Fas-induced apoptosis in human malignant melanoma cell lines is associatedwith the activation of the p34(cdc2)-related PITSLRE protein kinases

The Cdc2L locus encoding the PITSLRE protein kinases maps to chromosome ban d 1p36 and consists of two duplicated and tandemly linked genes, The purpos e of the present study was to determine whether diminution of PITSLRE kinas es leads to deregulation of apoptosis. The human melanoma cell lines A375 ( Cdc2L wild-type alleles) and UACC 1227 (mutant Cdc2L alleles) were tested w ith agonist anti-Fas monoclonal antibody. We found that exposure of these c ells to anti-Fas for 24, 48, or 72 h resulted in differential sensitivity t o Fas-induced apoptosis, In A375, cell death started at 24-48 h post-treatm ent, and it was maximal by 72 h. Conversely, UACC 1227 cells were resistant to Fas-mediated apoptosis. Induction of PITSLRE histone H1 kinase activity was observed in A375 anti-Fas treated but not in UACC 1227 cells. Also, th e PITSLRE protein kinase activity in A375 anti-Fas-treated cells preceded m aximal levels of apoptosis. Finally, fluorescence confocal microscopy revea led a nuclear localization of PITSLRE proteins in normal melanocytes and A3 75 cells but a cytoplasmic localization in UACC 1227 cells. The differences in PITSLRE protein and cellular localization between A375 and UACC 1227 ce lls appear to account for the differences in sensitivity of the two cells l ines to anti-Fas and staurosporine. These observations suggest that alterat ions in PITSLRE gene expression and protein localization may result in the loss of apoptotic signaling.