Clonal Attenuation Research Articles

Clonal Attenuation of Somatic Cells in Aging Mammals: A Review of Supportive Evidence and Its Biomedical Significance

Clonal attenuation can be defined as the gradual depletion of the replicative potentials of individual clones of mammalian somatic cells. Publications from the author's laboratory and from other laboratories are reviewed that support the proposition that clonal attenuation is a continuous process throughout the life course and that it occurs in vivo in primates. The puzzling discordance between the mass culture results from the laboratories of the late Vincent Cristofalo (tissue from living subjects) and those from the Martin laboratory (tissue predominately from autopsy subjects) is discussed. Finally, the implications of clonal attenuation and replicative senescence, for such major age-related pathologic processes as neoplasia, atherosclerosis, benign prostatic hyperplasia, and osteoarthritis, are addressed; these and other disorders of aging can be characterized as a mixture of atrophy and hyperplasia, presumably related to a failure of homeostatic cell-cell interactions in aging tissues. For the case of neoplasia, an argument can be made that such failures precede what is increasingly regarded as the most critical step in carcinogenesis-the evolution of a mutator phenotype.

Clonal attenuation and cell senescence: The next 30 years

Clonal attenuation and cell senescence: The next 30 years

Independent evidence for a commitment model of clonal attenuation

We previously reported a model of clonal attenuation which assumes three classes of cells: small highly replicative cells; intermediate size cells of limited replicative potential and large non-dividing cells. Computer simulations carried out with the model lead to predictions of how the relative proportion of each cell type varies throughout the in vitro replicative life span of a mass population. These predictions appear to be broadly confirmed by independent data recently reported by another laboratory.

Proliferative potential of human fibroblasts: an inverse dependence on cell size.

Human foreskin fibroblast-like cells were separated on the basis of DNA content and cell size by fluorescence-activated cell sorting. Subpopulations of "large" or "small" cells with the same (G1) DNA content were clonally expanded and found to contain predominantly nondividing or highly proliferative cells, respectively. From the rate of clonal growth, we deduce that small cells divide faster than large cells. Intermediate-sized cells were found to yield primarily smaller ("attenuated") clones. The clonal data can be incorporated into a previously reported kinetic model of clonal attenuation. This version of the model postulates that small "stem" cells yield larger daughters which have only a limited proliferative potential. We also postulate that a progressive increase in cell size can account for the decreasing concentration of DNA polymerase alpha, which has been reported in older cultures.

Evidence for a universal process underlying clonal attenuation

It is shown by computer simulation that an established commitment model of clonal attenuation can account for clone size distribution data obtained from three vertebrate species — chick, hamster and human — from two evolutionarily divergent classes. The different in vitro replicative lifespans of each cell strain can be explained simply by differences in cell kinetics. These results suggest that the process of clonal attenuation is qualitatively similar in fibroblasts from all vertebrate species.

Clonal attenuation in chick embryo fibroblasts. Experimental data, a model and computer simulations.

When cells from mass cultures of chick embryo fibroblasts are grown at very low density, some cells yield large clones while others produce smaller clones, and some cells fail to divide at all. The distribution of clone sizes is related to the number of population doublings which the donor mass culture has undergone: the more doublings which have occurred, the smaller the average clone size. In this report we describe a model which analyses this phenomenon, referred to as 'clonal attenuation', in detail. The model is based on the concept that a cell with hypothetically unlimited replicative potential--i.e. a 'stem' cell--can become 'committed' to a programme of limited replicative potential. This event is assumed to be stochastic and to have a fixed probability per stem cell division. The parameters of the model are: Pc, the probability of commitment; N, the number of differentiative divisions; and Tc, the cell-cycle times. By computer simulation, it is shown that Pc increases roughly exponentially at each successive stem cell division. According to the model, when the daughter of a stem cell becomes committed, its progeny proceed through N obligatory divisions before becoming terminally differentiated (post-mitotic). The best-fit value of N was found to be seven. The simulations also reveal that the absolute number of stem cells in the total population increases for most of the lifespan of the culture. When Pc becomes much greater than 0.5, the number of stem cells declines rapidly to zero, and the culture nears senescence. Sensitivity analysis shows that Pc can assume only a limited range of values at each stem-cell division.

Abnormal fibroblast aging and DNA replication in the Werner syndrome.

Cell and DNA replicative potentials were studied in 10 strains of skin fibroblasts from unrelated patients with the Werner syndrome (WS) and in a progeric CRL 1277 strain. The lifespans of all WS strains and of CRL 1277 cells are greatly abbreviated in vitro, due to large fractions of non-cycling cells and the basic process of progressive clonal attenuation during Phase II. In some WS strains, the population-doubling rate per day and the cloning efficiency fluctuated concurrently in random fashion during the cellular aging process, indicating alternating successions of adaptively well and poorly growing clones, probably resulting from various chromosome translocations. No detectable defect in excision repair was found in WS or CRL 1277 cells. However, the rate of increase in the molecular weight of pulse-chased DNA involving overall rates of chain elongation and replicon fusion was retarded in WS and CRL 1277 cells. Also, pulse-labelled DNA in WS fibroblasts was less enriched in the nuclear matrix and was more slowly chased out than in normal cells. These results led us to postulate a misfiring or delayed initiation due to the sticky attachment of replicating DNA to the nuclear matrix in the replisomes of WS fibroblasts. A suggested model for abnormal DNA replication is presented and discussed to explain the loss of DNA and the chromosome abnormalities in WS cells. The abnormal DNA-synthetic profiles so derived appeared to be normalized in SV40-transformed PSV811 (WS) cells as were in gamma ray-transformed wild-type WI38CT-1 cells.

Depletion of reserve in the hemopoietic system: I. Self-replication by stromal cells related to chronologic age

Transplanted devascularized mouse femurs and spleens or spleens ligated in situ all undergo a period of massive necrosis followed by a period of cellular regeneration and reconstitution of tissue components. This renewal of stromal tissue is endogenous to the organ and proliferates inward from a thin rim of surviving cells. Components of the femoral or splenic stroma constitute a hemopoietic microenvironment whose functions include the lodgment, commitment to differentiation and support of the proliferation of hemopoietic stem cells and their differentiating descendents. By using measures of the ability to support hemopoiesis, and the histological appearance of the reconstituted hemopoietic organs, it was shown that the degree of regeneration of the stroma was inversely related to its chronological age. Thus, although age-related changes in stromal cells are not readily demonstrable in hemopoietic tissue during a normal lifespan, they are in process and can be made obvious under anoxic conditions causing cell death followed by an unusual demand for cell replication. Key cellular components of such aged tissues apparently have either lessened resistance to anoxia or a reduction in replicative potential or both. The second alternative interpretation suggests an analogy in vivo to mesenchymal-cell clonal attenuation observed in vitro.

Evidence of clonal attenuation, clonal succession, and clonal expansion in mass cultures of aging Werner's syndrome skin fibroblasts.

Skin fibroblast-like (FL) cells from patients with Werner's syndrome (adult progeria) demonstrate multiple, stable, structural chromosome rearrangements (variegated translocation mosaicism) which can be used to identify cytogenetically marked clones of cells within a mass culture. We have cytogenetically followed eight FL cell strains (from two patients) throughout their entire in vitro replicative lifespans, and we show a correlation between the expansion and attenuation of individual clones and the growth of the mass cultures. One strain, which was aged several times, demonstrated a generally reproducible pattern of clonal succession but, surprisingly, also demonstrated, in two parallel derivative cultures, the late emergence of two relatively rapidly growing clones that had not been observed in the parental culture. These observations suggest that clonal succession and clonal attenuation occur in mass cultures, as had been predicted on the basis of dilute-plating cloning experiments. Our results may have implications for models of in vitro cellular senescence. In addition, there are interesting parallels with the tissue hyperplasia associated with in vivo aging, and this observation is compatible with the suggestion that skin FL cells in vitro provide a model for hyperplasia in vivo.

A model of clonal attenuation.

Two formal models of clonal attenuation [Kirkwood, T. B. L. & Holliday, R. (1975) J. Theor. Biol. 53, 481-496; Shall, S. & Stein, W. D. (1979) J. Theor. Biol. 76, 219-231] are considered in the light of recent data on the changing distribution of replicative potential among individual cultured fibroblasts on subcloning. The experimental data [Smith, J. R., Pereira-Smith, O. & Good, P. I. (1977) Mech. Ageing Dev. 6, 283-286] are shown to contradict both models. A new model, compatible with the subcloning data, is proposed. This model involves a gradual increase in the probability of commitment during cell growth in culture and a small number (about seven) of divisions following commitment. The gradual increase in commitment probability is shown to be compatible with the gradual accumulation of a gene product subject to autogenous regulation.

Evidence for clonal attenuation of growth potential in hela cells

The growth of primary clones and serial subclones of HeLa cells and of diploid human fibroblast-like cells were compared both in the presence and absence of feeder layers; the latter had no significant effects upon the results. Clones and subclones of both cell types displayed great heterogeneity in growth rates, typically with a bimodality of growth distributions. Serial passages of clones selected on the basis of superior rates of proliferation showed attentuation of growth potentials; the extent of such attentuations was much less in the case of HeLa cells, suggesting at least one possible basis for the differences in long-term growth potential between these two classes of cell lines.