Drift of Cell Function Through Serial Divisions

The classical experiments of Meselson and Stahl showing that the synthesis of DNA is semiconservative led to the belief that at each division each daughter cell receives the same kind of genetic information and hence that both sister cells are identical. However, when one follows a cell population during serial proliferation in vitro different parameters show that the cell functions evolve in many respects.

Human fibroblasts proliferating in vitro were extensively analyzed in this respect since they are used for the study of aging at the cellular level. During serial replications they are known to go through three phases (Hayflick and Moorhead 1961): phase I is suggested from the initial rise in the saturation densities and the morphological observations showing the appearance of a homogeneous population, phase II can be defined as the period where the fraction of non-dividing cells is relatively constant, phase

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Progress in Molecular and Subcellular Biology Alvaro Macieira-Coelho (Ed.) Asymmetric Cell Division © Springer-Verlag Berlin Heidelberg 2007

III is characterized by the slight increase in the number of cells which do not divide during a 24-h period and by the prolongation of the population doubling time. The existence of a final phase IV was later characterized (Macieira-Coelho and Taboury 1982) corresponding to the last three or four doublings with a rapid fall of the maximal cell density and of the fraction of cells synthesizing DNA during a 24-h period; the cells enter a very slow dividing state and eventually become post-mitotic (Fig. 1). Several metabolic events occur during the last three to four doublings, which support the idea of a final phase with distinct characteristics (Macieira-Coelho 1988).

It is obvious from the evolution of the kinetics of proliferation that something must take place during cell division, which progressively changes the cells and finally creates the conditions for the final abrupt steps.

Morphological observations also lead to the conclusion that there is a drift through cell division. There is an increase in cell, nuclear and nucleolar areas (Mitsui and Schneider 1976; Bemiller and Miller 1979); moreover, three types of fibroblasts can be distinguished, I, II and III, with an increasing volume with a shift to the larger volume during the cell population life span (Steinhardt 1985). At the end there is a sudden shift to a large type IV cell (Fig. 2) (Macieira-Coelho 1983) with a significant reorganization of the cytoskeleton and decreased motility (Raes et al. 1983).

Several enzymatic changes have been reported during serial proliferation of human fibroblasts. There is a decline in the ratio between the activities of two enzymes of the purine salvage pathway, hypoxanthine-guanine trans-ferase and adenine-phosphoribosyltransferase (Paz et al. 1981), a decline in ribonucleotide reductase (Dick and Wright 1985), and a progressive increase in the levels of the second enzyme of the pathway for de novo purine biosynthesis, glycinamide ribonucleotide synthetase (Hards and Patterson 1986).

Fig. 1. Maximal cell densities recorded before each cell subcultivation during the entire life span of a human embryonic lung fibroblast line. Each dot corresponds to a cell count from a different culture vessel. The population doublings corresponding to Phases I, II, III and IV are indicated
Fig. 2. A,B Morphology of the same cells used for the experiment illustrated in Fig. 3, in: A phase II; B phase IV

Another interesting enzymatic alteration is the progressive appearance of a new 3':5'-cyclic AMP-independent histone kinase, raising the possibility that new genes become progressively activated (Kahn et al. 1982).

Changes in glycolysis seem related to the kinetics of proliferation. Between population doubling 20 and 40 glucose uptake and lactate production decrease, thereafter both glucose uptake and lactate production increase (Bittles and Harper 1984); the critical change in the biphasic curve of glucose uptake/lactate production corresponds to the transition from phase II to phase III. The same investigators also observed an increase in the specific activity of piruvate kinase, a regulatory enzyme of the glycolytic pathway.

The relative proportion of cyclin decreases progressively (Celis and Bravo 1984), and during the second half of cell population life span the responsiveness to EGF decreases corresponding to the transition between phases II and III (Kaji and Matsuo 1983).

Progressive modifications of the cell surface became apparent with the reported decline in the negative surface charge and in the activity of a pH 7.8 protease (Bosmann et al. 1976), the increase of albumin transport (Berumen and Macieira-Coelho 1977), and the decrease of the binding, uptake and degradation of low density lipoproteins due to a reduction in the number of receptor sites (Lee et al. 1982).

Several data point to genetic modifications. There is an increased heterogeneity in DNA repeat lengths (Dell'Orco et al. 1986) and decreased hybridization of the probes for the a-globin and P-actin genes (Icard-Liepkalns et al. 1986). The mutation frequencies for diphtheria toxin and thioguanine resistance increase linearly during the first 2/3 of the life span of a human fibroblast population (Gupta 1980). The expression of the EPC-1 (early population doubling cDNA-1) gene declines gradually during the fibroblast proliferation life span (Pignolo et al. 1993). Furthermore, the activity of p53, a positive transactivator of p21 gene expression, was found to increase in a stepwise fashion through the different phases (Bond et al. 1996).

Other modifications occurring during the proliferation life span of human fibroblasts have been reviewed elsewhere (Macieira-Coelho 1988).

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