RESUMO
Culture and UV (254 nm) irradiation conditions that are suggested as appropriate for a study of the effect of UV on the limited in vitro lifespan of a normal human diploid fibroblast (HDF) strain are first described. An inoculation density at each subcultivation of 1.8 x 10(4) viable cells/cm-2 permits the decline in proliferative capacity to occur with kinetics similar to that observed using a 1:2 split and prevents cell overlap at the time of irradiation. Doses of 5 and 10 J/m2 have only a slight effect on initial growth rates and little or no effect on cell density achieved at confluence. With these conditions populations can be irradiated several times throughout the in vitro lifespan. No effect of UV on the limited division potential was observed. In the extreme, a population irradiated 14 times, once every second passage starting at P-18 with doses of 5 or 10 J/m2 had the same lifespan as controls, as measured by lifespan determinations and thymidine labeling index. Transformed cells were not detected in the multi-irradiated populations. Evidently no accumulation in the populations of damage induced by UV that affected life span, thymidine labeling index, growth rates or confluent cell densities occurred. No selection of a population with altered sensitivity occurred. An argument that genome hits may not be a prime reason for the limited proliferative capacity of HDF populations is presented.