Growth-factor-inducible gene expression in senescent human fibroblasts

Seshadri, Tara; Campisi, Judith

doi:10.1016/0531-5565(89)90057-0

Cited by 21 publications

(6 citation statements)

References 21 publications

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“…In addition, the fibronectin produced and secreted into the medium by high passage cells appears to be structurally and functionally different (Chandrasekhar et al, 1983;Sorrentino and Millis, 1984). On an RNA level, a 3-4-fold increase in fibronectin mRNA in high passage human fibroblasts has been observed (Kleinsek, 1989;Seshadri and Campisi, 1989;Kumazaki et al, 1991), observations that agree well with our data on fibronectin mRNA levels in Syrian hamster fibroblasts at the end of their proliferative life span in vitro. However, following completion of their proliferative life span, normal fibroblasts remain viable for extended periods of time (viable life span in vitro as opposed to proliferative life span in vitro), and during this postmitotic stage, our data indicate that the level of fibronectin mRNA drops.…”

Section: Discussionsupporting

confidence: 89%

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

Choi

Olsen

Cook

et al. 1992

Journal Cellular Physiology

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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.

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Section: Discussionsupporting

confidence: 89%

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

Choi

Olsen

Cook

et al. 1992

Journal Cellular Physiology

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“…Serum-stimulated senescent and quiescent human fibroblasts express many of the same genes regulating the cell cycle such as c-myc, c-jun and c-Ha-ras (Rittling et al, 1986;Seshadri and Campisi, 1989). However, senescent cells have lost the ability to express c-fos and cdc2-cyclin A kinase activity and to phosphorylate Rb protein (Seshadri and Campisi, 1990;Stein et al, 1990Stein et al, , 1991.…”

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confidence: 99%

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Fushimi¹,

Iijima²,

Gao³

et al. 1997

Int. J. Cancer

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In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273 Arg-His ) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In vitro cell transformation is a valuable model for studying the mechanisms of multistep carcinogenesis. The human cell system is useful for studying the various stages of in vitro transformation, because events in the transformation process, from normal to neoplastic phenotypes, are clearly defined by the successive stages of aging, immortalization, and neoplastic transformation (Namba et al., 1988). For normal human cells to undergo neoplastic transformation, they must first be immortalized. However, normal human cells have a finite capacity to replicate and eventually enter a state of irreversible growth arrest (for review, Vojta and Barrett, 1995). This feature has been termed cellular senescence or replicative senescence. Cellular senescence is thought to be a genetically programmed process rather than the result of a random accumulation of damage. In normal human cells, in contrast to rodent cells, cellular senescence is particularly stringent and spontaneous immortalization is an extremely rare event.The immortalization process of human cells is probably related to deregulation of cellular senescence. Serum-stimulated senescent and quiescent human fibroblasts express many of the same genes regulating the cell cycle such as c-myc, c-jun and c-Ha-ras (Rittling et al., 1986;Seshadri and Campisi, 1989). However, senescent cells have lost the ability to express c-fos and cdc2-cyclin A kinase activity and to phosphorylate Rb protein (Seshadri and Campisi, 1990;Stein et al., 1990Stein et al., , 1991. p53 regulates the cell-cycle machinery partially through the expression of p21, because p21 inhibits the action of multiple cyclin-dependent kinase (cdk) complexes and arrests growth (Noda et al., 1994).We obtained 3 human cell lines immortalized with either 4-nitroquinoline 1-oxide (4-NQO) or 60 Co gamma-rays (Namba et al., 1988...

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“…Senescent cells have intact DNA replication machinery and transduce most of signals during mitogenic stimulation (3,4,21,22). The loss of proliferation correlates with selective depression of certain mitogenic events such as inability to phosphorylate the retinoblastoma gene product (23) and failure to express c-fos, cdc2, or cyclins (24)(25)(26).…”

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confidence: 99%

Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.

Chen

Ames

1994

Proc. Natl. Acad. Sci. U.S.A.

567

312

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Human diploid fibroblast cells lose replicative potential after a certain number of population doublings. We use this experimental system to investigate the role of oxidative damage in cellular aging. Treating cells with H202 at <300 JAM did not affect the viability of the majority of cells when judged by morphology, trypan blue exclusion, and protein synthesis. However, the treatment caused a dosedependent inhibition of DNA synthesis. After a 2-hr treatment with 200 FM H202, the cells failed to respond to a stimulus of serum, platelet-derived growth factor, basic fibroblast growth factor, or epidermal growth factor by synthesizing DNA, and the loss of response could not be recovered by 4 days. Subcultivation showed that, as in senescent cells, division of the treated cells was inhibited. The life-time cumulative growth curve showed that the loss of replication due to H202 treatment was cumulative and irreversible. The H202 treatment decrased the number of the population doublings in the rest of the life span by 35.3 ± 10.3%. Enzymatic assays indicated that, like the cells in their senescent state, the treated cells were less able to activate ornithine decarboxylase and thymidine kinase. Furthermore, subcultivation after the H202 treatment showed that the cells developed the morphology of senescent cells. In conclusion, sublethal treatment of H202 "stuned" F65 cells and caused the cells to enter a state resembling senescence.One of the manifestations of aging is the accumulation of damage at both cellular and organismal levels. This damage is initiated by endogenous and exogenous toxicants. Oxidants appear to be the major source of damage and are produced during normal aerobic metabolism and inflammatory reactions (1, 2). High-dose exposure to oxidants may cause cytotoxicity and neoplastic transformation. We investigate here the role of oxidants in aging with human diploid fibroblast (HDF) cells as an experimental system. HDF cells lose replicative capacity in culture after a specific number ofpopulation doublings (PDs) and have been widely used as an in vitro model of aging (3)(4)(5)(6). Accompanying the loss of replicative potential, senescence is marked by several changes, including decreased activity of cellcycle-related enzymes (9, 10) and decreased efficiency of protein synthesis and degradation (11)(12)(13)(14). Nuclear and chromosomal aberrations are present at a higher frequency (15,16). The content of 5-methylcytosine in DNA and the length of telomeres are decreased (17)(18)(19) Amersham or 20 Ci/mmol from NEN) at 0.5 gCi per well or a 14C-labeled mixture of amino acids (54.4 mCi/mol atom carbon from NEN) at 0.2 uCi per well. The labeling medium was washed out and the cells were fixed in the well with 10% (wt/vol) ice-cold trichloroacetic acid. After two additional washes with 10o trichloroacetic acid, one wash with 70% ethanol, and one wash with phosphate-buffered saline (PBS), the precipitates were dissolved in 0.1 M KOH. Trichloroacetic acid-insoluble radioactivity was determined by liquid s...

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Growth-factor-inducible gene expression in senescent human fibroblasts

Cited by 21 publications

References 21 publications

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

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

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.

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