Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultrav iolet irradiation (UV) while UVr-1 cells are their variant with an increase d resistance to UV, Three days after UV at 10 J/m(2), the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%, This differe nt survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV , Addition of ALLN/calpain inhibitor I to the culture medium after UV resul ted in similar survival (14 -18%) between RSa and UVr-1 cells. Immunoblot a nalysis showed down-regulation of protein kinase CB, Src, Bar and mu-calpai n after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calp ain appeared within 1 h post-UV only in UVr-1 cells. The expression of calp astatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells, To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The resu lts showed that overexpression of calpastatin suppressed down-regulation of Src, p-calpain and Bar. Concomitantly, colony survival after UV was reduce d in calpastatin-transfected cells as compared to vector control cells, Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.