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Is cellular aging a stochastic process?
Mechanisms of Ageing and Development, 13 (1980) 393-395
393
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
IS C E L L U L A R A G I N G A S T O C H A S T I C PROCESS ?
ROBERT J. SHMOOKLER REIS and SAMUEL GOLDSTEIN Departments of Medicine and Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5 (Canada)
CALVIN B. HARLEY Division of Biological Sciences, University of Sussex, Sussex BN1 9QG (Great Britain)
(Received June 4, 1980)
The limited lifespan in vitro of human diploid fibroblasts may depend upon a deterministic mechanism common to all such cells, or it may arise in a population as a consequenc~ of stochastic events differentially affecting individual fibroblasts. Because random influences are inescapable, it is extremely difficult to distinguish between a deterministic process modified by stochastic elements, and a process which is itself inherently stochastic. Smith and Whitney [1 ] have recently presented elegant data attesting to the heterogeneity in proliferative potential within clones of human diploid fibroblasts, to confirm and extend earlier work of their own [2] and the Abshers [3,4], from which they conclude that a stochastic mechanism is the prime determinant of the limited replicative lifespan. This interpretation emphasizes only one aspect of the data, which in total best support a deterministic model containing stochastic elements. Smith and Whitney concede that their results cannot discriminate between these models of cellular aging, but would only exclude "a precise counting mechanism" [5] and the commitment theory of aging [6-8] in their present forms. However, they might also have pointed out that a purely stochastic model would not fit the data either, since it would predict no effect of previous clonal history on the maximum number of population doublings remaining within derived subclones. In fact, Smith and Whitney's pdrnary observation (Fig. 1 of ref. 1) indicates a rigid upper limit of 72-76 total population doublings regardless of the passage level at which subclones were taken. This strongly implies that some "counting mechanism" must exist and hence invokes a broader range of interpretations. That stochastic processes are also involved is evidenced by the variability and bimodality of division potential they found arising de novo in clones and subclones, with an increasing fraction of "short-lived" cells at late passage. Four possible sources of such heterogeneous distributions may be considered as examples. (i) Unequal partition o f cytoplasmic components. If some essential organdies, such as mitochondria, replicate more slowly than the cells themselves, then the number of these organelles per cell would decline with passage number and hence the probability would increase of a daughter cell receiving a subviable allotment. (if) Imperfect duplication o f a nuclear division counter. We have recently reported that the cellular content of certain reiterated DNA sequences declines with passage in vitro [9, 10]. If cells require a minimal complement of such re-
394 peated sequences for viability, then unequal recombination within these arrays would introduce new heterogeneity in division potential. (iii) lhfferential response to experimental manipulations. Cells at different phases of the cell cycle may react differently to such procedures as trypsinization, replating and re feeding, thereby generating the observed bimodal distributions. (iv) Distribution of generations in the parent clone. High cell density at the centre of the parent clone contact-inhibits division so that central cells accumulate fewer replications than peripheral cells. We have shown [11] that circular outgrowths which are densely packed at the center have only a thin outer rim of dividing cells, thus generating a radial distribution of population doubling levels. Since clones vary greatly in their interior cell densities, it is imperative to know the size and degree of confluence of each clone at the time of subculture. Smith and Whitney's assertion that they found little contact inhibition of DNA synthesis ([3H] thymidine incorporation) in the center of the original parent clone is less than reassuring in the absence of details explaining how the same clone was both harvested and autoradiographed. Under any of these four hypotheses, the data of Smith and Whitney are compatible with a deterministic mechanism for cellular aging, with stochastic events involved in the first two examples. In these two instances, the probability of transition to the "shortlived" state would depend on the age of the culture, rather than remaining constant as in a purely stochastic model. The stochastic elements of hypotheses (i) and (ii) would also account for disparities in sister colony sizes observed by Smith and Whitney after two weeks' growth. These short-term assays are in any case considerably less compelling than measurements of total proliferative capacity because variations of 2- to 5-fold in interdivision time are not uncommon among the cells of a single clonal lineage [3,4]. In conclusion, the data of Smith and Whitney demonstrate a pronounced stochastic component in the senescent loss of fibroblast replicative capacity, while confirming that a strict proliferative limit is determined through a counting process. At this time it is not clear whether the stochastic elements are internal and associated with the counter as in (i) and (ii), or externally induced and independent of the counter as in (iii) and (iv). The interest generated by this important paper should encourage testable hypotheses on the nature of the cellular counting mechanism.
REFERENCES 1 J. R. Smith and R. G. Whitney, Intraclonal variation in prOliferative potential of human diploid fibroblasts. Science, 207 (1980) 82-84. 2 J. R. Smith and L. Hayflick, Variation in the lffespan of clones derived from human diploid cell strains. J. Cell BioL, 62 (1974)48-53. 3 P. M. Absher, R. G. Abshet and W. K. Barnes, Geneologies of clones of diploid fibroblasts. Exp. Cell Res., 88 (1974) 95-104. 4 P. M. Abshet and R. G. Absher, Clonal variation and aging of diploid fibroblasts. Exp. CellRes., 103 (1976) 247-255. 5 P. I. Good and J. R. Smith, Age distribution of human diploid fibroblasts. A stochastic model for in vitro aging. Biophys. J., 14 (1974) 811-823.
395 6 T. B. L. Kirkwood and R. Holliday, Commitment to senescence: a model for the finite and infinite growth of diploid and transformed human fibroblasts in culture. J. Theor. BioL, 53 (1975) 481-496. 7 R. Holliday, L. I. Huschtscha, G. M. Tarrant and T. B. L. Kirkwood, Testing the commitment theory of cellular aging. Science, 198 (1977) 366-372. 8 C. B. Harley and S. Goldstein, Retesting the commitment theory of cellular aging. Science, 207 (1980) 191-193. 9 R. J. Shmookler Reis and S. Goldstein, Genomic content of reiterated DNA: effect of aging and mutants. Fed. Proc., 38 (1979) 535. 10 R.J. Shmookler Reis and S. Goldstein, Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts. Cell, (1980) in press. 11 C. B. Harley and S. Goldstein, Cultured human fibroblasts: distribution of cell generations and a critical limit. J. Cell Physiol., 9 7 (1978) 509-516.
393
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
IS C E L L U L A R A G I N G A S T O C H A S T I C PROCESS ?
ROBERT J. SHMOOKLER REIS and SAMUEL GOLDSTEIN Departments of Medicine and Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5 (Canada)
CALVIN B. HARLEY Division of Biological Sciences, University of Sussex, Sussex BN1 9QG (Great Britain)
(Received June 4, 1980)
The limited lifespan in vitro of human diploid fibroblasts may depend upon a deterministic mechanism common to all such cells, or it may arise in a population as a consequenc~ of stochastic events differentially affecting individual fibroblasts. Because random influences are inescapable, it is extremely difficult to distinguish between a deterministic process modified by stochastic elements, and a process which is itself inherently stochastic. Smith and Whitney [1 ] have recently presented elegant data attesting to the heterogeneity in proliferative potential within clones of human diploid fibroblasts, to confirm and extend earlier work of their own [2] and the Abshers [3,4], from which they conclude that a stochastic mechanism is the prime determinant of the limited replicative lifespan. This interpretation emphasizes only one aspect of the data, which in total best support a deterministic model containing stochastic elements. Smith and Whitney concede that their results cannot discriminate between these models of cellular aging, but would only exclude "a precise counting mechanism" [5] and the commitment theory of aging [6-8] in their present forms. However, they might also have pointed out that a purely stochastic model would not fit the data either, since it would predict no effect of previous clonal history on the maximum number of population doublings remaining within derived subclones. In fact, Smith and Whitney's pdrnary observation (Fig. 1 of ref. 1) indicates a rigid upper limit of 72-76 total population doublings regardless of the passage level at which subclones were taken. This strongly implies that some "counting mechanism" must exist and hence invokes a broader range of interpretations. That stochastic processes are also involved is evidenced by the variability and bimodality of division potential they found arising de novo in clones and subclones, with an increasing fraction of "short-lived" cells at late passage. Four possible sources of such heterogeneous distributions may be considered as examples. (i) Unequal partition o f cytoplasmic components. If some essential organdies, such as mitochondria, replicate more slowly than the cells themselves, then the number of these organelles per cell would decline with passage number and hence the probability would increase of a daughter cell receiving a subviable allotment. (if) Imperfect duplication o f a nuclear division counter. We have recently reported that the cellular content of certain reiterated DNA sequences declines with passage in vitro [9, 10]. If cells require a minimal complement of such re-
394 peated sequences for viability, then unequal recombination within these arrays would introduce new heterogeneity in division potential. (iii) lhfferential response to experimental manipulations. Cells at different phases of the cell cycle may react differently to such procedures as trypsinization, replating and re feeding, thereby generating the observed bimodal distributions. (iv) Distribution of generations in the parent clone. High cell density at the centre of the parent clone contact-inhibits division so that central cells accumulate fewer replications than peripheral cells. We have shown [11] that circular outgrowths which are densely packed at the center have only a thin outer rim of dividing cells, thus generating a radial distribution of population doubling levels. Since clones vary greatly in their interior cell densities, it is imperative to know the size and degree of confluence of each clone at the time of subculture. Smith and Whitney's assertion that they found little contact inhibition of DNA synthesis ([3H] thymidine incorporation) in the center of the original parent clone is less than reassuring in the absence of details explaining how the same clone was both harvested and autoradiographed. Under any of these four hypotheses, the data of Smith and Whitney are compatible with a deterministic mechanism for cellular aging, with stochastic events involved in the first two examples. In these two instances, the probability of transition to the "shortlived" state would depend on the age of the culture, rather than remaining constant as in a purely stochastic model. The stochastic elements of hypotheses (i) and (ii) would also account for disparities in sister colony sizes observed by Smith and Whitney after two weeks' growth. These short-term assays are in any case considerably less compelling than measurements of total proliferative capacity because variations of 2- to 5-fold in interdivision time are not uncommon among the cells of a single clonal lineage [3,4]. In conclusion, the data of Smith and Whitney demonstrate a pronounced stochastic component in the senescent loss of fibroblast replicative capacity, while confirming that a strict proliferative limit is determined through a counting process. At this time it is not clear whether the stochastic elements are internal and associated with the counter as in (i) and (ii), or externally induced and independent of the counter as in (iii) and (iv). The interest generated by this important paper should encourage testable hypotheses on the nature of the cellular counting mechanism.
REFERENCES 1 J. R. Smith and R. G. Whitney, Intraclonal variation in prOliferative potential of human diploid fibroblasts. Science, 207 (1980) 82-84. 2 J. R. Smith and L. Hayflick, Variation in the lffespan of clones derived from human diploid cell strains. J. Cell BioL, 62 (1974)48-53. 3 P. M. Absher, R. G. Abshet and W. K. Barnes, Geneologies of clones of diploid fibroblasts. Exp. Cell Res., 88 (1974) 95-104. 4 P. M. Abshet and R. G. Absher, Clonal variation and aging of diploid fibroblasts. Exp. CellRes., 103 (1976) 247-255. 5 P. I. Good and J. R. Smith, Age distribution of human diploid fibroblasts. A stochastic model for in vitro aging. Biophys. J., 14 (1974) 811-823.
395 6 T. B. L. Kirkwood and R. Holliday, Commitment to senescence: a model for the finite and infinite growth of diploid and transformed human fibroblasts in culture. J. Theor. BioL, 53 (1975) 481-496. 7 R. Holliday, L. I. Huschtscha, G. M. Tarrant and T. B. L. Kirkwood, Testing the commitment theory of cellular aging. Science, 198 (1977) 366-372. 8 C. B. Harley and S. Goldstein, Retesting the commitment theory of cellular aging. Science, 207 (1980) 191-193. 9 R. J. Shmookler Reis and S. Goldstein, Genomic content of reiterated DNA: effect of aging and mutants. Fed. Proc., 38 (1979) 535. 10 R.J. Shmookler Reis and S. Goldstein, Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts. Cell, (1980) in press. 11 C. B. Harley and S. Goldstein, Cultured human fibroblasts: distribution of cell generations and a critical limit. J. Cell Physiol., 9 7 (1978) 509-516.