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.

Abstract

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...

Making Omelets without Breaking Eggs: E2F-Mediated Induction of Cardiomyoycte Cell Proliferation without Stimulation of Apoptosis

“…16,17 This technique allows a retrograde assessment of cell cycle history: quiescent G 0 /G 1 -cells (normal PI and normal Hoechst fluorescence) can be distinguished from cells in G 1 -phase, which have completed a cell cycle (normal PI but reduced Hoechst fluorescence). Interestingly, E2F1 and E2F2 but not E2F4 were able to induce mitosis of cardiomyocytes although all of them induced S-phase.…”

Divergent Siblings

“…It remained possible that cardiomyocytes arrested either in G1- 16 or in G2-phase and thereby mimicked a hypertrophic phenotype as described earlier for E2F1. 17 We therefore used the flowcytometric BrdU-Hoechst assay, which allows retrospective assessment of G1, S, and G2/M cell cycle phases of synchronous and asynchronous cell populations 11,12 (Figure 4). In the absence of BrdU, all cells within G1-phase (either growth arrested or after mitosis) showed the same intensity of fluorescence for PI and Hoechst 33258 ( Figure 4B).…”

“…11 The cell cycle profile was calculated using MultiCycle software (Phoenix Flow Systems). Proliferation of cardiomyocytes was determined using the BrdU-Hoechst method, 11 which allows enumeration of cells at different stages of three consecutive cell cycles including mitotic cell division. 12 All FACS analyses were restricted to MF20-positive cells to exclude contaminating fibroblasts.…”

Analysis of cell proliferation in Drosophila wing imaginal discs using mosaic clones

“…Smith and Martin model considered here has sound experimental confirmation (Robinson et al, 1976;Shields, 1978;Shields et al, 1978;Ronning and Seglen, 1982;Rabinovich, 1983) and served as a basis for theoretical models describing the curves of labeled mitoses (Cain and Chau, 1997). We have demonstrated that the model of Smith and Martin is less accurate in describing our experimental data than is our model ascribing the major role in the heterogeneity of proliferation rates to the dispersion of the rates of cell-cycle progression in a deterministic phase.…”