro. Treatment of cells with vitamin E succinate (6 micrograms/ml] for a period of 24 hours was sufficient to reduce MSH-stimulated AC activity. Other forms of vitamin E, such as d1-alpha tocopheryl nicotinate, d1-alpha tocopheryl acetate, and d1-alpha tocopherol, which did not affect growth or morphology of melanoma cells, were relatively less effective in altering basal and MSH-stimulated AC activity. Retinoic acid, which inhibited the growth of B-16 melanoma cells, also reduced basal and MSH-, NaF-, and forskolin-stimulated AC activity in vitro. Prostaglandin A2, which inhibited growth and altered morphology, did not change basal or MSH-stimulated AC activity. These results show that one of the mechanisms of action of vitamin E succinate and retinoic acid on melanoma cells may involve reduction of basal and MSH-sensitive AC activity, and this vitamin effect is not necessarily related to growth inhibition.     

D‐tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity

Although L‐tyrosine is well known for its melanogenic effect, the contribution of D‐tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L‐tyrosine, D‐tyrosine dose‐dependently reduced the melanin contents of human MNT‐1 melanoma cells and primary human melanocytes. In addition, 500 μM of D‐tyrosine completely inhibited 10 μM L‐tyrosine‐induced melanogenesis, and both in vitro assays and L‐DOPA staining MNT‐1 cells showed that tyrosinase activity is reduced by D‐tyrosine treatment. Thus, D‐tyrosine appears to inhibit L‐tyrosine‐mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D‐tyrosine inhibited melanogenesis induced by α‐MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D‐tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D‐tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte‐derived cells.     

Structural/functional relationships between internal and external MSH receptors: modulation of expression in Cloudman melanoma cells by UVB radiation

Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al: J. Cell. Physiol., 142:129-136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bolognia et al: J. Invest. Derm., 92:651-656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.     

Decreased dopachrome oxidoreductase activity in yellow mice

Dopachrome oxidoreductase (DCOR) is a newly characterized enzyme in the melanin synthetic pathway, active in the conversion of dopachrome to 5,6-dihydroxyindole. DCOR and tyrosinase activity were measured in skin anagen hairbulbs from lethal yellow (Ay/a), sienna yellow (Asy/a) and recessive yellow (e/e) mice with and without treatment with melanocyte-stimulating hormone (MSH). DCOR activity was low (Asy/a) or absent (Ay/a, e/e) in yellow mice without MSH treatment, and increased dramatically in the lethal and sienna yellow mice with MSH. There was no increase in DCOR activity in recessive yellow mice with MSH. Corresponding tyrosinase activity was reduced in lethal yellow and sienna yellow mice without MSH, and increased with MSH. Tyrosinase activity was normal in recessive yellow mice without MSH and did not change with MSH. We conclude that DCOR is an MSH-sensitive enzyme and that DCOR activity is absent in recessive yellow melanocytes. The latter finding suggests that the extension locus may be the DCOR locus.     

Different susceptibilities of melanoma cells to retinoic acid-induced changes in melanotic expression

The effect of retinoic acid on murine B16 melanoma cell growth, tyrosinase activity and melanin synthesis was investigated. Retinoic acid inhibited the growth of B16F1, B16F10 and B16BL6 melanoma cells, but enhanced melanin synthesis only in the B16F1 cells. The B16F10 and B16BL6 cells exhibited retinoic acid-induced suppression of tyrosinase activity and melanin synthesis, which was most apparent in the B16F10 cell variant. For comparison, Cloudman S91 melanoma cells proved to be particularly sensitive to retinoic acid-induced growth inhibition and stimulation of the expression of their melanotic phenotype. These results suggest considerable heterogeneity in the B16 melanoma with respect to their response to retinoic acid.     

Isolation and characterization of a variant of B16-mouse melanoma resistant to MSH growth inhibition

A variant of B-16 F₁ mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition by MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F₁ cells. MSH induced a twofold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F₁ cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F₁ kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F₁ and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F₁ cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.