Altered Enzymes in Ageing Human Fibroblasts

Holliday, Robin; Tarrant, G. M.

doi:10.1038/238026a0

Cited by 406 publications

(74 citation statements)

References 22 publications

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“…Our results are consistent with the predictions made by a number of authors that alterations in the fidelity of DNA polymerase could be an important factor in cell aging (4,5,(23)(24)(25), at least for MRC-5 fibroblasts. In this system it was already shown by DNA fiber autoradiography that the rate of replicon elongation is significantly reduced in the senescent phase, suggesting that faulty DNA polymerase molecules are present (26).…”

Section: Fidelity Of the Dna Polymerasesupporting

confidence: 82%

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Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Linn¹,

Kairis²,

Holliday³

1976

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

113

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DNA polymerase (deoxynucleosidetriphosphate:DNA nucleotidyltransferase, EC 2.7.7.7 or DNA nucleotidyltransferase) activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared. The level of activity dropped with increasing passage. In addition, when the fidelity of polymerization was monitored with four synthetic templates under a variety of conditions, it was observed that the enzyme from late passage cells was more error-prone. The possible relation of these observations to "senescence" of the fibroblasts is discussed.Diploid human fibroblasts have a defined lifespan in culture that is somewhat related to the potential life expectancy of the individual from whom the cells were obtained (1, 2). This system has therefore been widely adopted as a model for studying the molecular bases of aging, physiological senescence, and cell death. Orgel has proposed that the fibroblast senescence results from an "error catastrophe" in macromolecular synthesis, which will inevitably lead to faulty and unregulated cellular metabolism (3, 4), and evidence has been obtained that defective enzyme molecules accumulate at increasingly higher levels in senescent fibroblasts, as well as in tissues from aging mice (5-10). Since the appearance of defective DNA polymerase could be an important component in any general breakdown of information transfer between macromolecules, we have compared the fidelity of DNA polymerase from cells in the early and late stages of cell culture during the replication of defined synthetic templates. MATERIALS AND METHODSGrowth of Cells. MRC-5 human male fetal lung fibroblasts (5) were grown in Eagle's basal medium containing 10% fetal calf serum, 100 units/ml of penicillin, 100 ,gg/ml of streptomycin, and 50,gg/ml of aureomycin. Cells were monitored to assure the absence of mycoplasmic contamination (11). For collection, cells that had not reached confluence, as judged by microscopic examination, were rinsed with 5 mM Tris-HCl (pH 7.5), 0.15 M NaCl, scraped off of the glass with a piece of soft plastic, then harvested and washed by centrifugation in the same buffer. The pellet could be stored at -70°or used immediately with no effect upon the subsequently fractionated activity.Assays for Enzyme Activity. Reaction mixtures (0.1 ml) contained 50 mM Tris-HCl (pH 8.5), 7.5 mM MgCl2, 0.1 M KCl, 0.5 mM dithiothreitol, 0.5 mg/ml of bovine serum albumin (Sigma, Fraction V), 0.13 ,gmol of "activated" salmon sperm DNA (12), and 5 nmol each of dATP, dGTP, dCTP, and dTTP. One of the triphosphates was labeled with 3H at 50-100 cpm/pmol. After 30 min at 37°, the reaction mixtures were chilled, then mixed with 0.2 ml of 0.1 M Na4P207 and 0.7 ml of 10% trichloroacetic acid. After at least 5 min, 3 ml of 1 M HCl, 0.1 M Na4P2O7 were added. The contents were filtered through a Whatman GF/C glass filter that had been soaked in 0.1 M Na4P207, then washed with the HCl-Na4P207. The filter was then successively washed 12 times with 3 ml of the HClNa4P2O7 and then with eth...

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Section: Fidelity Of the Dna Polymerasesupporting

confidence: 82%

“…Reaction mixtures (0.1 ml) contained 50 mM Tris-HCl (pH 8.5), 7.5 mM MgCl2, 0.1 M KCl, 0.5 mM dithiothreitol, 0.5 mg/ml of bovine serum albumin (Sigma, Fraction V), 0.13 ,gmol of "activated" salmon sperm DNA (12), and 5 nmol each of dATP, dGTP, dCTP, and dTTP. One of the triphosphates was labeled with 3H at 50-100 cpm/pmol.…”

Section: Activitymentioning

confidence: 99%

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Linn¹,

Kairis²,

Holliday³

1976

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

113

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“…However, the close linkage of the pathways of transcription, translation, replication, and repair of genetic information (Fig. 6) suggests that a more general error theory, which includes the possibility of error feedback at all levels of information transfer, is more plausible (Holliday and Tarrant 1972;Orgel 1973;Brash and Hart 1978;Kirkwood 1980). Evidence to support the hypothesis of a general accumulation of errors in human fibroblasts has been reported (Holliday and Tarrant 1972;Linnet al 1976), but contrary results have also been claimed (Evans 1977;Harley et al 1980).…”

Section: Mechanisms Of Cellular Ageingmentioning

confidence: 94%

“…6) suggests that a more general error theory, which includes the possibility of error feedback at all levels of information transfer, is more plausible (Holliday and Tarrant 1972;Orgel 1973;Brash and Hart 1978;Kirkwood 1980). Evidence to support the hypothesis of a general accumulation of errors in human fibroblasts has been reported (Holliday and Tarrant 1972;Linnet al 1976), but contrary results have also been claimed (Evans 1977;Harley et al 1980). From theoretical studies of the error theory (Kirkwood 1980;Rosenberger and Kirkwood 1981) it appears that the predictions are more complex than was originally recognised, and it may be some time before relevance of the theory to ageing is unambiguously resolved.…”

Section: Mechanisms Of Cellular Ageingmentioning

confidence: 99%

Cytogerontology since 1881: A reappraisal of August Weismann and a review of modern progress

1982

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Summary. Cytogerontology, the science of cellular ageing, originated in 1881 with the prediction by August Weismann that the somatic cells of higher animals have limited division potential. Weismann's prediction was derived by considering the role of natural selection in regulating the duration of an organism's life. For various reasons, Weismann's ideas on ageing fell into neglect following his death in 1914, and cytogerontology has only reappeared as a major research area following'the demonstration by Hayflick and Moorhead in the early 1960s that diploid human fibroblasts are restricted to a finite number of divisions in vitro.In this review we give a detailed account of Weismann's theory, and we reveal that his ideas were both more extensive in their scope and more pertinent to current research than is generally recognised. We also appraise the progress which has been made over the past hundred years in investigating the causes of ageing, with particular emphasis being given to (i) the evolution of ageing, and (ii) ageing at the cellular level. We critically assess the current state of knowledge in these areas and recommend a series of points as primary targets for future research.

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“…Proteins may age during the course of normal cellular functioning (4)(5)(6). Errors in protein synthesis at the transcriptional and translational level can generate abnormal proteins (7).…”

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confidence: 99%

Selective Degradation of Abnormal Proteins in Mammalian Tissue Culture Cells

Capecchi

Hughes

et al. 1974

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

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AB STRACTThe degradation rates of several missense mutants of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) in mouse L cells are compared to those of the wild-type enzyme. Although the rates of total protein breakdown in the mutant cell lines are identical to that of the parental L cell line, defective molecules of hypoxanthine-guanine phosphoribosyltransferase present in the mutant cell lines are degraded much faster than the wild-type enzyme. The level of defective phosphoribosyltransferase molecules present in the mutant cell lines is inversely proportional to the breakdown rate. This observation indicates that the major factor determining the concentrations of the defective phosphoribosyltransferases is their specific degradation rate. These results strongly support the hypothesis that abnormal proteins are selectively degraded in mammalian cells.

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Altered Enzymes in Ageing Human Fibroblasts

Cited by 406 publications

References 22 publications

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Cytogerontology since 1881: A reappraisal of August Weismann and a review of modern progress

Selective Degradation of Abnormal Proteins in Mammalian Tissue Culture Cells

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