Long-term growth of diploid human fibroblasts in low serum media

Hayflick and Moorhead demonstrated that diploid human fibroblasts have a limited life span when grown in media containing 10% bovine calf sera. Recent experiments have suggested that antigrowth factors in serum may be a potential contributor to the limited proliferative capacity of normal diploid cells. To reduce the concentration of inhibitory serum factors 10-fold, MRC-5 diploid fibroblasts were cultured in media with only 1% serum. Long-term culture in 1% serum requires the addition of purified growth factors to sustain proliferation. Although there are dramatic changes in cell morphology, we find that the long-term division potential of MRC-5 cells cultured in media containing 1% serum and growth factors differs little from that found with cells cultured in 10% serum. In contrast, MRC-5 cells cultured in 10% serum and added growth factors have a somewhat extended life span. These results suggest that negative growth factors are not responsible for the limited proliferative capacity of in vitro cultured human fibroblasts. Moreover, the evidence that human fibroblasts can undergo major changes in cell morphology and still retain a normal life span raises questions about the validity of using morphological changes as indicators of cellular senescence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28800/1/0000634.pd

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

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.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105817/1/9208695.pdfDescription of 9208695.pdf : Restricted to UM users only

An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fosand junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29861/1/0000209.pd

Chemistry: Matter and Change

A comprehensive course of study designed for a first-year high school chemistry curriculum, this program incorporates features for strong math support and problem-solving development