This proposal aims to investigate the molecular mechanisms determining the protection from programmed cell death (apoptosis) in senescent human fibroblasts. Our repeated attempts show that in vitro aged fibroblasts are resistant to the induction of apoptosis by serum deprivation; furthermore we have shown that, in mouse 3T3 fibroblasts, apoptotic cells lose their statin presence and gain en bloc expressions of c-fos, c-jun, c-myc, cdc2, PCNA, and RB phosphorylation. Apoptosis is reversible until terminin protein 30 kda (Tp30) appears at maximal levels; cultures ar then committed to death, and further rescue by adding serum back is no longer possible. We suggest that the process of apoptosis may be composed by two stages: the early death-initiation stage, identified by the en block expression of the above genes, and the late death-commitment stage, identified by novel protein products such as Tp30. We suggest that senescent human fibroblasts are incapable of undergoing either of these two stages, due to: i. the dysregulation of key early cell cycle events, and ii. the absence of Tp30. These two unique features, compounded by our other finding of the inability to down-regulate the bcl2 protein level in senescent fibroblasts, may lock these cells into a """"""""three-pronged anti-death"""""""" phenotype, and endow them with an exceptionally efficient molecular mode of protection from death. The focus of this project is to investigate how the three prongs of this anti-death mode are interrelated: are all three needed to establish the protection from apoptosis? And if so, is there an order of hierarchy among the three in protecting cells from self-destruction, or is one of them alone sufficient, leaving the other two as merely down-stream consequences? And finally, might molecular manipulations remove the blockade from apoptosis, by introducing Tp30 and/or SV-40 T antigen to lift the repression of c-fos expression and RB phosphorylation, etc., or by removing survival gene expressions such as bcl2? Specific aims include: (1.) characterizing in human fibroblasts the initiation and commitment stages by studying early cell cycle gene expressions, Tp30 presence and the transcriptional regulation of bcl2; (2.) characterizing biochemical properties affecting the presence of terminin 30 kda during apoptosis; (3.) purifying terminin polypeptides for protein sequencing and molecular cloning and sequencing; (4.) investigating the functional role of Tp30 in the commitment to programmed cell death; (5.) investigating whether, in senescent fibroblasts, removing the inhibition to DNA synthesis by adding SV-40 T antigen can also remove the block to apoptosis; and (6.) functional analysis of how regulation of the survival factor, bcl2, is related to the processing of terminin protein into the Tp30 form, and to the commitment to programmed cell death. Answers obtained here will advance our knowledge of the regulation of programmed cell death, a fundamental mechanism pivotal to the well-being of organisms.
Showing the most recent 10 out of 16 publications
