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J Pawelek

Publications and source records attributed to J Pawelek.

At least 19 recordsLinked to original sources
PubMed1995

Ultraviolet B and melanocyte-stimulating hormone (MSH) stimulate mRNA production for alpha MSH receptors and proopiomelanocortin-derived peptides in mouse melanoma cells and transformed keratinocytes.

Cell lines of cutaneous origin, namely melanocytes and keratinocytes, were previously demonstrated to exhibit functional melanocyte-stimulating hormone (MSH) receptors that are up-regulated by ultraviolet (UV) radiation and by MSH itself. In this study, it is demonstrated that UVB irradiation, exposure to MSH, or exposure to N6,O2-dibutyryl cyclic adenosine monophosphate stimulates production of mRNAs for both alpha MSH receptors and proopiomelanocortin in cultured mouse Cloudman S91 melanoma cells, and that UVB stimulates production and release of MSH and adrenocorticotropin peptides in both melanoma cells and transformed PAM 212 mouse keratinocytes. The results add support to the hypothesis that the effects of UVB on cutaneous melanogenesis are mediated through a series of coordinated events in which MSH receptors and proopiomelanocortin-derived peptides play a central role.

Animals↗
PubMed1992

Defect of insulin receptor in insulin-resistant variants of Cloudman S91 mouse melanoma cells.

To study the mechanisms by which insulin regulates proliferation, we have compared wild-type Cloudman melanoma cells, whose growth in inhibited by insulin (insinh) to variant lines that were genetically selected for resistance to insulin (insres). Scatchard analysis of insulin binding to five insres) lines and six insres variants revealed a marked reduction in the number of high-affinity binding sites for insulin in the insres lines, and insres lines displayed an abnormal beta-subunit of the insulin receptor. During autophosphorylation of the wheat germ agglutinin-purified receptor, the beta-subunit apparently underwent proteolytic degradation. This proteolysis was ATP-dependent and was prevented by bovine pancreatic trypsin inhibitor, and phenylmethylsulphonyl fluoride, but not by aprotinin or leupeptin. Receptor proteolysis was not observed in wild-type lines. The results suggest that insulin resistance in the mutant Cloudman melanoma cells is apparently due to proteolysis of the beta-subunit of insulin receptor which, in turn, alters insulin binding capacity of the cells and blocks their anti-proliferative response to the hormone.

Animals↗
PubMed1991

Interactions between ultraviolet light and interleukin-1 on MSH binding in both mouse melanoma and human squamous carcinoma cells.

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.

Animals↗
PubMed1989

L-tyrosine stimulates induction of tyrosinase activity by MSH and reduces cooperative interactions between MSH receptors in hamster melanoma cells.

L-tyrosine, a precurosr to melanin, has recently been shown to be a regulator of the melanogenic pathway in some cultured melanoma cell lines. In this paper we demonstrated that L-tyrosine, besides increasing binding capacity for MSH, decreased cooperativity between MSH receptors and increased the level of tyrosinase induction by MSH. Apparently, regulation of MSH receptor activity by L-tyrosine involves specific changes in the interactions between the receptors and modification of the cellular responsiveness to MSH.

Animals↗
PubMed1989

UVB-induced melanogenesis may be mediated through the MSH-receptor system.

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.

Animals↗
PubMed1988

Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, L-tyrosine and L-dopa.

We describe results demonstrating the positive regulation of melanogenesis by two substrates of the melanogenic pathway. We have found that L-tyrosine and L-dihydroxyphenylalanine (L-dopa), whose metabolic fates are affected by the activity of that pathway, can also act as its regulators. In living pigment cells, tyrosinase (EC 1.14.18.1), a crucial and rate-limiting enzyme of melanogenesis, acts in subcellular organelles known as melanosomes. Melanin is laid down only in these organelles. We demonstrate that supplementing Ham's F-10 medium with additional L-tyrosine or L-dopa during the culture of amelanotic Bomirski hamster melanoma cells results in a rapid increase in melanin formation, which is not simply due to greater availability of substrate. There is a rapid increase in tyrosinase activity and a large scale synthesis of melanosomes. The effects of L-tyrosine and L-dopa are prevented by the addition of cycloheximide. The actions of L-tyrosine and L-dopa are specific in that under similar conditions D-tyrosine, D-dopa, N-acetyl-L-tyrosine, L-phenylalanine, L-tryptophan and L-valine have little or no effect. The two substrates, L-tyrosine and L-dopa, appear to act through related but distinct mechanisms. Our findings provide an example of a little-known phenomenon: regulation of a differentiated eukaryotic phenotype through positive control by substrates in the pathway.

Animals↗
PubMed1987

MSH binding in Bomirski amelanotic hamster melanoma cells is stimulated by L-tyrosine.

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.

Animals↗
PubMed1984

Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol-wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin-resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.

Binding, Competitive↗
PubMed1983

Interactions between insulin and the cyclic AMP system of Cloudman S91 mouse melanoma cells.

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.

Adenylyl Cyclases↗
PubMed1982

Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin.

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.

Catechol Oxidase↗
PubMed1981

Assay of melanotropic peptides in an in vitro mammalian system.

A reproducible and sensitive assay for melanotropic agents is described employing mouse melanoma cells in culture and measuring tyrosinase activity in terms of production of tritiated water from L-(ring-3,5-3H)-tyrosine. Molar concentrations of peptides inducing one-half maximal stimulation of tyrosinase activity were: beta-MSH, 1 +/- 2 x 10(-9); alpha-MSH and Beta h-LPH, 1 +/- 2 x 10(-8); ACTHp, 1 +/- 2 x 10(-7). Beta p 9-18-MSH and melanotropin potentiating factor, beta s 88-91-LPH exhibited no activity at concentrations as high as 10(-5)M.

Animals↗
PubMed1980

Inhibition of proliferation of cloudman S91 melanoma cells by insulin and characterization of some insulin-resistant variants.

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.

Aminoisobutyric Acids↗
PubMed1979

Melanoma cells.

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Animals↗
PubMed1978

Phenylalanine hydroxylase in melanoma cells.

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.

Animals↗