As normal human cells approach the end of their proliferative lifespan they fail to respond to a variety of growth factors by replicating their DNA. We have examined growth factor responses extensively in WI-38 cells and have found that they do not result from the loss of necessary growth factor receptors or recognizable changes in ligand-receptor interactions. In fact, many of the normal mitogen stimulated cell cycle dependent events and activities are induced in senescent cells just as they are in young cells which do respond by entering the S phase. This has led to the speculation that senescent cells become blocked in late G1 near the beginning of the S phase. Recently we and others (Campisi and co-workers personal communication) have observed that the mRNA for the protooncogene c-fos appears to be undetectable in mitogen stimulated senescent cells. The expression of c- fos is a very early event following mitogen stimulation, reaching maximum levels after approximately 0.5 hr in young cells. We have subsequently observed that c-fos is actually expressed in senescent WI-38 cells following either serum or epidermal growth factor stimulation (EGF) but it is only detectable if protein synthesis is simultaneously inhibited with cycloheximide. This is consistent with the disproportionately rapid degradation of this mRNA in senescent cells. We intend to study the regulation of c-fos expression and that of two other early response genes, c-jun and the serum response factor (SRF) in young and senescent WI-38 cells. We will examine transcription, protein synthesis and posttranslational modification by phosphorylation. We will also directly test whether c-fos of c-jun can increase the proliferative life span of WI-38 cells by transforming young cells with plasmids containing these normal genes under the control of the metallothionein promotor.
