Melanosomal and lysosomal alterations in murine melanocytes following transfection with the v-rasHa oncogene.
Melanomas exhibiting mutated ras genes are frequently invasive and amelanotic. Transfecting melanocytes with ras oncogenes causes transformation and a loss of visible pigmentation. We analyzed murine melanocytes rendered amelanotic by transfection with the v-rasHa oncogene. Consistent with previous reports, tyrosinase and tyrosinase-related protein-1 (TRP-1) were not expressed by transformed cells. In addition, lack of expression of TRP-2 and the product of the silver locus was documented. Levels of melanosomal matrix antigens, the pink-eyed dilution locus protein and lysosome-associated membrane protein-1 were markedly reduced. Residual matrix antigens were localized by immunofluorescence to large vacuoles distributed peri-nuclearly in transfected cells. Electron microscopy demonstrated the absence of typical melanosomes and the presence of large vacuolar structures, also in a peri-nuclear distribution. Although levels of lysosomal hydrolases, such as beta-glucuronidase and cathepsin D, were diminished, marked elevations were observed in the expression of cathepsins B and L, 2 thiol proteases implicated in the acquisition of invasiveness. Our data demonstrate that transfection of melanocytes with v-rasHa is sufficient to disrupt the biogenesis of melanosomes and to up-regulate thiol protease synthesis, providing insights into the amelanotic and invasive nature of melanomas exhibiting mutations in ras genes.
Donatien
P D
PD
Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, USA.
Diment
S L
SL
Boissy
R E
RE
Orlow
S J
SJ
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D019084:Fluorescent Antibody Technique, Indirect; D011905:Genes, ras; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism* / Q000648:ultrastructure*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism; D014162:Transfection
D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D011506:Proteins; D010088:Oxidoreductases; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D002402:Cathepsin D; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase