A fundamental difference between human normal and transformed or tumor cells is that the former undergo an aging process in culture, while the latter apparently have unlimited lifespans in the same culture environment. This project will investigate two aspects of cellular aging, namely, (1) the genetic expression of limited lifespan in somatic cell hybrids constructed from the fusion or normal and tumor cell, and (2) the accuracy of cell-free protein synthesis in extracts from normal cells of varying ages, tumor cells, and from cells of patients with the premature aging Werner's syndrome. Hybrids will be constructed from genetically marked cells, facilitating clonal, quantitative analysis of: (I) hybrid lifespan; (II) the frequency and timing of transformation, or """"""""escape from senescence,"""""""" in hybrids; (III) the time of appearance of certain transformed cell properties, in particular the loss or retention of specfic chromonsomes; (IV) the influence of each parental cell nucleus in cybrids from enucleation fusions; and (V) the effect of mutagens on the rate of aging and the frequency of transformation. The accuracy of protein synthesis will be measured by using synthetic (poly(U) and poly(C)) and natural (sheep globin and sunn-hemp mosaic virus) mRNAs which lack codons for specific amino acids in an in vitro translation system. The detection these labelled amino acids in purified protein products of the reaction represents translational errors. This assay will enable a direct test of the Orgel error calastrophe theory of cell aging, and comparisons of the relative accuracies of cytoplasmic and mitochondrial protein synthesis in normal, tumor, and Werner's syndrome cells, to be made.
