The objective is to study the mechanism of regulation and the functional significance of polyamine metabolism during cellular aging. Our previous studies demonstrated significant changes of polyamine metabolism in senescence of IMR-90 human diploid fibroblasts. These changes include a 5-fold decrease of serum-induced ornithine decarboxylase (ODC) activity and a 10-fold increase of the catabolic conversion of putrescine to amino acids with the net result of an absence of serum-induces putrescine accumulation in senescent IMR-90 cells. Our results also demonstrated no difference in levels of c-myc mRNA and ODC mRNA, but a marked attenuation of thymidine kinase (TK) gene expression in senescent IMR-90 cells as compared to that of the young cells, suggesting that senescent cells are blocked at G1/S boundary upon re-entering the cell cycle after serum stimulation. Based on these results and the recognized importance of polyamines in growth regulation, we formulated a working hypothesis concerning the role of polyamines in aging. We propose that the lack of putrescine accumulation may directly or indirectly subserve the attenuation of the expression of TK gene (and/or other G1/S genes) in senescent cells which in turn leads to the failure of DNA synthesis in senescent cells. The mechanism for the decrease in ODC induction in senescent cells will be investigated; an ODC cDNA probe will be used to determine the distribution of ODC mRNA in polysomes, potent antibodies against OCD and affinity label [3H]difluoromethyl ornithine will be used to quantitate the amount of ODC molecules. The biochemical basis for the increase of putrescine catabolism in senescent cells will be studied by comparing various enzyme systems involved in polyamine catabolic pathways in young and old cells. To evaluate the physiological significance of putrescine accumulation in cellular aging, the relationship between ODC induction/putrescine accumulation and TK gene expression will be examined. We will examine whether the attenuation of gene expression in senescent cells is limited to TK or is a global phenomenon involving other G1/S genes such as dihydofolate reductase and thymidylate synthetase. The study should yield fundamental knowledge concerning the physiological significance of polyamines in cellular aging. It will also shed light on the control mechanism of gene regulation in senescent cells.
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