Over-expression of MSG1 transcriptional co-activator increases melanin in B16 melanoma cells: a possible role for MSG1 in melanogenesis.

MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase.

Transient expression of high molecular weight, heat sensitive, trypsin-resistant form of tyrosinase in B-16 melanoma cells.

High molecular weight forms of tyrosinase have been found to be expressed during spontaneous remelanization of the amelanotic B-16 melanoma cells in culture as well as in melanotic tumors formed from amelanotic melanoma cells grown in C57BL/6J mice. Overnight extraction of the crude melanosomal fractions from such tumors and cultured melanoma cells reveal the presence of an additional DOPA-MBTH positive band well below the stacking gel. This band has been found to be alpha-PEP7 (antibody specific for tyrosinase) positive and alpha-PEP1 (antibody specific for TRP-1) negative on Western blot analysis. Heat treatment at 60 degrees C for 60 min results in the loss of this band and considerable loss of activity of the melanosomal extract. Trypsin treatment of these melanosomal extracts resulted in a minor change in the mobility of the high molecular weight band. SDS-PAGE under reduced conditions followed by Western blotting revealed that the high molecular weight band was lost and not detected by alpha-PEP7 or alpha-PEP1. These findings indicate that high molecular weight, heat sensitive and trypsin resistant forms of tyrosinase are transiently expressed in B-16 melanoma cells and tumors that are initiating remelanization following phenotypic drift towards the amelanotic state.

Viability testing of homograft valves using methyl thiazol tetrazolium assay.

Viability status before and after treatment with antibiotics was investigated in a total of 104 homograft valves using MTT (3-[4, 5-dimethyl thiazol-2-y1]-2, 5-diphenyl tetrazolium bromide). The valves with warm ischaemic time above 11 hours, were found to be non-viable. Increase in storage time directly decreases cell viability. Methyl thiazol tetrazolium (MTT) assay can be used as a reliable, simple, rapid and economic method for assessing the viability status based on mitochondrial respiration even for homograft valves. Basal media with and without nutrients i.e., DMEM and Hanks BSS showed no difference in viability of the cells.

In vitro growth-stimulatory property of pigeon milk.

Five cell lines were employed to test the growth-stimulating property of pigeon milk in vitro. All the cell lines except A431 showed good growth response to crude homogenates of pigeon milk. Enhancement of DNA synthesis in quiescent Chinese hamster ovary (CHO) cells by pigeon milk was dose dependent up to a concentration of 1%. In vitro growth stimulation by 1% pigeon milk was approximately equal to that by 2% foetal bovine serum (FBS) when CHO cells were used, growth stimulation of Vero cells by 1% pigeon milk was roughly three times of that by 2% FBS. In contrast, 1% pigeon milk was only half as active as 2% FBS on NIH/3T3 cells and five times less active than 2% FBS on human foetal lung fibroblast cells. After dialysis using a relative mass (M(r)) cutoff of 3500, the pigeon milk mitogenic activity was retained in the dialyzed solution, although it decreased by 40-60% when dialyzed with M(r) cutoffs of 8000 and 12,000-14,000. The growth-stimulating activity of pigeon milk was resistant to heat, acid, alkali, and the action of urea, guanidine hydrochloride, dithiothreitol, and trypsin. We suggest that pigeon milk is a new source of growth factor(s) capable of stimulating in vitro the growth of many mammalian cell types.

Studies on the growth-stimulatory activity of pigeon milk--comparison and synergistic effects with serum.

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.

Etiology of vitiligo. A new hypothesis.

Serum from actively repigmenting human vitiligo subjects had maximum mitogenic effect on the growth of melanocytes in culture, followed by the serum from normal donors, and from untreated vitiligo subjects in that order. Based on these findings, a new hypothesis is suggested for the etiology of vitiligo.

Growth defects of melanocytes in culture from vitiligo subjects are spontaneously corrected in vivo in repigmenting subjects and can be partially corrected by the addition of fibroblast-derived growth factors in vitro.

Melanocytes cultured from uninvolved skin of untreated vitiligo subjects have decreased initial seeding capacities, manifest a lag period for the onset of the growth phase, and cannot be passaged. In contrast, melanocytes obtained from uninvolved and perilesional skin of vitiligo subjects actively repigmenting under 8-methoxy psoralen plus sunlight (PUVA) therapy have higher initial seeding capacities, grow faster without a lag period, and can be passaged to more than 12 passages. Extracts of a fetal lung fibroblast cell line (PMR-GF) that promote the growth rates and passage capacities of melanocytes from normal adult donors have been found also to promote the growth rates and passage capacities of melanocytes from the uninvolved skin of vitiligo subjects. Extracts of a fetal lung fibroblast cell line (PMR-GF), however, did not have any further stimulatory effect on the growth of melanocytes obtained from repigmenting vitiligo subjects. Melanocytes cultured from normal and untreated vitiligo subjects grew individually dispersed in the absence of PMR-GF, but tended to grow in clusters in its presence. Melanocytes from the repigmenting vitiligo subjects, however, tended to grow in clusters even in the absence of PMR-GF. These results indicate that the defective in vitro growth characteristics of melanocytes from vitiligo subjects may be related to the pathogenesis of this disease. It is possible that growth factors may be involved in the process of repigmentation in vitiligo subjects.

In vitro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects.

The in vitro growth characteristics of melanocytes obtained from uninvolved and perilesional skin of vitiligo vulgaris subjects have been investigated in comparison to those from healthy adult donors. Normal human melanocytes have been found to grow exponentially in the presence of 10(-11) M cholera toxin and 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in routine tissue culture media. They could be trypsinized up to 3-4 passages. Melanocytes of the uninvolved skin of vitiligo subjects manifested a lag of 8-11 days for the onset of growth and they could not be passaged. Melanocytes obtained from both hypo- and hyper-pigmented perilesional skin failed to grow under these conditions. Only in a few cases where the perilesional skin was normally pigmented did the melanocytes manifest some growth after a lag of 15 days. The initial seeding capacity of the melanocytes from uninvolved and perilesional skin of vitiligo patients were, respectively, 50% and 25% of the normal individuals. Vitiligo lesions themselves gave rise to unidentified dendritic cells that survived for 10-15 days without manifesting any growth. Our results suggest that melanocytes of individuals with vitiligo are defective. This fact has to be taken into account in any theory on the etiology of vitiligo.

Time-dependent suppression of melanoma metastases and natural killer cell activation by interferon.

The effect of murine alpha/beta interferon (IFN) on experimental metastasis was investigated using B16-F10 melanoma cells. Since the outcome of metastasis of blood-borne tumor cells is mainly determined within the first 24 h after i.v. inoculation of tumor cells, i.p. injection of IFN was focused on this critical early phase. The inhibition of pulmonary metastases by IFN was found to be maximal when given 3 h prior to tumor cell inoculation, while mice with 24-h and 12-h pretreatment and simultaneous IFN treatment also showed a reduction in metastases, but to a lesser extent. However, mice receiving IFN 2 h after tumor cell inoculation did not show any reduction. Tumor cells cultured for 24 h in IFN-containing medium showed no reduction in metastases. Administration of anti-asialo GMl prior to IFN treatment was found to eliminate the inhibitory effect of IFN 3 h pretreatment. However, natural killer (NK) cell activity in vitro measured at 3 h, 13 h and 24 h after IFN administration was enhanced to the same extent, not paralleling the inhibitory effect on pulmonary metastases. These data indicate that prepared host status against blood-borne tumor cells is established by IFN pretreatment, being maximal when injected several hours prior to tumor cell inoculation, and that this effect is substantially dependent on NK cell activity, though the implication of other factors is not excluded.

gamma-Glutamyl transpeptidase, tyrosinase, and 5-S-cysteinyldopa production in melanoma cells.

The distribution of gamma-glutamyl transpeptidase (gamma-GTP), tyrosinase, and 5-S-cysteinyldopa (5-S-CD) within melanoma cells has been studied in vitro as well as in vivo. Sodium periodate treatment of intact B-16 melanoma cells has been found to inhibit gamma-GTP present as an ectoenzyme. However, these periodate-treated cells in the presence of 10(-5) M dopa and glutathione have been found to continue to secrete large quantities of 5-S-CD in their medium. The large-granule fraction of Greene's melanotic melanoma contains substantial amounts of both tyrosinase and gamma-GTP. However, further separation of the large-granule fraction into sub-fractions indicates that tyrosinase and gamma-GTP seem to co-exist with premelanosome. It is suggested that glutathione-dependent t-S-CD genesis proceeds within premelanosomes through the formation of glutathione-dopa. The excess of glutathione-dopa and 5-S-CD, unutilized for pheomelanin formation overglow into the cytoplasm.

Effect of DOPA-loading on glutathione-dependent 5-S-cysteinyldopa genesis in melanoma cells in vitro.

The effect of dopa, cysteine, and glutathione on 5-S-cysteinyldopa (5-S-CD) genesis in melanoma cells cultured in normal and tyrosine- and cysteine-free media has been studied. In normal media only melanotic melanoma cells have been found to secrete 5-S-CD into the medium. In the presence of dopa and cysteine, both, media incubated with and without cells have been found to produce 5-S-CD. In the presence of dopa and glutathione, however, cell-free media did not show the presence of 5-S-CD. In contrast melanoma cell-cultured media has been found to contain large quantities of this amino acid. The optimum condition for maximum production of 5-S-CD via glutathione-dependent pathway has been found to be at the dopa concentration of 10(-5) M when glutathione is present at the concentration of 10(-5) M in the culture medium. Thus dopa concentration with regards to glutathione is 1:1 on the molar basis which is twice the dopa concentration required in in vitro noncellular tyrosinase system. It is suggested that higher dopa requirement in our melanoma cell culture system reflects the co-existence of eu- and pheomelanin synthesis taking place according to their genetically predetermined proportions.