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Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20-30 mJ/cm2 for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M "injury responses" of other cell types might be expected, consistent with their differentiated phenotypes.

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.

Receptors for melanotropin (MSH) were found to be expressed by immortalized primary human epidermal keratinocytes (RHEK-1). Using 125I-beta MSH as a probe, the MSH receptors from mouse melanoma cells and human keratinocytes were found to be remarkably similar. In each cell line, there were high and low affinity receptors, with the high affinity classes showing positive cooperativity. Competition of 125I-beta MSH for binding with non-radioactive MSH revealed similar profiles. Cross-linking studies, followed by gel electrophoresis and autoradiography, showed almost identical gel migration patterns. Both cell types expressed internal as well as plasma membrane binding sites. MSH receptors on both cell types were up-regulated by ultraviolet light and by MSH itself. Although the function of MSH receptors expressed by the immortalized keratinocytes is unknown, the results are consistent with recent reports that proliferation of epidermal keratinocytes is stimulated by MSH and that proopiomelanocortin genes are expressed in the epidermis. These results support a model in which keratinocytes and melanocytes, interacting in an "epidermal-melanin unit," each respond to UV light signals with increased MSH receptor activity.

MSH can up-regulate MSH binding capacity of cultured Cloudman melanoma cells in a dose- and time-dependent fashion. Binding is mediated through proteins exhibiting an apparent molecular weight of 50-53kDa, consistent with previous studies implicating them as the principal MSH receptors on Cloudman cells. Pre-incubation of cells with MSH stimulates expression of the receptor proteins both on the plasma membrane surface as well as in internal sites associated with coated vesicles. The effects of MSH are additive with those of UV light, suggesting that UV and MSH might stimulate receptor expression through separate mechanisms.

When human skin is exposed to ultraviolet (UV) light, a highly complex cascade of events ensues that culminates, among other things, in increased skin melanin content. From analyses at the tissue and cellular level, it has been shown that following exposure to UV light there is an increase in the number of active melanocytes in the basal layer of the epidermis, and individual melanocytes are stimulated to produce more melanin. In addition, the rate of transfer of melanosomes from melanocytes to keratinocytes is apparently increased, although the role of UV light in this process remains to be demonstrated. Recent biochemical evidence is reviewed on factors that regulate these processes. A plausible explanation for the effects of UV on pigmentation is that there are mechanisms in the skin for the orderly, regulated reception of UV signals that are then transduced to initiate the cascade. The signals involve both melanocytes and keratinocytes, and available evidence supports a model in which melanotropins and their receptors play a central role in the process.

Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 micron filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.).(ABSTRACT TRUNCATED AT 250 WORDS)

Dopachrome tautomerase (DT) (EC 5.3.2.3) is a melanocyte-specific, membrane-associated, heat-labile, non-dialyzable, protease-sensitive factor which catalyzes the isomeric rearrangement of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), apparently through a tautomerization reaction. Metal ions such as Cu, Ni, Co, Zn, Mn, Ca, Al, and Fe can also catalyze the dopachrome/DHICA isomerization. How is the reaction regulated in vivo? An attractive possibility would be that DT is a metalloenzyme. Here we present evidence that this may indeed be the case. Purified preparations of DT and tyrosinase, obtained from Cloudman S91 mouse melanoma cells, were assayed in the presence of a variety of metal chelators including EDTA (predominantly Ca and Mg), EGTA (predominantly Ca), phenylthiourea (PTU) (predominantly Cu), 2,2'-dipyridyl (predominantly Fe); 1,10-phenanthroline (predominantly Fe), and 2,3-dihydroxybenzoic acid (predominantly Fe). In addition, DT activity was assayed in the presence of two non-chelating structural analogs of 1,10-phenanthroline. Results were as follows: (i) iron chelators inhibited DT activity with no effects on tyrosinase activity; (ii) inhibition by the chelators was reversible with the addition of ferrous iron; (iii) 1,10-phenanthroline pre-complexed to ferrous iron was not inhibitory to DT; (iv) non-chelating analogs of phenanthroline were not inhibitory to DT; (v) PTU was inhibitory to tyrosinase but not DT; (vi) Ca2+ and Mg2+ chelators had little effect on either enzyme activity. Finally, studies with glycosylation inhibitors, glycosylase enzymes, and immobilized lectins, indicated that DT is a glycoprotein. The results suggest that DT is a metal-containing glycosylated enzyme, possibly with ferrous iron at its catalytic center.

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.

Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: 1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; 2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; 3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and 4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1.

Dopachrome, an intermediate in melanin biosynthesis, exhibits some unusual properties. At physiologic pH (e.g., pH 6-8) it is unstable and spontaneously loses its carboxyl group to form 5,6-dihydroxyindole (DHI) and CO2. However, over this same pH range, if various metals or a melanocyte-specific enzyme are present, it rapidly rearranges to its isomer form--5,6-dihydroxyindole-2-carboxylic acid (DHICA)--which is far more stable than dopachrome in its ability to retain the carboxyl group. Whether or not the carboxyl group is retained could have important implications for the regulation of melanogenesis, since in the presence of oxygen DHI spontaneously forms a black precipitate, whereas DHICA forms a golden-brown solution. The solubility of "DHICA-melanin" is due to the presence of carboxyl groups, which provide negative charges and hydrophilicity. Thus, in vivo, the extent to which dopachrome is converted to DHI or DHICA may well influence the solubility and color of the melanin formed. The purpose of this article is to review recent findings in these areas and to discuss the possible significance of dopachrome conversion in the regulation of melanogenesis and color formation.

Treatment of Cloudman S91 melanoma cells with retinoic acid (RA) inhibits MSH-induced tyrosinase activity and melanin formation [(1990) J. Invest. Dermatol. 94, 461-464]. We report here, however, that in spite of inhibiting MSH-induced pigmentation, RA treatment caused a marked increase in MSH binding capacity for both cell surface and internal MSH binding sites. The stimulation was dose- and time-dependent and reversible, with half-maximal effects seen at 2 microM RA. Stimulation of MSH binding was seen as early as 3 h after exposure of cells to RA. Cell surface and internal binding activity increased in concert. Scatchard analysis indicated that increased MSH binding resulted from a 3-4-fold increase in the number of sites with no significant difference in their affinity for MSH. It appears that in suppressing MSH-induced melanogenesis, RA elicited a compensatory up-regulation of the MSH receptor system.

Retinoic acid, hexamethylene bisacetamide, sodium butyrate, and dimethylsulfoxide, four compounds which modulate phenotypic expression in a variety of neoplastic cell lines, all inhibited the induction of tyrosinase activity and melanogenesis by the combination of melanocyte-stimulating hormone and isobutylmethyxanthine in Cloudman S91 melanoma cells. Results were the same in assays of whole cells or in extracts made from them. Only retinoic acid, however, was effective at inhibiting the activation of dopachrome isomerase, another regulatory enzyme in melanogenesis. Despite inhibiting the effects of melanocyte-stimulating hormone (MSH) and isobutylmethylxanthine on tyrosinase activity, all of the agents tested increased the binding of MSH to intact cells. Ultrastructural analysis of treated cells following DOPA cytochemistry revealed that both retinoic acid and hexamethylene bisacetamide arrested melanosomal maturation at stage I-II. Retinoic acid resulted in a derangement of melanosomal structure. The specificity of these agents for preventing the induction of melanogenesis makes them powerful tools for the dissection of this complex cellular process.

A stock of hairless pigmented guinea pigs was developed to facilitate studies of mammalian pigmentation. This stock combines the convenience of a hairless animal with a pigmentary system that is similar to human skin. In both human and guinea pig skin, active melanocytes are located in the basal layer of the interfollicular epidermis. Hairless albino guinea pigs on an outbred Hartley background (CrI:IAF/HA(hr/hr)BR; designated hr/hr) were mated with red-haired guinea pigs (designated Hr/Hr). Red-haired heterozygotes from the F1 generation (Hr/hr) were then mated with each other or with hairless albino guinea pigs. The F2 generation included hairless pigmented guinea pigs that retained their interfollicular epidermal melanocytes and whose skin was red-brown in color. Following UV irradiation, there was an increase in cutaneous pigmentation as well as an increase in the number of active epidermal melanocytes. An additional strain of black hairless guinea pigs was developed using black Hr/Hr animals and a similar breeding scheme. These two strains should serve as useful models for studies of the mammalian pigment system.

Melanocyte-stimulating hormone (MSH) induces melanogenesis in Cloudman mouse melanoma cells. The activities of two enzymes in the melanogenesis pathway, tyrosinase and dopachrome conversion factor, are increased as part of the induction process. Trans retinoic acid (RA), at concentrations as low as 0.1 nM, inhibited the induction of tyrosinase, dopachrome conversion factor, and melanogenesis, but had no effect on the basal levels of either enzyme or of cellular melanin content. Half-maximal effects of RA occurred at a concentration of 10 nM; maximal effects were observed at 1 microM. The effects of RA on melanogenesis were independent of its effects on cellular growth since one Cloudman line tested was growth-inhibited by RA and another was growth-stimulated by RA, but the induction of melanogenesis by MSH in both lines was inhibited by RA. Mixing experiments with cell lysates failed to demonstrate the induction of a tyrosinase inhibitor by RA. The effects of RA were not limited to MSH or to Cloudman melanoma cells since RA blocked cholera toxin-inducible melanogenesis in Cloudman cells, as well as the induction of tyrosinase activity by L-tyrosine in Bomirski hamster melanoma cells. The effects of RA were specific to melanogenesis, however, since RA did not interfere with MSH-induced changes in cellular morphology and growth. Thus, RA appears to be a new and potent tool for understanding mechanisms regulating induction of the pigmentary system.

Dopachrome conversion factor is an enzymatic activity associated with the pigmentary system which catalyzes the conversion of dopachrome, an intermediate in melanin biosynthesis, to dihydroxyindole-2-carboxylic acid (DHICA). To date, the mechanism of action of DCF has been unknown because all previous assays have employed a dopachrome substrate contaminated with L-dopa. It has therefore not been possible to determine whether L-dopa acts as a hydrogen donor in the reaction or whether the formation of DHICA occurs through an isomerization of dopachrome. In this study it is shown that DCF catalyzes the conversion of dopachrome to DHICA equally well in the presence or absence of L-dopa. The DCF-mediated reaction thus appears to be an isomeric rearrangement of hydrogen ions from one portion of the dopachrome molecule to another. The results indicate that the name "dopachrome isomerase" appropriately describes the function of DCF.

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient (8-80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: 1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. 2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. 3) Most of the internal binding sites were not as dense as fully melanized melanosomes. 4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. 5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) 6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.

Growth and melanization are intimately related in melanoma cells. MSH, by promoting elevated cyclic AMP levels, causes increases in melanization, cessation of growth, and gross morphologic changes in Cloudman S-91 melanoma cells. Growth inhibition results from high levels of cyclic AMP while growth stimulation occurs with lower levels. During melanization, oxidation products of tyrosine are generated which are toxic to the cells. Genetic studies have revealed that some of these processes are related through common biochemical pathways. This article reviews work of recent years on such regulatory mechanisms in melanoma.

The biosynthesis of melanin is initiated by the catalytic oxidation of tyrosine to dopa by tyrosinase in a reaction that requires dopa as a cofactor. Tyrosine then catalyzes the dehydrogenation of dopa to dopaquinone. The subsequent reactions can proceed spontaneously in vitro. Tyrosinase, purified from murine melanomas and the skins of brown mice, has now been shown to catalyze a third reaction in mammalian melanogenesis, namely the conversion of 5,6-dihydroxyindile to melanochrome. This reaction requires dopa as a cofactor and is inhibited by tyrosine. Conversely, 5,6-dihydroxyindole inhibits the oxidation of tyrosine to dopa, so that the relative concentrations of tyrosine and 5,6-dihydroxyindole within the mammalian pigment cell are capable of regulating melanogenesis in a previously unrecognized fashion. Tyrosinase has the unusual property of catalyzing three distinct reactions within a single biochemical pathway: the hydroxylation of a monophenol, the dehydrogenation of a catechol, and the dehydrogenation of a dihydroxyindole. The first and third of these reactions require dopa as a cofactor; in the second reaction, dopa is a substrate.

Ultraviolet B radiation (UVB) elicits an increase in melanin production in mammalian skin. The mechanisms regulating this process are not understood, although it is well documented that there is an increase in the number of melanin-producing melanocytes. The melanotropins (MSH) are a family of peptides that increase the melanin content of melanocytes through an interaction with high affinity receptors. We have obtained evidence that the effects of UVB on melanogenesis may be mediated through an increase in MSH receptor activity on melanocytes. First, exposure of Cloudman S91 mouse melanoma cells to UVB resulted in increased binding of 125I-MSH to cells within 24 h. In five separate experiments, UVB-irradiated cultures displayed 2-10-fold increases in MSH binding capacity over that of unirradiated control cultures (optimum doses 10-20 mJ/cm2). Second, UVB and MSH potentiated one another in promoting cutaneous melanogenesis in both mice and guinea pigs. In the areas of guinea pig skin that received both UVB and MSH, there was a fivefold increase in active melanocytes/mm2 over the sum of active melanocytes/mm2 in areas receiving either MSH or UVB separately. Our results suggest that UVB light causes an increase in MSH receptor activity on cutaneous melanocytes, thus increasing cellular responsiveness to MSH. Implicit in this mechanism is a transduction of radiant energy into chemical energy during the process of UVB-induced melanogenesis.

Bomirski Ab amelanotic melanoma cells have recently been shown to undergo striking phenotypic changes when precursors of the melanogenic pathway, L-tyrosine and L-dopa, are added to the culture medium. The changes include increased tyrosinase activity and de novo synthesis of melanosomes and melanin. L-tyrosine and L-dopa appeared to elicit these responses through separate but overlapping regulatory pathways. Here we show an additional effect of L-tyrosine: stimulation of MSH binding capacity. Cells cultured for 24-48 hours in the presence of 200 microM L-tyrosine display a 3-4 fold increase in their ability to bind 125I-beta-MSH. L-dopa did not stimulate MSH binding under the same conditions. In control experiments neither L-tyrosine nor L-dopa had any effect on insulin binding. The amelanotic cells respond to MSH with increased dendrite formation, increased tyrosinase activity without melanin production, and decreased growth rate.

A new class of compounds, termed "dopa phosphates," is described. The compounds contain phosphate ester linkages at positions 3 and/or 4 of the phenylalanine ring. Dopa phosphates are highly soluble compounds which are stable over a wide range of pH values and are not hydrolyzed by boiling in concentrated acid. Synthetic yields of greater than 90% can be obtained using dopa as starting material. Exposure to alkaline phosphatase results in hydrolysis of the phosphate moieties and production of dopa. Dopa phosphates do not inhibit dopa oxidase (tyrosinase, EC 1.14.18.1) activity. Dopa oxidase does not catalyze the conversion of dopa phosphates into melanin unless the dopa phosphates are first treated with alkaline phosphatase. Dopa phosphates, when compared to L-dopa, are stable in the presence of O2 and are not oxidized by serum proteins. In the presence of cultured melanoma cells, dopa phosphates are readily converted into melanin, indicating that the cells are able to produce dopa from dopa phosphates. At high concentrations, dopa phosphates are cytotoxic toward melanoma cells in culture. The cytotoxicity is enhanced at least 3-fold by pretreatment of cells with melanotropin and is prevented by phenylthiourea, an inhibitor of dopa oxidase activity. These results, combined with studies on the uptake of radioactive forms of dopa phosphates (32P and 14C), indicate that phosphorylated isomers of dopa are efficiently taken up by Cloudman melanoma cells and are readily converted by the cells into a melanin precursor, presumably L-dopa.

L-dopa is a key metabolite in the process of melanogenesis. However, it is difficult to use in biological experiments because it is subject to auto-oxidation and relatively insoluble at neutral pH. Dopa phosphates contain phosphate ester linkages at positions 3 and/or 4 of the phenylalanine ring of L-dopa, rendering them highly soluble and stable to auto-oxidation when compared to L-dopa. Dopa phosphates are readily taken up by melanoma cells in culture and converted to L-dopa and inorganic phosphate by cellular phosphatases, making them useful for studying L-dopa effects in vivo. Here we investigated the effects of dopa phosphates on receptors for MSH in cultured melanoma cells. We found that dopa phosphates caused a 3-fold stimulation of MSH binding capacity by the cells which probably occurred through an increase in the number of receptors for MSH with no apparent change in affinity of the receptors. The increased binding capacity for MSH was followed by increased cellular tyrosinase activity and melanogenesis. Thus dopa phosphates and/or L-dopa can act as regulators of the MSH receptor system. The observations suggest a novel mechanism for regulation of hormonal responsiveness: hormonal signal amplification by a metabolite in the target pathway.

Dopa phosphates, a new class of compounds, contain phosphate-ester linkages at the 3- and/or 4- positions of the phenylalanine ring of L-dopa. Dopa phosphates have been shown to increase pigment production in the epidermis of hairless mice. Groups of Skh-2 pigmented hairless mice were treated topically with various concentrations of dopa phosphates daily for five weeks. Half of each group received suberythemal UVB radiation three times weekly for four weeks from a bank of filtered FS20 lamps. UVB and dopa phosphates alone each caused a modest increase in epidermal pigmentation. However, treatment of mice with dopa phosphates plus UVB radiation resulted in a marked increase in pigmentation, greater than with either treatment alone. The optimal concentration of dopa phosphates was 0.01% (100 micrograms/ml Tris-glycerol buffer) whether or not they were applied in conjunction with UVB radiation. Histological analyses revealed that dopa phosphates and UVB radiation each caused an increase in the number of pigmented melanocytes in the epidermis. Control groups treated with Tris-glycerol buffer alone, or buffer containing L-phenylalanine or L-dopa showed no significant changes in pigmentation. Our results indicate that dopa phosphates stimulate the production of melanin and affect the development and distribution of melanocytes in the skin of Skh-2 mice. By these criteria, dopa phosphates and UVB act in a similar manner to increase melanin content in the skin. The processes may be related to those recently observed in cultured mouse melanoma cells where dopa phosphates are incorporated into melanin, presumably following enzymatic hydrolysis by cellular phosphatases with the resultant production of L-dopa and inorganic phosphate.

MELANOMA CELLS EXPRESS A PHENOTYPE THAT IS EASY TO RECOGNIZE: the synthesis of melanin. We used this marker to isolate clones of amelanotic variants from large populations of wild-type melanotic clones. Cloudman mouse melanoma (S91, clone M-3, CCL 53.1) was chosen as the parental line because the cells are highly pigmented, grow well as clones in soft agar, and fuse readily with Sendai virus. Subclones (10(7)) of this line were screened without prior mutagenesis, and nine amelanotic variants were isolated. The mutagen ethylmethanesulfonate increased the frequency of variants by three to four orders of magnitude. Wild-type cells had both basal and melanocyte stimulating hormone-inducible tyrosinase activities. The four amelanotic variants that we have examined to date all behaved similarly: they lost basal tyrosinase (EC 1.14.18.1; monophenol monooxygenase) activity but retained melanocyte stimulating hormone-inducible activity; they contained Stage-II melanosomes but no melanized melanosomes; they exhibited growth characteristics similar to those of wild-type cells in culture but produced fewer tumors in mice.

A review of the basics of pigment cell biology is followed by a discussion of the characteristics of several disorders of hypopigmentation. By determining such features as inheritance pattern, time of onset (congenital, childhood, adulthood), natural history (stable vs progressive), type of pigment loss (diffuse or circumscribed), distribution of lesions (generalized vs localized), degree of pigment loss (incomplete or complete), number of melanocytes, if any, in biopsy specimens of affected areas, type of melanocytic dysfunction, and associated inflammation or infection, one can classify the disorders of hypopigmentation. The proposed pathophysiology for each disorder of hypomelanosis is presented.

Three new regulatory factors in the melanogenesis pathway were recently described: dopachrome conversion factor accelerates the conversion of dopachrome to 5,6-dihydroxyindole; indole conversion factor accelerates the conversion of 5, 6-dihydroxyindole into melanin; and indole blocking factor retards the conversion of 5, 6-dihydroxyindole into melanin. Exposure of wild-type Cloudman melanoma cells in culture to melanotropin (MSH) removes blocking factor activity and increases indole conversion factor activity. The chemical nature of factors has not yet been determined. In this report we demonstrate that highly purified isozymes of tyrosinase from C57B1/6N murine hair bulbs and B16 murine melanoma are closely associated with conversion and blocking factor activities. The soluble isozymes. T1, T2, and T2 contain blocking factor activity, while isozyme T4, the major tyrosinase species found in melanosomes, contains activity that accelerates melanin formation from dopachrome. The results suggest that melanogenesis is regulated by the association of these different factors with the specific tyrosinase isozymes.

Diazepam was found to be the most surface active substance in aqueous solutions, chlorpromazine was less active and the lowest activity was found for haloperidol. The surface activity in blood was similar. The effect of pH on the surface activity of chlorpromazine solutions was observed.

Insulin is a potent, reversible inhibitor of proliferation in Cloudman S91 mouse melanoma cells. The inhibition seems to be unique to the Cloudman line since five other cell lines, including the mouse B16 and hamster Greene melanomas, were unaffected by insulin under the same culture conditions. Variants of Cloudman S91 cells were isolated which differed from wild-type (WT) cells in their response to insulin. Most of these variants were resistant to insulin (INSres) and had the same generation time independent of the presence of the hormone. One line (INSdep) was found to require insulin for growth. This line was about 15 times more sensitive to the proliferative effects of insulin than the WT. Revertants of the INSdep line were selected for their ability to proliferate in the absence of insulin. Five out of five such revertants were insulin resistant, suggesting that the INSdep line arose as a results of at least two separate mutations. Both WT and INSdep cells showed enhanced uptake of 14C-alpha-aminoisobutyric acid (AIB) when exposed to insulin. Dose-response curves of the stimulation of AIB uptake in WT and INSdep cells were superimposable. Stimulation of AIB uptake and stimulation of proliferation by insulin were not under coordinate control since AIB uptake was increased equally in the wild-type cells when proliferation was inhibited and in INSdep cells when proliferation was enhanced. Binding of 125I-insulin was used to demonstrate the presence of specific, high affinity insulin receptors on the cells. INSres variants generally had fewer receptors than WT, but in no case did the magnitude of this effect appear to be sufficient to explain the insensitivity to insulin. The INSdep variant showed a greater than two-fold increase in the number of insulin receptors per cell, compared to WT. Revertants of the INSdep line had the same number of receptors as WT. The specificity for both binding and for the effects on proliferation were the same in WT and INSdep cells. Since the effects of insulin on proliferation were opposite in the two lines, we propose at least two distinct sites of insulin action on the cells. Further isolation and analyses of Cloudman lines with unusual responses to insulin should be useful for understanding the molecular basis of action of this hormone.

Insulin inhibits the proliferation of wild-type Cloudman S91 mouse melanoma cells. The effects, which are mediated through specific, high-affinity receptors for insulin, appear to involve interactions with the cAMP system. Our evidence is as follows: 1) Cloudman cells have a cAMP requirement for proliferation and pigmentation. Exposure of cells to insulin results in a lowering of intracellular cAMP levels and inhibition of both cell division and pigment formation. 2) The effects of insulin are reversed by agents which raise cAMP levels, or by the cAMP analogue dibutyryl cAMP. 3) A mutant cell line with a temperature-dependent requirement for cAMP is most sensitive to the growth inhibitory effects of insulin when its requirements for cAMP are maximal. 4) Mutants selected only for alterations in their response to insulin frequently have concomitant alterations in their cAMP systems. 5) The melanotropin-responsive adenylate cyclase system is stimulated following prolonged exposure of cells in culture to insulin. Although we do not know the mechanism(s) for the interactions between the insulin and the cAMP system, our initial findings suggest that protein phosphorylation/dephosphorylation reactions are involved.

The biosynthesis of melanin occurs through sequential steps known as the Mason-Raper pathway. The initial steps are the conversion of tyrosine to dihydroxyphenylalanine (dopa) and of dopa to dopaquinone by the enzyme tyrosinase (EC 1.10.3.1). Until recently it was assumed that once these first two conversions were completed, the subsequent reactions occurred spontaneously. However, studies with mouse melanoma cells in culture revealed that subsequent steps in the pathway are also regulated. In this report, we demonstrate that these steps are also regulated in skins of fetal and newborn mice. The specific activities of the regulatory factors change during the first week after birth and differ in mice of different genotypes. The findings provide new insights into genetic and developmental regulation of the pigmentary system in mammals.

Cloudman S91 mouse melanoma cells respond in culture to B-melanocyte-stimulating hormone (B-MSH) with changes in morphology, growth rates, and melanin production. The effects of MSH appear to be mediated through a stimulation of the cyclic AMP system. It was reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle [Wong, G., Pawelek, J., Sansone, M., & Morowitz, J. (1974) Nature (London) 248, 351-354] and that the apparent reason for this cell cycle restriction is that receptors for MSH are most active in the G2 phase [Varga, J. M., DiPasquale, A., Pawelek, J., McGuire, J., & Lerner, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593]. In this report, we found that by two separate methods of obtaining populations of cells in the G2 phase of their cycle--centrifugal elutriation or synchronization with thymidine--we observed increased binding of MSH by cells in the G2 and possibly late S phases of their cycle. However, cultures of cells passing through their cycle in synchrony were quite different from nonsynchronized (random) cultures. Both synchronized and random cultures expressed receptors for MSH in the G2 and possibly late S phases of their cycle, but synchronized cultures bound severalfold more MSH per cell than random cultures. This increased binding of MSH by synchronized cells was accompanied by an increase in tyrosinase activity and pigment production.(ABSTRACT TRUNCATED AT 250 WORDS)

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A review of the studies done at Yale on the role of MSH in regulating pigmentation and growth of Cloudman (S91) melanoma cells is presented. The areas covered include the isolation and analyses of mutant cell lines unresponsive to MSH; the role of cyclic AMP, cyclic AMP-dependent protein kinases, and protein phosphorylation reactions in the response of MSH; new regulators of the melanogenesis pathway; the cytotoxicity of melanin precursors; the development of methodology for synthesizing 125I-beta-MSH; the use of this ligand to study receptors for MSH; and the chemical and biological properties of phosphorylated isomers of L-dopa, a new class of compounds exhibiting potent bio-activity toward melanocytes. All of the experiments described were carried out in the Department of Dermatology at the Yale University School of Medicine during the tenure of Dr. Aaron B. Lerner as chairman.