Evidence contrary to the protein error hypothesis for in vitro senescence.
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 glucose-6-phosphate dehydrogenase (G-6-PD) 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 +/- 1%, S.E.), in comparison with that in 19 old cultures (4.9 +/- 1%, S.E.). Old cultures had ceased active cell division (49-60 doublings); DNA replication, measured by [3H]thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5-22 doublings) had a [3H]thymidine labeling index of 75-85%. Titration of G-6-PD activity in extracts of young and old cells with neutralizing antibody directes specifically against G-6-PD failed to detect an increment of enzymatically defective G-6-PD in old cells. The thermostability studies were capable of detecting altered G-6-PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G-6-PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G-6-PD from the thermolabile mutant and G-6-PD inactivated with N-ethylameimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.
D000328:Adult; D001383:Azetidinecarboxylic Acid / Q000494:pharmacology; D002455:Cell Division; D002478:Cells, Cultured / Q000201:enzymology / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007118:Immunoassay; D007231:Infant, Newborn; D008297:Male; D008954:Models, Biological; D009154:Mutation; D014176:Protein Biosynthesis
D001383:Azetidinecarboxylic Acid; D004247:DNA; D003513:Cycloheximide; D005954:Glucosephosphate Dehydrogenase; D005033:Ethylmaleimide