Regulation of senescence in human diploid fibroblasts: Alterations in gene expression and effects of serum factors.

Abstract

Cultured diploid human fibroblasts have a limited proliferative life span. The work presented here tests two of the possible mechanisms by which replicative senescence may occur: (1) growth inhibition by serum factors, especially in serum from older animals, or (2) changes in gene expression, especially increased expression of genes involved in growth inhibition. To determine whether the concentration of growth inhibitors present in serum increases as a function of donor age, the life spans of MRC-5 cells cultured in serum from cows of various ages were compared to those cultured in fetal calf serum. Fibroblasts cultured using the different sera exhibited nearly identical proliferative capacities. These data suggest that the balance of serum factors that stimulate and inhibit growth does not vary significantly as a function of donor age. To examine serum for the presence of growth inhibitors, the life spans of MRC-5 cells cultured in 10% serum plus growth factors and 1% serum plus growth factors were compared. Long-term division potential was found to be similar under the two conditions. These data suggest that growth inhibitors found in serum are not responsible for the limited proliferative capacity of cultured human fibroblasts, since a 10-fold reduction in the concentration of serum does not result in an increased life span. To identify genes preferentially expressed in senescent fibroblasts, a differential hybridization screening was carried out using labeled cDNA from early- and late-passage MRC-5 cells to probe a cDNA library made from senescent MRC-5 cells. A Senescence-Associated Gene (SAG) whose mRNA level is up-regulated 3-fold with senescence was identified. Data showing that the increases in SAG mRNA parallel the slowing of proliferative rate and that the apparent magnitude of changes in SAG mRNA levels are not due to cell cycle position indicate that SAG is a good marker for growth potential during replicative senescence. Further studies demonstrate that SAG is a novel gene active in cells from many different tissue types and that it is highly conserved. DNA sequencing data indicate that the SAG protein contains a potential DNA binding domain, suggesting that SAG may have a regulatory function.