A re‐evaluation of the changes in proliferation in human fibroblasts during ageing <i>in vitro</i>

Macieira‐Coelho, A.; Taboury, F.

doi:10.1111/j.1365-2184.1982.tb01039.x

Cited by 26 publications

(22 citation statements)

References 54 publications

(16 reference statements)

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“…In several cell types, immunochemistry has allowed a qualitative and interpretative evaluation of the cytoskeleton complex during in vitro aging (7,16).…”

Section: Discussionmentioning

confidence: 99%

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

Ronot¹,

Gaillard‐Froger²,

Hainque³

et al. 1988

Cytometry

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In vitro senescence of chondrocytes, characterized by a decline in the proliferation rate during late passages, resulted from a rapid growth rate in early subcultures to a complete loss of division after seven to nine passages. One senescent-associated phenotypic change was the apparent increase in the density of cytoplasmic cytoskeletal proteins. We examined the relationship between tubulin content and growth (measured by DNA and total protein contents and cell volume), using flow cytometry, in the assessment of cytoskeleton analysis during in vitro aging. In contrast with previous microscopic observations of tubulin organization, flow eytometry revealed a tubulin content that was modulated as a function of protein content and/or cell volume.Key terms: Chondrocyte, in vitro aging, cytoskeleton, DNA, protein content, cell size A goal of flow cytometry in cell biology and cell culture is to explain the behavior of the cell in terms of the functional properties of its individual components. Some cellular components that have been studied successfully by flow cytometry include nucleic acids, proteins, and mitochondria, for which quantitation using fluorescent probes and/or monoclonal antibodies have been directly applicable (13,17,18). One major challenge for flow cytometry is to describe satisfactorily in vitro aging by analyzing cell morphology and structure and the related problems of cell growth, metabolism, and differentiation; all of these parameters are thought to depend on an intractably large number of specific interactions between cell compartments.During in vitro aging, physiological cell parameters are modified such that there are: a) a gradual decline in proliferative capacity, a lengthened interphase interval, and a reduction of the number of cells actively synthesizing DNA; b) increased mean cell size and mass; c) a reduction in saturation density observed in monolayers; d) an increased number of binucleate/multinucleate cells or cells with lobed nuclei; and e) an increased number of lysosomes per cell (19).These senescence-associated phenotypic changes have been correlated with a remarkable increase in the density and organization of the three cytoskeletal components, including actin-containing microfilaments, intermediate filaments, and microtubules in the enlarged cells (6, 22, 23).The cytoskeleton is an extensive, highly ordered structure with important functional roles in the determination of cell shape, cell and chromosome movements, disposition and movement of organelles, and cell division (2,4,5). Much of the current interest in the cytoskeleton has been triggered by technical developments, including high-voltage electron microscopy and deepetching and immunofluorescence microscopy (1). The controversial and confusing information regarding its structure and functions has stimulated research interest in the cytoskeleton, and details of its structure, functions, and organization have been related to in vitro senescence in several model systems.This report examines the senescence-associated phen...

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“…In several cell types, immunochemistry has allowed a qualitative and interpretative evaluation of the cytoskeleton complex during in vitro aging (7,16).…”

Section: Discussionmentioning

confidence: 99%

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

Ronot¹,

Gaillard‐Froger²,

Hainque³

et al. 1988

Cytometry

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“…Since, as in most other studies, no attempts were made here to first remove residual cycling cells, we cannot formally distinguish true reentry of arrested cells into the cycle from the alternative of a contraction of G 1 phase duration in a very slowly cycling compartment (39). However, the vast majority of cells which synthesized DNA as a result of PAb1801 and DO-1 (or SV oriϪ ) injection were derived from cells which, prior to injection, displayed the conventional criteria of terminal senescence observed in cultures from which cycling cells have been selectively depleted (12, 24, 52), i.e., large, flattened shape and absence of labelling by BrdU.…”

Section: Discussionmentioning

confidence: 99%

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

Gire

Wynford-Thomas

1998

Molecular and Cellular Biology

152

115

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In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four-to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21 sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.Normal human fibroblasts are capable of only a finite number of cell divisions even under optimum culture conditions, after which they enter a state of viable but permanent growth arrest (29). This phenomenon of cellular senescence has been observed in many other normal cell types (15,23,54) and represents a natural obstacle to clonal expansion (14, 58) which is thought to be an important restriction on the progression of many (although probably not all) human cancers (19).One currently popular model for senescence proposes that an intrinsic cell division clock, possibly based on telomere erosion (4), triggers one or more signal pathways which inhibit key components of the cell cycle regulatory machinery. Two candidate inhibitors whose levels increase with proliferative life span are p16 (2, 25) and p21 sdi1/WAF1 (46); these proteins inhibit the cyclin-dependent kinases CDK4/6 and CDK2, which are required for passage through and exit from the G 1 phase of the cell cycle (51). A related protein, p24 (40), may represent a third inhibitor. One consequence (55) of this inhibition is the failure of senescent cells to phosphorylate a major downstream target of these enzymes-the product of the retinoblastoma (Rb) sensitivity gene, pRb-which in its unphosphorylated form sequesters transcription factors needed for G 1 -S progression (61). Not surprisingly, therefore, escape from senescence is often associated with deregulation of the Rb pathway, either directly throu...

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“…Studies by time-lapse cinematography (4,5) have been inconclusive and have been interpreted as both consistent (6,7) and inconsistent (8)(9)(10) with the Smith and Martin model. Studies based upon clone size analysis indicate an increased number of nondividing cells with advancing population doubling level (PDL) (11,12); however, this result has been criticized as possibly related to cloning conditions not present in mass culture (13). Autoradiographic analysis of HDFC cultures has yielded results that have been interpreted as showing no noncycling population (14), noncycling cells arising only in the last 10 population doubling levels (15), or proportions of noncycling cells progressively increasing with age (16).…”

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confidence: 99%

Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry.

Rabinovitch¹

1983

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

110

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Growth of human diploid fibroblasts in the presence of 5-bromodeoxyuridine, followed by flow cytometric analysis of DNA-specific fluorescence with Hoechst 33258 dye, allows quantitation of the proportion of cells that have not cycled, as well as those in G1 and G2 of two subsequent cell cycles. This technique allows rapid and accurate quantitation of the growth fraction and G1/S transition rate of these cells. The cell cycle kinetics of human diploid fibroblasts at all population doubling levels reveal two components: cycling cells showing a probabilistic rate of G1/S transition, and a variable proportion of noncycling cells. Both the transition probability (rate of exit from G1) and the noncycling proportion of cells change systematically as a function of serum concentration and as a function of population doubling level. The data suggest the existence of an underlying heterogeneity in the population of human diploid fibroblasts with respect to the capacity to divide in the presence of a given concentration of mitogen. Models of cell cycle kinetics must be modified to include regulation of growth by changes in the fraction of cycling cells, as well as by changes in the rate of emit from G1.Heterogeneity in interdivisional times is a common and welldocumented feature of the proliferative behavior of cultured mammalian cells, and whereas S, G2, and M phases are of relatively constant duration, the G1 phase length is highly variable. Of the proposals advanced to explain this variability, one that has been shown to closely fit most experimental data is the transition probability model of Smith and Martin (1). In this model cells remain in a subset of G1 (the "A phase") for a variable length of time, leaving this state with a constant probability of transition per unit time (P), to then complete G1 (the "B phase"). (17), who compensated for cell proliferation by counting cell numbers at the start and end of the experiment. They concluded that noncycling cells are indeed present and progressively increase with culture age. With the objective of performing a more detailed study of these cellular kinetics, we have adapted a technique of flow cytometric assay of growth kinetics based upon Hoechst dye staining of cells grown in 5-bromodeoxyuridine (BrdUrd) (18)(19)(20). The increased ease and accuracy of this technique compared to conventional methods allows an analysis demonstrating that changes in the proliferative rate of HDFC cultures are a result of alterations in both the fraction of noncycling cells and the transition probability of the remaining cycling cells. MATERIALS AND METHODSCell Strains and Culture. HDFC strain 79-81 was explanted from a skin biopsy sample of a normal 27-year-old male, and strain 78-18 was derived from a skin biopsy sample of a 20-week gestational age, karyotypically normal female abortus. Cells were grown as described (21) 2951The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accord...

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A re‐evaluation of the changes in proliferation in human fibroblasts during ageing in vitro

Cited by 26 publications

References 54 publications

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry.

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