Preparation of purified tyrosinase devoid of dopachrome tautomerase from mammalian malignant melanocytes

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60 degrees C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.     

Inhibition of N-glycan processing in B16 melanoma cells results in inactivation of tyrosinase but does not prevent its transport to the melanosome

Tyrosinase is the key enzyme in melanin biosynthesis, catalyzing multiple steps in this pathway. The mature glycoprotein is transported from the Golgi to the melanosome where melanin biosynthesis occurs. In this study, we have investigated the effects of inhibitors of N-glycan processing on the synthesis, transport, and catalytic activity of tyrosinase. When B16 mouse melanoma cells were cultured in the presence of N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum-processing enzymes alpha-glucosidases I and II, the enzyme was synthesized and transported to the melanosome but almost completely lacked catalytic activity. The cells contained only 2% of the melanin found in untreated cells. Structural analysis of the N-glycans from N-butyldeoxynojirimycin-treated B16 cells demonstrated that three oligosaccharide structures (Glc3Man7-9) predominated. Removal of the glucose residues with alpha-glucosidases I and II failed to restore enzymatic activity, suggesting that the glucosylated N-glycans do not sterically interfere with the enzyme's active sites. The mannosidase inhibitor deoxymannojirimycin had no effect on catalytic activity suggesting that the retention of glucosylated N-glycans results in the inactivation of this enzyme. The retention of glucosylated N-glycans does not therefore result in misfolding and degradation of the glycoprotein, as the enzyme is transported to the melanosome, but may cause conformational changes in its catalytic domains.     

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-beta1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-beta1 on the melanogenic activity of B16/F10 cells.     

Capsaicin inhibits plasma membrane NADH oxidase and growth of human and mouse melanoma lines

Hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane is thought to be involved in the control of normal cell proliferation. The aim of this study was to determine the effect of the naturally occurring quinone analogue capsaicin (8-methyl-N-vanillyl-6-noneamide) on the NADH oxidase activity of plasma membranes and cell growth of human primary melanocytes, the A-375 and SK-MEL-28 human melanoma cell cultures. NADH oxidase activity was inhibited preferentially in the A-375 melanoma cells but not in the primary melanocytes, by capsaicin. Inhibition of growth and the NADH oxidase by capsaicin could be induced in resistant SK-MEL-28 melanoma cells by co-administration of capsaicin with t-butyl hydroperoxide, a mild oxidising agent. Death of the inhibited cells was accompanied by nuclear changes suggestive of apoptosis. With B16 mouse melanoma, capsaicin inhibited both the NADH oxidase activity and growth in culture. Growth of B16 melanoma, transplanted in C57BL/6 mice, was significantly inhibited by capsaicin injected directly into the tumour site when co-administered with t-butyl hydroperoxide. The findings correlate the inhibition of cell surface NADH oxidase activity with inhibition of growth and capsaicin-induced apoptosis, and also suggest that the extent of inhibition may relate to the oxidation state of the plasma membrane.     

Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production

The role of growth factor networks in regulating the progression of human melanocytes towards tumorigenicity and ultimately the malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix degradative enzymes of melanoma cells remain undefined at the molecular level. We report here that nerve growth factor (NGF) can modify some metastasis-associated cellular properties of human and mouse melanoma cells. Treatment of early-passage human metastatic melanoma cells (MeWo) or their variants (3S5, 70W) with biologically active 2.5S NGF resulted in (a) delayed density-dependent inhibition of melanoma cell growth; (b) increased in vitro invasion through a reconstituted basement membrane; and (c) time- and dose-dependent induction of heparanase, a heparan-sulfate-specific endo-beta-D-glucuronidase associated with human melanoma metastasis. These effects of NGF were most marked in the 70W brain-colonizing cells (70W > MeWo > 3S5). The NGF enhancement of heparanase secretion was not species-specific, since it was also observed in murine B16 melanoma cells; the highest NGF stimulation of heparanase was found in brain-colonizing murine B16-B15b variant (B16-B15b > B16-BL6, B16-F10, B16-F1). NGF also increased the invasive capacity of the human 70W and murine B16-B15b sublines in a chemoinvasion assay performed with filters coated with purified heparan sulfate proteoglycan (HSPG). The enhancement of chemotactic response and heparanase production was detected at NGF concentrations sufficient to fully saturate both low- and high-affinity NGF receptors (NGFR), the neurotrophin receptor (p75) and the trkA gene product, respectively. The results suggest that, in addition to the effects of NGF on cellular development and differentiation within the peripheral and central nervous systems, NGF can exert changes in the invasive properties of neuroectoderm-derived melanoma cells.     

Focal adhesion kinase-dependent apoptosis of melanoma induced by tyrosine and phenylalanine deficiency

We found previously that restriction of tyrosine (Tyr) and phenylalanine (Phe) inhibited growth and metastasis of B16BL6 murine melanoma and arrested these cells in the G0-G1 phase of the cell cycle. Here, we report that deprivation of these two amino acids in vitro induces apoptosis in B16BL6 and in human A375 melanoma cells but not in nontransformed, neonatal murine epidermal cells or human infant foreskin fibroblasts. Four days after deprivation of Tyr and Phe in vitro, 37% of B16BL6 and 51% of A375 melanoma cells were undergoing apoptosis. Apoptosis was not associated with elevation in intracellular calcium or alteration in p53 or c-myc protein expression. Expression and Tyr phosphorylation of focal adhesion kinase (FAK) were inhibited in both melanoma cell lines by deprivation of Tyr and Phe but not by deprivation of glutamine or serum. Tyr phosphorylation of FAK in Tyr- and Phe-deprived melanoma cells was enhanced within 30 min of refeeding with complete DMEM. FAK protein expression recovered within 60 min, and cell viability recovered within 24 h. Genistein, a tyrosine kinase inhibitor that specifically inhibits Tyr phosphorylation of FAK, did not induce apoptosis in A375 melanoma cells at a concentration of 50 microM. Genistein prevented the recovery of cell viability upon refeeding with Tyr and Phe to previously deprived A375 melanoma cells. These data collectively indicate that apoptosis induced by Tyr and Phe deprivation is FAK-dependent.     

A new mouse model of experimental melanoma for vaccine and lymphokine therapy

The annual incidence of malignant melanoma is estimated at 10-12 per 100,000 inhabitants in countries of central Europe and the United States, and alarmingly there has been a dramatic upward trend in that estimate. The B16 murine melanoma is a rapidly growing metastatic tumor of spontaneous origin, as are human malignant melanomas. Melanoma cells produce specific antigens which are uniquely different from normal cellular antigens, and the expression of such antigens is the cornerstone for preparation of anti-melanoma vaccines. One major problem in evaluating the effectiveness of vaccination and other biologic therapies is the variability of experimental tumor models. A new metastatic model of experimental melanoma which was developed in our laboratory imitates the major clinical stages of malignant metastatic melanoma: stage I, primary (local) tumor growth and bone marrow invasion; stage II, regional lymph node involvement; and stage III, metastasis to distant organs, such as the lungs. This model has been used successfully for screening vaccines constructed in our laboratory. Immunization with formalinized vaccines (of extracellular antigens, intact melanoma cells, or B700 antigen) or irradiated vaccines (of intact melanoma cells) partially inhibit primary melanoma tumor growth, reduce metastasis to regional lymph nodes and lungs, and significantly increase mean survival time. These anti-tumor effects were improved when polyvalent and monovalent vaccines were combined with IL-2 therapy. We also compared the immunogenic activity of vaccines made from B16 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF, and effects on tumor bearing mice were compared with or without therapy using the corresponding lymphokines. In sum, comparison of antibody production, growth of primary melanoma tumors, number of surviving mice, mean survival time, and percent of mice with lung metastases, showed that the best course of immunotherapy involves vaccination of mice with irradiated B16 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.     

Response of Cloudman S91 melanoma cells to melanocytestimulating hormone: enhancement by cytochalasin B

Cloudmann S 91 mouse melanoma cells treated for 5 days with melanocyte-stimulating hormone (MSH), cytochalasin B (CB), or both, exhibited changes in cell volume, population, nucleation, and pigment production. Cells treated with CB or CB in combination with MSH were eight to nine times larger, rounder, multinucleated, and heavily pigmented. CB alone increased melanin and DNA per nucleus threefold. CB in combination with MSH increased melanin per nucleus 30-fold. Data on DNA per nucleus suggest that CB-treated cells remained in the G phase longer than did control cells. MSH alone caused a reduction in cell population and a fourfold increase in melanin per nucleus.     

Up-regulation by ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) of Fas/Apo-1 expression on B16 melanoma cells: implications for the antitumor effects of AS101

It was recently reported that human and mouse melanoma cells express Fas ligand (FasL) but almost no Fas, which may contribute to their immune privilege. AS101 (ammonium trichloro(dioxoethylene-0,0')tellurate), a synthetic immunomodulator with minimal toxicity, was found to have antitumor effects in various tumor models. Our present study shows that AS101 has direct and indirect effects on tumor cells; AS101 inhibits the clonogenicity of B16 melanoma cells in vitro. Moreover, wild-type P53 expression, which is required for induction of Apo-1 expression, increased significantly in AS101-treated cells. We therefore investigated Fas expression in AS101-treated B16 cells and found that Fas, but not FasL, expression was significantly increased; moreover, Fas receptors were functional. Longer incubation with AS101 resulted in spontaneous apoptosis triggered by the Fas-FasL system. To explore the relationship of these results to the antitumor effects of AS101, we injected B16-F10 mouse melanoma cells into syngeneic C57BL/6 mice carrying the lpr mutation in the Fas gene and to gld mutant mice that lack functional FasL. Tumor development in control groups was lowest in the lpr mice, while no difference was observed between gld and wild-type mice. Among the AS101-treated groups, the most pronounced effect appeared in the lpr mice, while the lowest was seen in the gld mutant mice. Our study suggests that AS101 may render melanoma tumor cells more sensitive to Fas/FasL-induced apoptosis and may therefore have clinical potential.     

Smad7 is an activin-inducible inhibitor of activin-induced growth arrest and apoptosis in mouse B cells

Members of the transforming growth factor-beta (TGF-beta) family, which includes the activins, relay signals from serine/threonine kinase receptors in membrane to nucleus via intracellular Sma- and Mad-related (Smad) proteins. Inhibitory Smad proteins were found to prevent the interaction between the serine/threonine kinase receptors and pathway-restricted Smad proteins. Smad7 was identified as a TGF-beta-inducible antagonist of TGF-beta signaling, and it may participate in a negative feedback loop to control TGF-beta signaling. Here we demonstrate that the mRNA expression of Smad7 is induced by activin A in mouse B cell hybridoma HS-72 cells, which undergo growth arrest and apoptosis upon exposure to activin A. The ectopic expression of mouse Smad7 in HS-72 cells suppressed the activin A-induced cell cycle arrest in the G1 phase by abolishing the activin A-induced expression of p21(CIP1/WAF1) and hypophosphorylation of retinoblastoma protein. Furthermore, Smad7 expression suppressed activin A-induced apoptosis in HS-72 cells. Thus, our data indicate that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis of B lineage cells.     

Anti-cell death activity promotes pulmonary metastasis of melanoma cells

Bcl-2 inhibits apoptosis from a variety of stimuli, and a Bcl-2-binding protein BAG-1 also functions in protection from apoptosis in concert with Bcl-2. Here, we provide evidence that prolonged cell survival introduced by overexpression of Bcl-2 or BAG-1 proteins strongly promotes experimental pulmonary metastasis of melanoma B16-BL6 cells. In murine melanoma cell line B16-BL6, gene transfer-mediated expression of the Bcl-2 or BAG-1 led to prolonged cell survival against serum-starved apoptosis in vitro. The Bcl-2-expressing B16 cells, B16-Bcl-2 and the BAG-1-expressing B16 cells, B16-BAG-1 strongly enhanced pulmonary metastasis in allogenic BALB/c nude mice and whole lung weights were increased by 2.4-fold and 1.4-fold, respectively, compared with control transfectants, suggesting that Bcl-2 is a stronger positive modulator of metastasis. When the viable B16-Bcl-2 and control transfectants were injected subcutaneously into BALB/c nude mice, the colony numbers of pulmonary metastasis of the B16-Bcl-2 transfectant increased by 5.6-fold compared with the control transfectants. These enhanced metastatic potentials in the B16-Bcl-2 and the B16-BAG-1 transfectants were well correlated with anti-cell death activity against serum-starvation and enhanced cell viability on limiting dilution. Analysis of the transfectants however revealed that their growth rates, invasive ability and cell motility were not significantly altered by overexpression of either Bcl-2 or BAG-1 proteins. Taken together, these studies demonstrate that prolonged cell survival is a crucial factor to promote metastasis of melanoma, thereby contributing to tumor progression.     

Terson syndrome. Results of vitrectomy and the significance of vitreous hemorrhage in patients with subarachnoid hemorrhage

Although the induction of pigmentation following exposure of melanocytes to ultraviolet light in vivo and in vitro is well documented, the intracellular mechanisms involved in this response are not yet fully understood. Exposure to UV-B radiation leads to the production of DNA damage, mainly cyclobutane pyrimidine dimers, and it was recently suggested that the thymidine dinucleotide pTpT, mimicking small DNA fragments released in the course of excision repair mechanisms, could trigger melanin synthesis. We now report that the thymidine dinucleotide pTpT induces melanogenesis both in human normal adult melanocytes and in human melanoma cells. Thus, the SOS-like response suggested by Gilchrest's work to be evolutionary conserved, based primarily on work in murine cells and guinea pigs, is also apparently present in the human. Thymidine dinucleotide is nontoxic to melanoma cells and does not induce apoptosis in these cells, but induces S phase cell cycle arrest and a proliferation slow down. Because thymidine excess in culture medium leads to the synchronization of cells in S phase, we investigated whether this phenomenon was involved in the increase in melanin synthesis. We show that melanin synthesis is specifically triggered by the dimeric form of the thymidine and not by the monomeric form pT. Thus, our data strongly support that thymidine dinucleotides pTpT mimic at least part of the effects of ultraviolet irradiation, and may hence represent an invaluable model in the study of the molecular events involved in melanogenesis induction triggered through DNA damage.     

Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site ''A'': effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN

This study was performed to evaluate the relative efficacy of Maharishi Amrit Kalash ambrosia (MAK-5) and Maharishi Amrit Kalash nectar (MAK-4) on murine (B-16) and human (SK-Mel) melanoma cells in culture. Ethanol extract (EE) of MAK-5 (EE-MAK-5) induced morphological differentiation (enlargement of soma and nuclei and formation of long dendritic processes) and growth inhibition in mouse melanoma cells, whereas EE-MAK-5 inhibited only growth in human melanoma cells. Murine melanoma cells were more sensitive (about 3 times) than human melanoma cells in culture to EE-MAK-5; the aqueous extract (AE) of MAK-5 (AE-MAK-5) was ineffective in both cells. Boiling EE-MAK-5 for 10 minutes or exposing it to light at room temperature for 72 hours did not alter growth-inhibiting potency. Ethanol extract of another herbal agent, MAK-4 (EE-MAK-4), inhibited growth in human melanoma cells but not in mouse melanoma cells. AE-MAK-4 was ineffective for both cells. These results suggest that murine and human melanoma cells respond differently to MAK-5 and MAK-4 and that human melanoma growth-inhibiting agents are present in both EE-MAK-5 and EE-MAK-4.