Specificity and mechanism of serotonin stimulation of adenylate deaminase activity]

Adenylate deaminating activity was stimulated in the liver mitochondria of rats in vivo not only by serotonin or synthetic indolylalkylamines, but also by phenyl- and imidazolalkyamines. Actinomycin D and cycloheximide protein biosynthesis inhibitors prevented stimulation of adenylate deaminating activity. Theophylline, phosphodiesterase inhibitor, produced a similar effect only when the extent of stimulation of adenylate deaminating activity was comparatively high.     

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

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

A similar pool of cyclic AMP phosphodiesterase in Xenopus oocytes is stimulated by insulin, insulin-like growth factor 1, and [Val12,Thr59]Ha-ras protein

Pharmacological analysis of in vivo cAMP phosphodiesterase in Xenopus oocytes using the nonselective enzyme inhibitors 3-isobutyl-1-methylxanthine (IBMX), theophylline, and papaverine, demonstrated inhibition of insulin- and insulin-like growth factor-1-induced maturation at concentrations that were 17-60-fold lower than those required to inhibit progesterone-induced germinal vesicle breakdown. The abilities of the phosphodiesterase inhibitors to block the maturation response showed the same rank order of potencies for each hormone: papaverine greater than IBMX greater than theophylline. Insulin-induced oocyte maturation that was accelerated by 0.01 microM progesterone was also inhibited by low micromolar concentrations of IBMX, demonstrating that the accelerated time course was due to a synergistic potentiation of insulin action by progesterone. Both insulin-induced maturation and insulin-stimulated phosphodiesterase activity displayed similar sensitivities to inhibition by IBMX, suggesting that hormone-stimulated phosphodiesterase activity is required for the peptide hormone action. Furthermore, microinjection of the transforming ras gene product [Val12,Thr59]Ha induced oocyte maturation and stimulated oocyte phosphodiesterase activity by approximately 50%, and both of these actions were inhibited by IBMX. These results suggest that oocyte maturation induced by insulin, insulin-like growth factor 1, and transforming ras protein involves stimulation of a similar phosphodiesterase.     

Inhibition of -tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that -tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM -tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. -Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of -tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high -tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of -tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM -tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from -tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of -tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

Inhibition of L-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD-2) cloned from the C-1300 tumor. 8Br-cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta-hydroxylase activity. In contrast, catechol O-methyltransferase, monoamine oxidase and aromatic-l -amino-acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta-hydroxylase were similar with respect to dose response relationships and with respect to time course of onset. Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20-1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta-hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD-2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta-hydroxylase activity. The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta-hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta-hydroxylase in adrenergic cells.     

The opposite effects of cyclic AMP-protein kinase a signal transduction pathway on renal cortical and medullary Na+,K+-ATPase activity.

Cyclic AMP-protein kinase A (PKA) pathway plays an important role in signal transduction in renal tubular cells, however, its role in transport regulation is not completely established. The aim of this study was to investigate in vivo the effect of PKA on renal Na, K-ATPase activity. The study was performed in male Wistar rats. The animals were anaesthetized with pentobarbital and investigated drugs were infused through the catheter inserted into the abdominal aorta. Na+,K+-ATPase activity was assayed in an isolated microsomal fraction of the renal cortex and medulla. Cell-permeable cAMP analogue, dibutyryl-cAMP (db-cAMP), dose-dependently stimulated Na+,K+-ATPase in the renal cortex and inhibited in the renal medulla. Maximal stimulation (+38.5%) and inhibition (-46.8%) were observed at a dose of 10(-6) mol/kg/min. Measurement of Na+,K+-ATPase activity at different Na' concentrations revealed that in the renal cortex db-cAMP increased Vmax of the enzyme without any effect on sodium affinity, whereas in the renal medulla decrease in Vmax was accompanied by decreased sodium affinity, evidenced by elevated K(0.5) for sodium. The effect of db-cAMP was mimicked by the infusion of either adenylate cyclase activator, forskolin, or inhibitor of phosphodiesterase, IBMX. Both stimulatory and inhibitory effects of db-cAMP were prevented by pretreatment with protein kinase A inhibitor, KT 5720 (10(-8) mol/kg/min) but not by inhibitor of protein kinase G, KT 5823. The inhibitory effect in the renal medulla was partially blocked by pretreatment with either ethoxyresorufin or 17-ODYA - two nonspecific inhibitors of cytochrome P450-dependent arachidonate metabolism, whereas an inhibitor of epoxygenase, miconazole, was not effective. Infusion of 20-hydroxyeicosatetraenoic acid (20-HETE) at a dose of 10(-10) mol/kg/min decreased medullary Na+,K+-ATPase activity by 24.2%. Exogenous protein phosphatases inhibitor, okadaic acid (OA, 10(-8) - 10(-7) mol/kg/min) caused dose-dependent decrease in renal medullary Na+,K+-ATPase activity, maximally by 31.9%, but had no effect in the renal cortex. The effects of OA and db-cAMP in the renal medulla were not additive. When OA administration (10(-7) mol/kg/min) was followed by 20-HETE (10(-10) mol/kg/min), medullary Na+,K-ATPase activity decreased by 48.6% and was similar as after db-cAMP. We conclude, that cAMP-PKA pathway activates Na+,K+-ATPase in the renal cortex and inhibits in the renal medulla. The inhibitory effect is partially mediated by cytochrome P450-dependent arachidonate metabolites and possibly also by PKA-dependent inhibition of protein phosphatases.     

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB)

The effects of 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB), one of the benzimidazole derivatives designed for a nucleic acid analogue, on melanogenesis of murine B16-F10 melanoma cell lines were investigated. MPB (40 microM) induced a striking dendricity in B16 melanoma cells within 12 h and maximal dendricity between 48 and 72 h. The stimulation of melanin synthesis was observed after only 2 days of treatment together with a dose-dependent growth inhibition. Moreover, MPB increased the activity of tyrosinase through the expression of tyrosinase mRNA without increasing the intracellular cyclic AMP content. MPB-induced melanogenesis was inhibited by novel protein kinase A inhibitors, KT-5720 and H-85. These findings indicate that MPB stimulated B16 cells to terminally differentiate and may be a useful drug in studying the regulation of melanogenesis.     

Regulation of Melanogenesis Induced by 5-Methoxypsoralen Without Ultraviolet Light in Murine Melanoma Cells

Melanogenesis in melanoma cells can be enhanced by psoralens in the absence of UV light. Melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase, DOPAchrome tautomerase (DCT), and tyrosinase-related protein-1 (TRP-1, gp75). To get more insight on the molecular mechanisms involved in psoralens-induced melanogenesis, we determined tyrosinase and DCT activities as well as mRNA and protein levels of tyrosinase, DCT, and TRP-1 in S91 mouse melanoma cells treated by 5-MOP. High concentration of 5-MOP (5 × 10^-5 M) induced a time-dependent increase of tyrosinase activity and melanin content, which was correlated to an increase of both mRNA and protein levels of tyrosinase. These results demonstrate that the 5-MOP stimulation of melanogenesis is related to increased tyrosinase synthesis. In addition, 5-MOP stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without any modification in the expression of the protein. We explored then the signalling pathways involved in 5-MOP-induced melanogenesis and, particularly, the role of cyclic AMP and protein kinase C (PKC). A small stimulation of cyclic AMP production was observed in presence of 5-MOP. Furthermore, 1-oleoyl-2-acetylglycerol (OAG), a PKC activator, potentiated the 5-MOP stimulation of tyrosinase activity, while calphostin, a specific PKC inhibitor, inhibited the 5-MOP induction of tyrosinase activity. Phorbol-myristate acetate (PMA), described as a strong activator of PKC, inhibited also the effect of 5-MOP when used at long term. Taken together, these results demonstrate that in murine melanoma cells 5-MOP stimulates melanogenesis by increasing activity and synthesis of tyrosinase. Tyrosinase and TRP-1 expression are coordinately regulated by 5-MOP Furthermore, a negative correlation between melanogenesis and DCT activity was observed under 5-MOP stimulation. At least, PKA and PKC systems appear to play an important role in the melanogenic effect of 5-MOP.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites.     

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.     

The effects of histamine H2 receptor antagonists on melanogenesis and cellular proliferation in melanoma cells in culture

B16-C3 murine melanoma, A375P human melanotic melanoma, and C32 human amelanotic melanoma cells were incubated in the presence of (0-4 mM) H2-antagonists, ranitidine and cimetidine. Cell proliferation, tyrosinase activity and melanin content were monitored. H2-antagonists stimulated tyrosinase activity and melanin accumulation in B16-C3 cells in a dose- and time-dependent manner. Stimulation of enzyme activity and pigment production was accompanied by inhibition of cellular proliferation in B16-C3 cells. The inhibitory concentration of cimetidine was approximately 2-fold higher than that of ranitidine. H2-antagonists failed to stimulate melanogenesis in A375P or C32 cells, but inhibited cellular proliferation in both cell lines. These results are the first demonstration of H2-antagonist induced phenotypic changes in malignant melanoma cells in vitro, and represent a novel mechanism for the previously described in vivo antitumor effects of these agents.     

Increased level of p27 subunit of proteasomes and its co-localization with tyrosinase in amelanotic melanoma cells indicate its direct role in the regulation of melanin biosynthesis

Proteasomes have been shown to be involved in the regulation of melanin biosynthesis in melanoma cells. Here we report on the correlation between proteasome subunits and Tyrosinase (Tyr) activity in different cell phenotypes, and thereby regulation of melanin biosynthesis in B16F10 mouse melanoma cells. Our results indicated that the quantity of proteasome subunit p27 is higher and that of the enzyme Tyr and its activity are lower in amelanotic melanoma cells, while the reverse is true in melanotic melanoma cells. Proteasome subunit p27, compared to another subunit p31, shows increased co-localization with Tyr and Tyrosinase related protein 1 (Trp1) in amelanotic cells to a greater extent than that in melanotic cells. On exposure to cycloheximide, increased Tyr degradation was seen in amelanotic cells, as indicated by increased co-localization of p27 and Tyr. Further, exposure of amelanotic melanoma cells with proteasome-specific inhibitor MG132 resulted in an increased Tyr activity, increased levels of Tyr and Trp1, leading to increased melanin synthesis. These results therefore suggest that proteasomes, particularly p27 subunit, are directly involved in the regulation of melanin biosynthesis in mouse melanoma cells.     

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.     

High-resolution magic angle spinning nuclear magnetic resonance analysis of metabolic changes in melanoma cells after induction of melanogenesis

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with ¹³C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.     

Stimulation of melanin synthesis of B16-F10 mouse melanoma cells by bufalin.

Bufalin, which is one of prominent components of Chinese toad venom, was found to decrease the rate of cell proliferation of mouse melanoma clone B16-F10 cells and a concomitant stimulation of expression of its melanotic phenotype. The effect of bufalin on melanogenesis included stimulation of tyrosinase activity and increase of cellular melanin content. These effects became apparent after 48 hr exposure to 10(-4) M bufalin and increased thereafter. Other cardiotonic steroids, such as cinobufagin and ouabain, at the concentration of 10(-4) M for 6 days, also showed the stimulatory effect on melanin synthesis of B16-F10 cells, but not digitoxigenin.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

Biochemical characterization of three hamster melanoma variants--II. Glycolysis and oxygen consumption

Lactate production and oxygen consumption were studied in single cell suspension prepared from solid tumours of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) melanomas of hamster. Aerobic lactase production was about 5 times higher in the fast growing Ab melanoma than in the slow growing Ma and MI melanomas. Aerobic lactate production in both melanotic hamster melanomas was stimulated by 3-(3,4-dihydroxyphenyl)-L-alanine. This compound was without effect on the cells isolated from amelanotic hamster melanoma. L-Phenylalanine, a known competitive tyrosinase inhibitor reduced the stimulatory effect of L-DOPA on the the lactate production. Oxygen consumption was similar in all three melanomas. The oxygen consumption was inhibited completely by 1 mM potassium cyanide in the Ab melanoma but only in about 1/3 in the Ma and MI melanomas. The Pasteur effect was higher in relative terms and lower in absolute terms in the melanotic melanomas than in the Ab melanoma only . The Crabtree effect was present in the Ab melanoma only. Thus glycolysis measured by aerobic and anaerobic lactic acid formation, and cell respiration, measured by oxygen consumption sensitive to KCN, were both higher in the more malignant, less differentiated Ab melanoma than in the Ma and MI melanomas. The suggestion is presented that the process of melanogenesis influences both aerobic glycolysis and the KCN insensitive consumption in the melanotic hamster melanomas.