Proc. NatL Acad. Sci. USA Vol. 80, pp. 2951-2955, May 1983 Cell Biology Regulation of human fibroblast growth rate by both noncycling cell fraction and transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry (cell cycle Idnetics/growth control/human diploid cells/life-span in vitro) PETER S. RABINOVITCH Department of Pathology, SM-30, University of Washington, Seattle, Washington 98195 Communicated by Earl P. benditt, February 18, 1983 ABSTRACT Growth of human diploid fibroblasts in the pres- toradiographic analysis of HDFC cultures has yielded results ence of 5-bromodeoxyuridine, followed by flow cytometric anal- that have been interpreted as showing no noncycling popula- ysis of DNA-specific fluorescence with Hoechst 33258 dye, allows tion (14), noncycling cells arising only in the last 10 population quantitation of the proportion of cells that have not cycled, as well doubling levels (15), or proportions of noncycling cells pro- as those in G1 and G2 of two subsequent cell cycles. This technique gressively increasing with age (16). The inconsistencies are pre- allows rapid and accurate quantitation of the growth fraction and sumably related to problems with method (13) and may be ex- G1/S transition rate of these cells. The cell cycle kinetics of hu- plained in part by the use of [3H]thymine pulse periods that are man diploid fibroblasts at all population doubling levels reveal two too short to label slowly dividing cells, or the proliferation of components: cycling cells showing a probabilistic rate of G1/S labeled cells the interval. More this transition, and a variable proportion of noncycling cells. Both the during labeling recently, transition probability (rate of exit from G1) and the noncycling problem was addressed by Matsumura et al (17), who com- proportion of cells change systematically as a function of serum pensated for cell proliferation by counting cell numbers at the concentration and as a function of population doubling level. The start and end of the experiment. They concluded that noncy- data suggest the existence of an underlying heterogeneity in the cling cells are indeed present and progressively increase with population of human diploid fibroblasts with respect to the ca- culture age. With the objective of performing a more detailed pacity to divide in the presence of a given concentration of mi- study of these cellular kinetics, we have adapted a technique togen. Models of cell cycle kinetics must be modified to include offlow cytometric assay of growth kinetics based upon Hoechst regulation of growth by changes in the fraction of cycling cells, as dye staining of cells grown in 5-bromodeoxyuridine (BrdUrd) well as by changes in the rate of emit from G1. (18-20). The increased ease and accuracy of this technique compared to conventional methods allows an analysis demon- Heterogeneity in interdivisional times is a common and well- strating that changes in the proliferative rate of HDFC cultures documented feature of the proliferative behavior of cultured are a result of alterations in both the fraction of noncycling cells mammalian cells, and whereas S, G2, and M phases are of rel- and the transition probability of the remaining cycling cells. atively constant duration, the G1 phase length is highly vari- able. Of the proposals advanced to explain this variability, one MATERIALS AND METHODS that has been shown to closely fit most experimental data is the transition probability model of Smith and Martin (1). In this Cell Strains and Culture. HDFC strain 79-81 was explanted model cells remain in a subset of G1 (the "A phase") for a vari- from a skin biopsy sample of a normal 27-year-old male, and able length of time, leaving this state with a constant proba- strain 78-18 was derived from a skin biopsy sample of a 20-week bility of transition per unit time (P), to then complete G1 (the gestational age, karyotypically normal female abortus. Cells were "B phase"). The proliferative rate of a culture is, according to grown as described (21) at 37°C in modified Eagle's minimal this theory, modulated by alterations in this transition proba- essential medium (GIBCO) supplemented with 26 mM sodium bility, not by changes in the number of cells in a noncycling bicarbonate and the indicated concentration offetal bovine serum compartment (2, 3). (GIBCO). Tests for mycoplasma were uniformly negative by Human diploid fibroblast-like cells (HDFC) have been ob- staining with 4',6-diamidino-2-phenylindole (22). served to exhibit pronounced heterogeneity of interdivisional Kinetic Analysis with BrdUrd and Hoechst 33258 Dye. Log- times that increases dramatically in later passages as the cells arithmic-phase cells were plated at 200,000 cells per 25-cm2 flask approach the limits of their proliferative life-span. Interpre- (Coming) in media containing 0.1% fetal bovine serum. Above tations of this phenomenon, however, have been conflicting. PDL 50 the HDFC had markedly increased surface areas and Studies by time-lapse cinematography (4, 5) have been incon- only 100,000 cells per 25-cm2 flask were plated in order to min- clusive and have been interpreted as both consistent (6, 7) and imize contact-mediated inhibition of growth. After 5 days fresh inconsistent (8-10) with the Smith and Martin model. Studies medium containing BrdUrd and fetal bovine serum at the in- based upon clone size analysis indicate an increased number of dicated concentrations was added. The medium was replaced nondividing cells with advancing population doubling level (PDL) every second day thereafter and the cells were exposed only to (11, 12); however, this result has been criticized as possibly re- 580- to 590-nm light (model DUB safelight, Thomas Instru- lated to cloning conditions not present in mass culture (13). Au- ment, Charlottesville, VA). At the times described the cells were treated with trypsin, pelleted, and resuspended in 1 ml of a The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: HDFC, human diploid fibroblast-like cells; BrdUrd, 5- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. bromodeoxyuridine; PDL, population doubling level. 2951 Downloaded by guest on September 27, 2021 2952 Cell, Biology: Rabinovitch Proc. Natl. Acad. Sci. USA 80 (1983) buffer containing 0.146 M NaCl, 0.1 M Tris HCl (pH 7.4), 0.6% Nonidet P-40 (Sigma), and Hoechst 33258 dye (Calbiochem) at A 5.8 ,tg/ml, then Vortex mixed for 5 sec and kept for 2 hr at 40C. 9 81 Cells were then analyzed immediately, or after addition of 10% u (vol/vol) dimethyl sulfoxide (Schwarz/Mann) they were frozen r., at -700C. Later flow cytometric analysis is unaltered by this ~~~~~~~~~ci u.4 freezing. Cells analyzed 16 and 24 hr after feeding were, in- 410. stead.of the above, fixed with ethanol, stained with ethidium 0 bromide and mithramycin, and analyzed as described (23). All *.; 210 162 time points were examined with duplicate culture flasks. Flow W., cytometry was performed on an ICP-22 cytophotometer (Ortho Diagnostic Systems, Westwood, MA) interfaced to a PDP 11/ 20 40 60 80 100 120 140 03 computer (Digital Equipment, Maynard, MA). UG1 excita- tion and GG 435 emission filters were used for-Hoechst 33258 fluorescence analysis. BrdUrd-Hoechst fluorescence histograms were analyzed by computerfitting of Gaussian curves with use of the nonlinear least-squares technique of Marquardt (24). The numbers of S phase cells were approximated as the unfit portion of the counts between G1 and G2 peaks or, at 48 hr and earlier, the S phase component was determined by the method of Dean and Jett (25). Ethidium bromide/mithramycin fluorescence histograms were also analyzed by the latter technique. The nonlinear least- squares technique was also used to fit the kinetic data to the Channel number model of Smith and Martin, modified to include a noncycling fraction of cells: FIG. 1. BrdUrd-Hoechst fluorescence histograms of middle-PDL (PDL 30) HDFC grown for 30 hr (A) and 11 days (B) in media with 16% a = (1 - f)o1'P(t-TB) + f (for t TB; a = 1 for t < TB), fetal bovine serum and 150 ,.tM BrdUrd. The abscissa shows the his- togram channel number, which is proportional to fluorescence inten- in which a is the proportion of the initial population remaining sity. The G1 ofcells that did not incorporate BrdUrd is indicated as well in G1 at time t, fis the fraction of absolutely nondividing cells, as cell cycle phases after one (prefix B) or two (prefix BB) rounds of and TB is the length of the Smith and Martin B-phase lagbefore DNA synthesis in the presence of BrdUrd. the onset of S. Autoradiography. In one experiment 250,000 and 125,000 into their DNA show decreased fluorescence intensity with cells per 25-cm2 flask were plated, synchronized, and stimu- successive cell divisions, up to and including the second sub- lated with serum as described above, and duplicate sets of flasks sequent G1. The fluorescence intensity of the G1 cells after were either analyzed with BrdUrd-Hoechst 33258 or contin- growth for one cycle in BrdUrd (BG1) is approximately 35% of uously exposed to [3H]thymidine (40-60 Ci/mmol, New En- the original G1 under the conditions shown, with a further de- gland Nuclear; 1 Ci = 3.7 x 1010 Bq) at 0.05 ,uCi/ml. In both crease in the G1 peak after the next mitosis (BBG1). The shifts cases, cultures were refed every second day and were har- are sufficiently great so that the five cell cycle phases shown are vested 16 hr and 1, 2, 4, 7, and 11 days after serum stimulation.