PIG3V, an immortalized human vitiligo melanocyte cell line, expresses dilated endoplasmic reticulum.

Vitiligo is an enigmatic pigmentary disorder of the skin. Factors potentially involved in the progressive loss of melanocytes from the basal layer of the epidermis include genetically determined aberrancies of the vitiligo melanocyte. It follows that analysis of melanocytes cultured from vitiligo donors can contribute to a further understanding of the etiopathomechanism. A setback for vitiligo research has been the limited availability of vitiligo-derived melanocytes. To overcome this limitation, we have generated a vitiligo melanocyte cell line according to a protocol established previously for the immortalization of normal human melanocytes. Vitiligo melanocytes Ma9308P4 were transfected with HPV16 E6 and E7 genes using the retroviral construct LXSN16E6E7. Successful transformants were selected using geneticin and subsequently cloned to ensure genetic homogeneity. The resulting cell line PIG3V has undergone more than 100 cell population doublings since its establishment as a confluent primary culture, whereas untransfected melanocytes derived from adult skin senesce after a maximum of 50 population doublings. Cells immortalized by this transfection procedure retain lineage-specific characteristics and proliferate significantly faster than parental cells. In this study, the phenotype of PIG3V resembled melanocytes rather than melanoma cells in culture. Tyrosinase was processed properly and melanosomes remained pigmented. Importantly, ultrastructural characterization of PIG3V cells revealed dilated endoplasmic reticulum profiles characteristic of vitiligo melanocytes. An explanation for this dilation may be found in the retention of proteins with molecular weight of 37.5. 47.5, and 56.5 kDa, as determined by gel electrophoresis of microsomal proteins isolated from radiolabeled cells.

Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein.

The opportunistic pathogen Pseudomonas aeruginosa produces several potential virulence factors, including the ADP-ribosylating toxin, exotoxin A (PE). Studies using a burned mouse model have shown that PE consistently inhibits protein synthesis and depletes elongation factor 2 in mouse liver and variably in other organs. One reason for toxin sensitivity could be the presence of a PE receptor on the surface of cells. Therefore we examined detergent extracts of mouse tissues for the presence of toxin-binding proteins. Proteins which specifically bind PE were present in extracts from liver, kidney, lung, spleen, and heart. Because liver appears to be a prominent target for the toxin in a burned animal, we choose to isolate the PE-binding protein from mouse liver and compare this protein to the recently characterized toxin-binding protein from toxin-sensitive mouse LM fibroblasts. The toxin-binding proteins from both sources have a molecular mass of approximately 350 kDa, share similar protease digestion profiles, and are glycosylated. However the glycosylation patterns for the two species are quite different. Both glycoproteins bind toxin with high avidity. The toxin-binding moiety is located, at least in part, on the plasma membrane and thus could represent the receptor involved in internalization of toxin molecules responsible for cell death.