Differential radiosensitivity in cultured B-16 melanoma cells following interrupted melanogenesis induced by glucosamine

The relationship between cell pigmentation and radiosensitivity was investigated in a cell model in which melanogenesis was suppressed by a glycosylation inhibitor. It was found that X-irradiation of melanotic B-16 melanoma cells and their amelanotic counterparts, obtained by glucosamine treatment, showed an inverse correlation between radiosensitivity and melanin contents. Since melanogenesis interruption by glucosamine does not affect the DNA repair capacity of nonpigmented cells, it is likely that intracellular melanins play a role in the relative resistance of pigmented cells to X-irradiation.     

Delayed alteration of membrane fluidity in intact cultured B-16 melanoma cells affected by ultraviolet irradiation

Lipid peroxidation in the plasma membrane has been reported to decrease membrane fluidity. We examined membrane fluidity in relation to lipid peroxidation processes after UV-B exposure of cultured B-16 melanoma cells. UV exposure promptly increased TBA-positive material(s), but alteration of membrane fluidity was delayed. Plasma membrane fluidity increased significantly 6 hours after exposure when the TBA-value(s) had become under the control level. To examine the direct effect of lipid peroxides on the fluidity, tert-butyl hydroperoxide was added to B-16 melanoma cells. Similar results were obtained with respect to membrane fluidity. These results suggest that lipid peroxidation at UV doses maintaining cell viability does not directly induce a significant alteration of membrane fluidity, but may influence the fluidity either during metabolizing processes of UV-induced lipid peroxides or during repair processes following oxidative cell membrane damage.     

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.     

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.     

Relative biological effectiveness (RBE) of thermal neutron capture therapy of cultured B-16 melanoma cells preincubated with 10B-paraboronophenylalanine

An experimental study of the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) for melanoma cell inactivation using 10B1-paraboronophenylalanine (10B1-BPA) was carried out to demonstrate a high therapeutic effect of TNCT, compared with that of fast neutron. Cells preincubated with or without 10B1-BPA at a concentration of 50 micrograms/ml for 20 h were irradiated with 60Co gamma-ray, fast neutron or thermal neutron. The absorbed dose of the cells from thermal neutron was calculated by Kobayashi's model. The D0 value of fast neutron was 1.07 Gy, and the D0S of thermal neutron radiation with or without preincubation of the cells with 10B1-BPA were 0.46 Gy or 0.67 Gy, respectively. The RBEs of fast neutron, thermal neutron beams, and neutron capture therapy relative to 60Co gamma-ray were calculated as 2.78, 4.18, and 6.15 at 0.1 surviving fraction, respectively. These results indicate radiologically that thermal neutron capture therapy using 10B1-BPA is an excellent radiation therapy for malignant melanoma.     

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.     

Evidence that the small GTPase Rab8 is involved in melanosome traffic and dendrite extension in B16 melanoma cells

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion.This investigation was supported in part by Grants-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (grant 15591176)     

Temporal synthesis and presentation of antigens by cultured B16 melanoma cells

Previous studies have demonstrated the presence of two distinct antigens, B700 and B50, which are unique to murine melanoma. One of these, B700 has been studied in detail, and is present on 5 different murine melanomas; it can function as a transplantation antigen in at least 3 of them (B16, JB/RH and K1735). The synthesis and presentation of these antigens has been studied as a function of cell culture conditions. Direct immunofluorescence studies of cells in serial culture indicate that the expression of B700 and B50 antigens at the cell surface and in the cytoplasm increases as a function of time in culture, over 1-5 days. By day 5, when the cells are confluent, all cells show some degree of antibody binding. Parallel 35S-methionine pulse chase labeling experiments show that incorporation into Triton soluble proteins, and Triton insoluble SDS soluble proteins, increases to a peak at 3.5 days after subculturing, then decreases as the cells reach confluence. Incorporation into proteins shed into the culture supernatant continued throughout the time course of cell growth to confluence. However, as the cells become confluent, total protein synthesis shifts towards greater production of the antigens (both cellular and shed). The sum of the results suggest that tumor growth may succeed in vivo by the wholesale production of "decoy" antigens.     

Release into culture medium of membrane-associated, tumor-specific antigen by B-16 melanoma cells.

Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57Bl/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57Bl/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.     

RBE of a thermal neutron beam and the 10B(n, alpha)7Li reaction on cultured B-16 melanoma cells

The RBE of a thermal neutron beam and the 10B(n, alpha)7Li reaction were determined in cultured B-16 melanoma cells. The Kyoto University Research Reactor (KUR) was used as a thermal neutron source which had a very low contamination of gamma-rays and fast neutrons. The cells were irradiated with the beam in the presence or absence of 10B-boric acid. The absorbed dose from the neutron capture reaction to the cells was calculated by a method of Kitao (1975). Survival curves in both conditions had no shoulder and Do values were 0.506 or 0.604 Gy in the presence or absence of 5 micrograms 10B/ml-medium, respectively. The Do value of the 10B(n, alpha)7Li reaction was also estimated at 0.466 Gy, assuming each component of radiation was additive. The RBEs of the KUR thermal neutron beam and the 10B(n, alpha)7Li reaction relative to 60Co gamma-rays were estimated as 4.62 and 6.01 at 0.1 surviving fraction, respectively. Using these results, we calculated the absorbed dose from the 10B-compound and estimated the specific accumulation in the cultured cells of 10B-compounds which we have previously reported but not quantified (Nakanishi et al. 1980, Ichihashi et al. 1982).     

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.     

Citrate activates tyrosinase from B-16 murine melanoma and human skin

Citrate stimulates cresolase activity of tyrosinase from B-16 murine melanoma and human skin. Maximal stimulation by citrate was obtained at 2 mM, and stimulation was decreased at higher concentrations. Citrate stimulates tyrosinase not only from mammalian sources but also from mushroom. The stimulation was not due to reversal of inhibition of enzyme activity by excess tyrosine. On rapid decrease in pH of the enzyme solution from 6.8 to 5.0-5.2, the enzyme is no longer inhibited by excess tyrosine even when its activity was assayed at pH 6.8. Citrate also stimulates this form of enzyme. However, the stimulation is more at acidic pH than at pH 6.8. At higher concentrations of citrate the stimulatory effect decreases at both pH 5.0 and pH 6.8. Inhibition of this enzyme occurs at higher concentrations (22 mM) at pH 6.8. The physiological role of stimulation of cresolase activity of tyrosinase by citrate is yet to be unravelled.     

Transforming growth factor beta1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation

Transforming growth factor-beta1 (TGFbeta1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by alpha-melanocyte stimulating hormone (alphaMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFbeta1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFbeta1 (10(-10) M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFbeta1 and alphaMSH on melanosome number, volume density and maturation degree. alphaMSH (10(-7) M, 48 h) increased 7-fold melanosome volume density, whereas TGFbeta1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or alphaMSH-treated cells. Moreover, TGFbeta1 blocked the alphaMSH-elicited increase in the number of melanosomes. Control and alphaMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFbeta1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with alphaMSH and TGFbeta1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFbeta1 and with its hypopigmenting effect.     

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.     

Depigmentation and inhibition of cell growth of B-16 melanoma cells by compounds isolated from Paeonia suffruticosa callus

The anther segments of Paeonia suffruticosa Andrews were cultured for callus formation on MS medium supplemented with 2,4-D and BAP(each 1 mg/l) in the dark at 25°C for six weeks. Gallic acid, methyl gallate, ethyl gallate and 1,2,3,4,6-penta-O-galloyl--D-glucose were isolated from the ethanol extract of callus, and paeoniflorin was identified. A dose of 5 g/ml pentagalloyl glucose resulted in depigmentation of B-16 melanoma cells without inhibition of cell propagation.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.