1-oleoyl-2-acetylglycerol and Carcinoma--Squamous-Cell

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.

Topics: Biological Transport; Carcinoma, Squamous Cell; Cytoplasmic Granules; Diglycerides; Flow Cytometry; Humans; Keratinocytes; Lectins; Melanins; Melanoma; Microscopy, Electron; Tumor Cells, Cultured

Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2-3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF-stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2-acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor.

Topics: Calcium; Calcium Radioisotopes; Carcinoma, Squamous Cell; Cell Line; Diglycerides; Epidermal Growth Factor; Humans; Kinetics; Phosphatidylinositols; Protons; Sodium; Sodium Radioisotopes; Tetradecanoylphorbol Acetate; Vanadates; Vanadium