Transfection of nonmelanocytic cells with tyrosinase gene constructs for survival studies

To better understand the role of melanin in the response of cells to radiation, the vector pcTYR containing the tyrosinase cDNA and a control vector pcTYW with no tyrosinase cDNA were transfected and expressed in nonpigmented CHOK1-A(L) 1282B5 cells. A pigmented clone was selected from the pcTYR transfectants and an antibiotic-resistant clone was selected from the controls. Melanin was assessed qualitatively by electron paramagnetic resonance (EPR) and quantitatively by a 14C-based assay. The EPR signal detectable in pcTYR-containing cells was at least twice that of pcTYW and parental CHOK1-A(L) cells and the tyrosinase activity was found to be at least six times greater. Melanin was classified to be eumelanin. Survivals of the transfectants were compared to those of the parent cells after irradiation by UVC from a germicidal lamp, UVB from TL01 lamps, UVA from Alisun lamps, UVB/UVA from FS20 lamps, and by gamma-rays from a 137Cs source. Compared to the pcTYW-containing cells, the pigmented cells were more sensitive to killing by UVC, and resistant to killing by UVA and gamma-rays. There were no significant differences in survival after the other irradiations. These results suggest that the pigment synthesized by the activity of tyrosinase alone, unmodified by the activities of TRP1 and TRP2, is protective against the types of reactive oxygen species produced by UVA and gamma-rays but not protective against lethal damage from photons in the UVB range and sensitizes to UVC photons.