Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Microheterogeneity of melanosome-bound tyrosinase from the harding-passey murine melanoma

• 1.1. This study was directed towards the characterization of the origin of the microheterogeneity displayed by mammalian tyrosinase, the enzyme responsible for pigmentation in mammals.
• 2.2. Tyrosinase was purified from the Harding Passey murine melanoma, fractionated into a continuous series of subisozymic forms, and analyzed using various chemical and immunological probes.
• 3.3. Treatment with neuraminidase revealed that all the forms had similar amounts of sialic acid, and reactivity with various carbohydrate specific lectins showed that the isozymes also contained subterminal galactose, N-acetylglucosamine, and mannose, but lacked α-fucose.
• 4.4. Amino acid composition data indicated that the polypeptides of all the forms had identical residue contents.
• 5.5. The sum of the evidence further supports the theory that the isozymic forms demonstrable for mammalian tyrosinase represent intermediate processing stages of the enzyme from the nascent protein chain to the fully glycosylated, high molecular weight form of tyrosinase that is localized within melanin granules.

     

ET-1 inhibits B-16 murine melanoma cell migration by decreasing K(+) currents

Cell migration is mediated by ion channels and transporters, and plays crucial roles in a variety of physiological and pathological processes. Previously, our studies have shown that a Ca(2+)-regulated K(+) current exists in B-16 murine melanoma cells, and that endothelin-1 (ET-1) inhibits the K(+) current via a PKC-dependent pathway. In the present study, patch-clamp whole-cell recording and transwell migration assays were used to examine the effects of ET-1 on B-16 murine melanoma cell migration. ET-1 (100 nM in the injection pipette and 10 nM in the incubation medium) decreased the K(+) current amplitude by 33.0 +/- 2.5% and inhibited migration of B-16 cells by 57.4 +/- 9.4%. Similarly, the Ca(2+)-regulated K(+) channel blockers, BaCl(2) and quinidine, decreased the K(+) current by 20.5 +/- 1.0% and 36.6 +/- 1.2%, respectively, and slowed migration of B-16 melanoma cells by 37.1 +/- 8.6% and 42.7 +/- 8.8%, respectively. The effect of ET-1 on the K(+) current and cell migration was simulated by ET-3. In contrast, the K(+) channel opener, diclofenac, increased the K(+) current by 128.8 +/- 11.7%, 257.4 +/- 35.8% at concentrations of 1 and 5 mM, respectively. Likewise, the migration of B-16 murine melanoma cells dramatically increased by 75.6 +/- 12.7% in the presence of 100 microM diclofenac in incubation medium. Furthermore, the ET-1- and ET-3-induced inhibition of K(+) current and migration was abrogated by diclofenac. In the presence of diclofenac, ET-1 only reduced the K(+) current amplitude by 10.6 +/- 1.1%, and slowed B-16 cell migration by only 10.8 +/- 8.9%. The results suggest that the K(+) channel-dependent migration of B-16 melanoma cells is modulated by ET-1. Cell Motil.     

Mechanism and inhibitory effect of galangin and its flavonoid mixture from Alpinia officinarum on mushroom tyrosinase and B16 murine melanoma cells

The whitening effects of the flavonoid constituents of Alpinia officinarum Hance were investigated on melanin biosynthesis in B 16 mouse melanoma cells, tyrosinase inhibition and UV absorption. The melanin content was reduced to 1.276 microg /10(5) cell for flavonoid mixture and 1.161 microg /10(5) cell for galangin while the melanin control was 1.632 microg/10(5) cell. Both flavonoid mixture and galangin reduced melanin production with an inhibition of 21.81% and 28.86% at a concentration of 26.5 microg/mL and 29 microg/mL (107.4 microM), respectively. Tyrosinase inhibition by the flavonoid mixture and galangin were higher at lower concentrations and galangin showed competitive inhibition at a concentration less than 21.23 microg/mL which was soluble. In addition, the flavonoid mixture and galangin showed a broad absorption band at 270 approximately 290 nm related to the UV-B area. These observations suggest that galangin may be a whitening agent and a promising candidate for prevention of skin cancer. This is the first full scale report on the evaluation of the whitening effect of galangin.     

Proteolysis with trypsin of mammalian tyrosinase isoforms from B16 mouse melanoma.

In spite of the central role of tyrosinase in mammalian pigmentation, few data are available on its structure and structure-function relationships based on direct analysis of the protein. A number of reasons have been invoked to account for this situation, including the problems for its purification and its resistance to proteases. However, no study on the effects of proteases on purified tyrosinase has been reported. We have purified the melanosomal and cytosolic tyrosinases from B16 mouse melanoma and analyzed their susceptibility to trypsin digestion. Both isoforms are sensitive to trypsin, and display similar peptide maps and kinetics of proteolysis, suggesting that they are products of the same gene. The peptide maps and the kinetics of appearance of the fragments were consistent with the sequential removal of N-terminal peptides, leading to a core of 55.3 kDa for the melanosomal form and 48.6 kDa for the cytosolic enzyme. This core was apparently resistant to further proteolysis and catalytically inactive. The difference in molecular weight for the core of the cytosolic and melanosomal forms is the same as that calculated for the native isoforms. The kinetics of enzyme inactivation indicate that the tyrosine hydroxylase and Dopa oxidase activities of tyrosinase are lost at the same rate, and should therefore display similar if not identical structural requirements. The results are discussed in terms of the relationship of both isoforms and of the putative protein sequences deduced from the cDNA clones proposed for tyrosinase.     

Oxidation of carbidopa by tyrosinase and its effect on murine melanoma

Oxidation of the anti-Parkinsonian agent carbidopa by tyrosinase was investigated. The products of this reaction were identified as 3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid and 6,7-dihydroxy-3-methylcinnoline. These results demonstrate that after oxidation of the catechol moiety to an o-quinone either a redox exchange with the hydrazine group or a cyclization reaction occur. The cyclization product underwent additional oxidation reactions leading to aromatization. The cyclization reaction is undesired in the case of hydrazine-containing anti-melanoma prodrugs and will have to be taken into account in designing such compounds. Carbidopa was tested against B16(F10) melanoma cells in culture and showed cytotoxicity significantly higher than either of its oxidation products and l-dopa. This effect, however, was not specific to this cell line.     

pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.     

Heparin and heparan sulfate binding sites on B-16 melanoma cells

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.     

Variation in melanosome numbers in cultured B-16 melanoma cells

Melanosomes from B-16 mouse melanoma cells in culture were isolated by treatment of pigmented cells with 2% SDS, sonication, and heating at 100°C. The total number of melanosomes in cultures of B-16 mouse melanoma cells increased exponentially during the rapid phase of sigmoid growth. The numbers of melanosomes per cell decreased during rapid phase of growth, and repigmentation was observed only when the cultures attained the stationary growth phase. BUdr at a minimum concentration of 0.5 μg/ml decreased both cell growth and numbers of melanosomes per cell, and completely inhibited repigmentation following a period of active growth. Cells cultured in 0.1 μg/ml BUdr grew at the same rate as untreated cells but contained fewer melanosomes/cell and lower total numbers of melanosomes during the late stages of the growth cycle.     

Membrane association of collagenase stimulatory factor(s) from B-16 melanoma cells

Past studies have shown that contact between tumor cells and fibroblasts results in stimulation of collagenase production by the fibroblasts. Membrane fractions prepared by differential centrifugation of sonicated B-16 melanoma cells were shown here to contain a collagenase stimulatory factor(s) (CSF). Trypsin treatment of intact B-16 cells prior to membrane fractionation led to loss of 90% of the total activity, indicating that CSF is localized on the outer surface of the cells. Stimulation of fibroblast collagenase production was also observed with dialyzed octylglucoside extracts of the B-16 membranes. Additional of exogenous lipid, ie, a mixture of phosphatidylcholine and phosphatidylserine, to the detergent extract of the membranes followed by dialysis and centrifugation at 100,000g resulted in 80% recovery of the factor activity in the pellet containing reconstituted lipid vesicles. Fractionation of tritium-labeled, reconstituted lipid vesicles on a Sephacryl S-300 column revealed that the collagenase stimulatory factor coeluted with the radioactive lipid vesicles. The fractionated lipid vesicles lost stimulatory activity completely after trypsin treatment or heating at 65 degrees C, indicating that the factor is a protein.     

Effect of UV-B irradiation on release of arachidonic acid from B-16 melanoma cells

B-16 melanoma cells in culture were prelabeled with (3H)-arachidonate, and exposed to UV radiation. Immediately after irradiation the cells released labeled materials. This UV-stimulated release was inhibited by mepacrine (20 microM) and calmodulin inhibitor W7 (0.5 microM). To determine the influence of extracellular Ca2+ on the UV-stimulated release, experiments were made with media containing various concentrations of Ca2+. The release decreased significantly at lower Ca2+ concentrations. These results suggest that Ca2+-calmodulin-dependent phospholipase A2 was involved in UV-stimulated release of radiolabeled materials, possibly arachidonic acid and its metabolites, from the cells.     

Effects of salicylic acid on mushroom tyrosinase and B16 melanoma cells

Salicylic acid slightly inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase noncompetitively without being oxidized. In contrast, 4-hydroxybenzoic acid did not inhibit this enzymatic oxidation if a longer reaction time was observed, although it suppressed the initial rate of the oxidation to a certain extent. Neither acid showed noticeable effects on cultured murine B16-F10 melanoma cells except weak cytotoxicity.     

Ca(2+)-inactivated K+ current is modulated by endothelin-1 in B-16 murine melanoma cells

It is well established that endothelin-1 (ET-1) plays a role in differentiation and proliferation in a variety of cells such as fibroblasts and human melanoma cells via a receptor-mediated mechanism. However, whether ET-1 modulates ion channel activity in these cell types is still unknown. In this report, we recorded the voltage-dependent outward K+ current in cultured B16 melanoma cells using the patch-clamp technique. Biophysical and pharmacological properties of the K+ current, and the effect of ET-1 on the K+ current were investigated. When cells were loaded with a Ca(2+)-chelating agent (EGTA or BAPTA), the K+ current amplitude gradually increased with time after establishment of the whole cell configuration. Replacement of Ca2+ with Co2+ in the extracellular medium caused no significant modulation of the K+ current amplitude. Addition of BaCl2 or quinidine to the extracellular solution reduced the K+ current amplitude, whereas the K+ current was insensitive to tetraethylammonium. ET-1 (10 nM) reversibly decreased the K+ current amplitude and accelerated the decay of the K+ current. The ET-1-induced inhibitory effect displayed no desensitization following repeated ET-1 application. Pretreatment with pertussis toxin (PTX) or perfusion of cells with the protein kinase C (PKC) inhibitor H-7 abolished the inhibitory effect of ET-1 on the K+ current. We conclude that the outward K+ current recorded in murine B-16 melanoma cells represents a Ca(2+)-inactivated K+ current, and that the inhibitory effect of ET-1 on the K+ current may reveal a novel mechanism to control the differentiation and proliferation of melanoma cells.     

Decreased expressions of c-myc and H-ras oncogenes in vitamin E succinate induced morphologically differentiated murine B-16 melanoma cells in culture

Treatment of B-16 melanoma cells in culture with d-alpha-tocopheryl succinate (vitamin E succinate) at concentrations of 11.3 and 15.1 microM inhibited growth and induced cell differentiation in culture. Vitamin E succinate treatment decreased the levels of c-myc and H-ras specific mRNAs in melanoma cells. Similar results were obtained by the vitamin retinoic acid and the nonvitamin agents R020-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), an inhibitor of cyclic nucleotide phosphodiesterase (0.72 mM), and sodium butyrate (1 mM), which induced differentiation and (or) inhibited growth of melanoma cells in culture. The extent of inhibition of c-myc mRNA was greater than that of H-ras mRNA. These results indicate that vitamin E succinate induced reduction of the levels of c-myc and H-ras mRNAs is related to growth inhibition of melanoma cells in culture.     

The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells.

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

Cytofluorometric analysis of thymic lymphocytic subpopulations in mice with B-16 melanoma

Two colour flow cytometric analysis of the Lyt2 and L3T4 T-cell surface antigens on thymocytes from B16 melanoma bearing mice, reveal an altered cell phenotype. At the early stage of the neoplasia and during the growth, the phenotypes Lyt2+/L3T4- (T suppressor) and Lyt2+/L3T4+ (T immature) decrease. Conversely, Lyt2-/L3T4+ (T helper) increase. These findings suggest the hypothesis that impairment of the immune functions in B16 melanoma bearing mice could be due to a primary thymic alteration.