MSH stimulates adenylate cyclase and tyrosinase in cultivated melanoma cells in the presence of cytochalasin B

MSH and cholera toxin increase intracellular levels of cyclic AMP (cAMP) and tyrosinase activity in cultivated mouse melanoma cells in the presence of cytochalasin B (CB) at a concentration sufficient to prevent pinocytosis and cytokinesis. The data suggest that MSH and cholera toxin exert their effects without entering the cell.     

Induction of a reversible G 1 block in human glia-like cells by cytochalasin B

In cultures of normal adult human glia-like cells, density-dependent cell cycle inhibition (topoinhibition) and contact inhibition of ruffling occur almost simultaneously, suggesting a functional coupling between activities of the cell surface and the initiation of DNA synthesis. The present paper examines whether cytochalasin B (CB), which reversibly inhibits ruffling, also blocks the glia cell cycle.The effects of the drug (2 μg/ml) were the following:

1. 1. Initiation of DNA synthesis of subcultivated stationary cells was inhibited.

2. 2. Stimulation of DNA synthesis in stationary cells by medium change was suppressed.

3. 3. Migration of cells into a wound in a confluent cell layer was blocked as well as the initiation of DNA synthesis in cells lining the wound.

4. 4. Initiation (but not continuation) of DNA synthesis of exponentially growing cells was inhibited leading to a population mainly arrested in G 1 as determined by microspectrophotometry on Feulgen-stained cells. Topoinhibited cells were also blocked in G 1. Since cytokinesis was blocked by CB, a fraction of binuclear cells appeared.

The cell cycle block induced by CB was reversible, even after several weeks of treatment, with the exception that binuclear cells more reluctantly entered the S phase after release of the block.In conclusion, CB efficiently induces a reversible and probably physiologic cell cycle block. This finding strengthens the notion of a connection between cell membrane and cell proliferation. The underlying mechanism is discussed.     

Ferritin light chain down-modulation generates depigmentation in human metastatic melanoma cells by influencing tyrosinase maturation

Recently, after the identification of ferritin light chain (L-ferritin) gene and protein over-expression in human metastatic melanoma cells, we engineered, starting from the LM metastatic melanoma cell line, clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. The present investigation started from the observation that L-ferritin down-regulated LM cells displayed a less pigmented phenotype, confirmed by a major decrease of total melanin, when compared to control LM cells. This finding was accompanied by a dramatic decrease in tyrosinase activity, which was not paralleled by a concomitant reduction of the amount of tyrosinase specific mRNA. Western blot analysis of tyrosinase in control LM cells displayed a pattern, which corresponds to the progressive glycosylation of the native protein up to the 80 kDa form, considered the functional one. Tyrosinase pattern assayed in L-ferritin down-regulated LM cells showed the remarkable absence of the 80 kDa form and a prevalence of endoglycosidase H (endo H)-sensitive immature (70 kDa) tyrosinase, accumulated in the endoplasmic reticulum (ER), as confirmed by confocal microscopy analysis. These results demonstrate that, in a human metastatic melanoma cell line, the stress condition promoted by L-ferritin down-modulation, can substantially influence proper maturation of tyrosinase.     

Molecular mechanism of tyrosinase regulation by L-dopa in hamster melanoma cells

Exposure of hamster amelanotic melanoma cells to L-dihydroxyphenylalanine (L-DOPA) resulted in a time dependent increase of cell pigmentation, tyrosinase concentration and activity with peak after 24 hours. Northern blot analysis showed a small but reproducible increase of tyrosinase mRNA after 3 hours and a decrease below the control level after 9 hours. After 24 and 48 hours tyrosinase mRNA was undetectable. It is suggested that L-DOPA or its oxidation products can stimulate intracellular tyrosinase concentration and regulate tyrosinase mRNA level both in positive and negative fashion.     

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.     

Synthesis and degradation of tyrosinase in cultured melanoma cells

The tyrosinase (EC 1.14.18.1) activity of cultured B-16 mouse melanoma cells (C2M) in the stationary phase depends greatly on whether the culture medium contains glucose or galactose. The activity in medium containing galactose was about ten times that in medium containing glucose at pH 7.2. This difference in tyrosinase activity was concluded to be due to a shift of balance between synthesis and degradation of the enzyme. Experiments were conducted with stationary phase cultures in the presence of cycloheximide. The melanoma cells did not synthesize tyrosinase in medium containing glucose in the stationary phase. But when they were cultured under identical conditions, except that glucose was replaced by galactose, they continued to synthesize tyrosinase. The rate of synthesis in medium containing galactose at pH 6.3 was one third of that in the same medium at about pH 7, in which the increase in specific activity of tyrosinase per day was about 30 nmoles/mg cell protein per hr. The rate of degradation of the enzyme was practically the same in medium containing glucose as in medium containing galactose, and largely depended on the pH of the culture medium. At pH 6.3, the half-life was about one third of that at pH 7.2, where it was about 1.8 days. The degradation at acidic pH values was much reduced by ammonium salt and was strongly inhibited by the protease inhibitor, leupeptin.     

Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents

The tyrosinase (EC 1.14.18.1) activity of cell-free extracts (TyH) of B16 melanoma cells cultured in the presence of 5 to 10 mM ammonium chloride was considerably higher than that of cells from control cultures. This increase in TyH in the presence of ammonium chloride seemed to be due to de novo synthesis of the enzyme, because it was inhibited by 1 microgram/ml of cycloheximide. In the presence of the latter, however, ammonium chloride did increase the tyrosinase activity of living cells in culture (TyC) resulting in about threefold increase in the TyC/TyH ratio, a measure of the extent of tyrosinase reaction exerted by the enzyme present in living cells. This higher TyC/TyH ratio induced by ammonium chloride was also observed in the absence of cycloheximide. Similar increases in TyH, TyC, and TyC/TyH occurred in the presence of methylamine or ethylamine instead of ammonium chloride, but not in the presence of tetraethylammonium chloride, and also in culture medium of higher pH. The apparently similar effects of lysosomotropic bases and medium of higher pH on the TyC/TyH ratio suggest that there are some mechanisms that control the intramelanosomal pH lower than the cytoplasmic pH.     

Blockage of prereplicative progression by cytochalasin D in serum-stimulated GC-7 cells

When growth-arrested GC-7 cells, a cell line from African green monkey kidney, are stimulated with 10% calf serum, they enter S phase 14-15 h later. Cytochalasin D at 0.6 micrograms/ml blocks the entrance into S phase, and inhibits, though only partially, the increase in protein synthesis after serum stimulation. Since partial inhibition of protein synthesis by cycloheximide interferes with accumulation of labile proteins and thus blocks the entrance of serum-stimulated cells into S phase, the effects of these two inhibitors are compared. Cytochalasin D at lower concentrations reduced the rate of entry into S phase without affecting the length of the prereplicative phase, whereas cycloheximide extended the prereplicative phase dose dependently without affecting the rate of entry into S phase. Cytochalasin D affected neither individual [35S]methionine-labeled spots on two-dimensional polyacrylamide-gel nor degradation of cellular proteins. These results indicate that cytochalasin D, though it interferes with protein synthesis, blocks prereplicative progression of serum-stimulated GC-7 cells in a different manner than cycloheximide.     

Activation of melanogenesis by vacuolar type H(+)-ATPase inhibitors in amelanotic, tyrosinase positive human and mouse melanoma cells

In this study, we describe the activation of melanogenesis by selective vacuolar type H(+)-ATPase inhibitors (bafilomycin A1 and concanamycin A) in amelanotic human and mouse melanoma cells which express tyrosinase but show no melanogenesis. Addition of the inhibitors activated tyrosinase within 4 h, and by 24 h the cells contained measurable amounts of melanin. These effects were not inhibited by cycloheximide (2 microgram/ml) which is consistent with a post-translational mechanism of activation. Our findings suggest that melanosomal pH could be an important and dynamic factor in the control of melanogenesis in mammalian cells.     

Effects of tyrosinase activity on the cytotoxicity of 3,4-dihydroxybenzylamine and buthionine sulfoximine in human melanoma cells

The rationale for melanoma specific dihydroxybenzene containing antitumor agents is based in part upon the ability of the enzyme tyrosinase to oxidize these pro drugs to toxic intermediates. In situ tyrosinase activity was demonstrated to be affected by both cell density and time from plating in pigmented melanoma cells. Phenylthiourea, which completely inhibited tyrosinase activity with minimal cytotoxicity was found to block the growth inhibitory activity of the antitumor dopamine analog 3,4-dihydroxybenzylamine (3,4-DHBA) (NSC 263475). The antioxidant dithioerythritol was also found to inhibit tyrosinase activity and to block the growth inhibitory effects of 3,4-DHBA in pigmented melanoma cell lines. Buthionine sulfoximine (BSO) was shown to be cytotoxic to melanoma cells and its growth inhibitory effects appears to correlate with tyrosinase levels. Furthermore, BSO was shown to potentiate the growth inhibitory effects of 3,4-DHBA on marginally pigmented human melanoma cell lines.     

Induction of apoptosis by Hax-1 siRNA in melanoma cells

HS1-associated protein X-1 (Hax-1) is a novel intracellular protein and recent studies suggested that it is an anti-apoptotic factor in different tumors. Hax-1 expression was upregulated in various metastatic tumors and cancer cell lines, including melanoma. To understand the role of Hax-1 in melanoma development and progression, we constructed Hax-1 short interfering RNA (siRNA) expression vectors to downregulate Hax-1 expression in a human melanoma A375 cell line. One of the two Hax-1 RNA interference (RNAi) constructs significantly reduced melanoma cell viability, which was due to induction of apoptosis in A375 cells. Molecularly, the induced apoptosis through downregulation of Hax-1 expression was mediated by activation of caspase-3 and poly-ADP-ribose polymerase (PARP) enzymatic activity in A375 cells. The data indicate that Hax-1 plays a role in suppression of apoptosis and promotion of melanoma cell growth, suggesting that this Hax-1 siRNA has a therapeutic indication in control of melanoma.     

Inhibition of tyrosinase activity and protein synthesis in melanoma cells by calcium ionophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. Ionophore at a concentration of 10(-6) g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. Ionophore A23187 also inhibits the PGE1 mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10(-4)M). Ionophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGE1, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. Ionophore causes a rapid and marked (greater than 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Effect of cytochalasin B and D

Summary The effect of cytochalasin B (CB) and cytochalasin D (CD) on the endocytotic uptake of horseradish peroxidase (HRP) by intestinal absorptive cells was investigated by morphometric methods. The results showed that CD inhibited endocytosis considerably, and without any detrimental side-effects. CB had hardly any effect on the endocytosis of HRP, but caused a significant decrease in the number of apical vesicles and tubules involved in the transport of cell-coat glycoproteins from the Golgi apparatus to the brush border.Electron-microscopic autoradiographic analysis of the effect of CD showed that although endocytosis is inhibited significantly by the drug, the amount of radiolabelled cell-coat material entering the lysosome-like bodies was unaltered compared with control cultures. These observations support our hypothesis that the cell-coat glycoproteins of the absorptive cells enter the lysosome-like bodies by a crinophagic rather than by an exocytotic-endocytotic mechanism.     

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.