Abstract
A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucoseโ6โphosphate dehydrogenase (Gโ6โPD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 ยฑ 1%, S.E.), in comparison with that in 19 old cultures (4.9 ยฑ 1%, S.E.). Old cultures had ceased active cell division (49โ60 doublings); DNA replication, measured by [^3^H] thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5โ22 doublings) had a [^3^H] thymidine labeling index of 75โ85%. Titration of Gโ6โPD activity in extracts of young and old cells with neutralizing antibody directed specifically against Gโ6โPD failed to detect an increment of enzymatically defective Gโ6โPD in old cells. The thermostability studies were capable of detecting altered Gโ6โPD in skin fibroblasts from a female heterozygous for a thermolabile mutant of Gโ6โPD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered Gโ6โPD from the thermolabile mutant and Gโ6โPD inactivated with Nโethylmaleimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.