Absence of three secreted proteins and presence of a 57-kDa protein related to irreversible arrest of cell growth.

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.

mentioning

confidence: 99%

Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence

Porter

Pereira‐Smith

Smith

1990

“…Although early studies using two-dimensional polyacrylamide gel electrophoresis identified a few proteins that appeared to be senescent cell-specific, the vast majority of the proteins detected were synthesized by both young and old fibroblasts (Sakagami et al, 1979;Engelhardt et al, 1979;Lincoln et al, 1984). Senescent-specific polypeptides of 57 kDa and 55 kDa have been described by Ching and Wang (1988) and Sottile et al (1987), respectively. West et al (1989) have suggested that these proteins may be forms of procollagenase, a protein overexpressed in senescent normal fibroblasts (West et al, 1989) and fibroblasts derived from Werner's syndrome patients (Bauer et al, 1986).…”

mentioning

confidence: 99%

Altered regulation of platelet‐derived growth factor A‐chain and c‐fos gene expression in senescent progeria fibroblasts

Winkles

O'connor

Friesel

1990

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.

“…If this were the case, one would expect genes, including those associated with the differentiated function of the cells, to be expressed differentially as the cells progress from phase I to phase 111. Several laboratories have identified proteins (Engelhardt et al, 1979;Wang, 1985;Sottile et al, 1987;Bayreuther et al, 1988;Ching and Wang, 1988;Cowles et al, 1989) or mRNAs (Lumpkin et al, 1986;Giordano and Foster, 1989) that are differentially expressed in low and high passage cells. However, the roles of these gene products in the differentiation or aging of the cells have not yet been fully defined.…”

mentioning

confidence: 99%

Differential extracellular matrix gene expression by fibroblasts during their proliferative life span in vitro and at senescence

Choi

Olsen

Cook

et al. 1992

Increasing evidence supports the idea that the finite proliferative life span of normal fibroblasts is a differentiation-like phenomenon. If this were correct, an ordered sequence of differential gene expression should be associated with the in vitro progression of cells from low passage to high passage (senescence). To define the pattern of expression of fibroblast differentiation-associated genes during this in vitro progression, we have determined the temporal pattern of expression of extracellular matrix (ECM) genes in Syrian hamster dermal fibroblasts as a function of passage level and percentage of proliferative life span in vitro. Steady-state mRNA levels were determined by Northern and dot blot analyses of total cellular RNA hybridized with cDNA probes specific for fibronectin, procollagen alpha 1III, and procollagen alpha 1I. Cells were analyzed at 24 hr postconfluence to minimize the presence of actively proliferating cells, and because maximal levels of fibronectin, alpha 1III, and alpha 1I mRNAs were observed 24 hr postconfluence. Unique, multiphasic patterns of expression of each of these ECM components were observed as the cells progressed from low passage to high passage. As the cells reached midhigh passage, fibronectin mRNA levels increased. This midpassage increase in fibronectin was followed by an increase in the level of alpha 1III mRNA as the cells reached the end of their in vitro proliferative life span, and then alpha 1I when the cells entered the postmitotic senescent phase, at which time the level of fibronectin mRNA also declined. A similar overlapping cascade pattern of up-regulation of these genes is seen during development and wound repair. This suggests that as cultured fibroblasts reach the end of their proliferative life span, they reinitiate a gene expression program used in tissue development and repair.