Robert J. Pignolo

Expression of hydrogen peroxide and glutathione metabolizing enzymes in human skin fibroblasts derived from donors of different ages

We have examined the activities and mRNA abundance of two hydrogen peroxide metabolizing enzymes (glutathione peroxidase and catalase), glutathione concentration, and the activities of several enzymes that influence glutathione concentration, including glutathione reductase (GR), glucose‐6‐phosphate dehydrogenase (G‐6‐PD), and γ‐glutamylcysteine synthetase (γ‐GCS), in 29 skin fibroblast lines derived from donors ranging in age from 14 gestational weeks to 94 years of age. H2O2 metabolizing enzyme activities and mRNA abundances were greater in skin fibroblast cultures established from postnatal donors than in fetally derived cultures. There were no significant differences in either of these parameters in cell lines established from postnatal donors of different ages. Total glutathione concentration decreased with age, but GR activity appeared to be unaffected by age. In order to estimate the ability of the cultures to produce NADPH (an important component of cellular redox status and a cofactor for GR), we determined glucose‐6‐phosphate dehydrogenase activity and mRNA abundance. We were unable to directly measure γ‐GCS activity or mRNA abundance in any of the skin lines or in fetal lung fibroblasts; however, we were able to indirectly demonstrate the presence of this enzyme by stimulating fetal lung fibroblasts with H2O2 following treatment with L‐buthionine‐S,R‐sulfoximine (BSO), an inhibitor of γ‐GCS activity. These results show that some, but not all, age‐associated differences in antioxidant defense levels are maintained in a culture environment and are consistent with the hypothesis that developmental stages of life are associated with lower antioxidant defense levels than are present in postnatal phases of life.

Differential gene expression between young and senescent, quiescent WI-38 cells

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.

Putative role for EPC‐1/PEDF in the G0 growth arrest of human diploid fibroblasts

EPC‐1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast‐like (HDF) cells and is diminished with proliferative senescence in vitro. EPC‐1 expression in HDF cells is induced under conditions of density‐dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC‐1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC‐1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC‐1 antibodies alone enhance the ability of near plateau‐phase early PDL WI‐38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC‐1 (rEPC‐1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC‐1 expression in WI‐38 and IMR‐90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC‐1 expression with SV40 transformation occurs at the level of steady‐state mRNA and protein accumulation with genomic EPC‐1 sequences grossly intact. Taken together, these results suggest that EPC‐1 may play a role in the entry of early passage fibroblasts into a G0 state or the maintenance of such a state once reached.

ALTERATIONS IN CONTACT AND DENSITY-DEPENDENT ARREST STATE IN SENESCENT WI-38 CELLS

SummaryNormal human WI-38 fibroblast-like cells in culture undergo a process of senescence, one feature of which is a gradual decline in proliferative capacity. As these cells reach the end of their replicative life span they exhibit decreases in the fraction of cells able to synthesize DNA, in the number of doublings per passage (constant seeding density), and in the cell harvest and saturation densities. They also display increased average cell cycle times, largely at the expense of longer G1 intervals. These alterations are accompanied by morphologic changes, including cell enlargement. Before the end of the replicative life span or phase-out, there is a highly reproducible (55/58 sublines) cell loss of approximately 50%; however, a stable population survives that can exist in a viable yet nonproliferative state for many months. This stable population maintains an extremely low saturation density, representing <5% of that achieved by early passage cultures. Further, we show that maximum harvest densities achieved by senescent cells are lower, irrespective of seeding densities, i.e. when placed at cell densities higher than those normally achieved by senescent cultures they display a net decline in cell number. This decline continues until the cell density approximates the density that would have been achieved had the cultures been seeded at standard density (1×104 cells/cm2). By measuring the accumulation of an mRNA species,EPC-1, that is expressed when early passage cultures reach a growth-arrested state via density-dependent contact inhibition, we also show that senescent cells are unable to produce this transcript at either their normal confluent density or at high cell density obtained by overseeding. The above results suggest that there are significant alterations in cell-to-cell contact sensitivity and arrest state of senescent cultures. These changes result in both a cytotoxic response to crowding and failure to express at least one molecular marker which is induced as young cells approach growth arrest by contact inhibition.