Proc. NatL Acad. Sci. USAVol. 80, pp. 2951-2955, May 1983Cell Biology

Regulation of human fibroblast growth rate by both noncycling cellfraction and transition probability is shown by growth in5-bromodeoxyuridine followed by Hoechst 33258flow cytometry

(cell cycle Idnetics/growth control/human diploid cells/life-span in vitro)

PETER S. RABINOVITCHDepartment 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-ence of 5-bromodeoxyuridine, followed by flow cytometric anal-ysis of DNA-specific fluorescence with Hoechst 33258 dye, allowsquantitation of the proportion of cells that have not cycled, as wellas those in G1 and G2 of two subsequent cell cycles. This techniqueallows rapid and accurate quantitation of the growth fraction andG1/S transition rate of these cells. The cell cycle kinetics of hu-man diploid fibroblasts at all population doubling levels reveal twocomponents: cycling cells showing a probabilistic rate of G1/Stransition, and a variable proportion of noncycling cells. Both thetransition probability (rate of exit from G1) and the noncyclingproportion of cells change systematically as a function of serumconcentration and as a function of population doubling level. Thedata suggest the existence of an underlying heterogeneity in thepopulation of human diploid fibroblasts with respect to the ca-pacity to divide in the presence of a given concentration of mi-togen. Models of cell cycle kinetics must be modified to includeregulation of growth by changes in the fraction of cycling cells, aswell as by changes in the rate of emit from G1.

Heterogeneity in interdivisional times is a common and well-documented feature of the proliferative behavior of culturedmammalian cells, and whereas S, G2, and M phases are of rel-atively constant duration, the G1 phase length is highly vari-able. Of the proposals advanced to explain this variability, onethat has been shown to closely fit most experimental data is thetransition probability model of Smith and Martin (1). In thismodel cells remain in a subset of G1 (the "A phase") for a vari-able length of time, leaving this state with a constant proba-bility of transition per unit time (P), to then complete G1 (the"B phase"). The proliferative rate of a culture is, according tothis theory, modulated by alterations in this transition proba-bility, not by changes in the number of cells in a noncyclingcompartment (2, 3).Human diploid fibroblast-like cells (HDFC) have been ob-

served to exhibit pronounced heterogeneity of interdivisionaltimes that increases dramatically in later passages as the cellsapproach the limits of their proliferative life-span. Interpre-tations of this phenomenon, however, have been conflicting.Studies by time-lapse cinematography (4, 5) have been incon-clusive and have been interpreted as both consistent (6, 7) andinconsistent (8-10) with the Smith and Martin model. Studiesbased upon clone size analysis indicate an increased number ofnondividing cells with advancing population doubling level (PDL)(11, 12); however, this result has been criticized as possibly re-lated to cloning conditions not present in mass culture (13). Au-

toradiographic analysis of HDFC cultures has yielded resultsthat have been interpreted as showing no noncycling popula-tion (14), noncycling cells arising only in the last 10 populationdoubling levels (15), or proportions of noncycling cells pro-gressively increasing with age (16). The inconsistencies are pre-sumably related to problems with method (13) and may be ex-plained in part by the use of [3H]thymine pulse periods that aretoo short to label slowly dividing cells, or the proliferation oflabeled cells during the labeling interval. More recently, thisproblem was addressed by Matsumura et al (17), who com-pensated for cell proliferation by counting cell numbers at thestart and end of the experiment. They concluded that noncy-cling cells are indeed present and progressively increase withculture age. With the objective of performing a more detailedstudy of these cellular kinetics, we have adapted a techniqueof flow cytometric assay of growth kinetics based upon Hoechstdye staining of cells grown in 5-bromodeoxyuridine (BrdUrd)(18-20). The increased ease and accuracy of this techniquecompared to conventional methods allows an analysis demon-strating that changes in the proliferative rate of HDFC culturesare a result of alterations in both the fraction of noncycling cellsand 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, andstrain 78-18 was derived from a skin biopsy sample of a 20-weekgestational age, karyotypically normal female abortus. Cells weregrown as described (21) at 37°C in modified Eagle's minimalessential medium (GIBCO) supplemented with 26 mM sodiumbicarbonate and the indicated concentration of fetal bovine serum(GIBCO). Tests for mycoplasma were uniformly negative bystaining with 4',6-diamidino-2-phenylindole (22).

Kinetic Analysis with BrdUrd and Hoechst 33258 Dye. Log-arithmic-phase cells were plated at 200,000 cells per 25-cm2 flask(Coming) in media containing 0.1% fetal bovine serum. AbovePDL 50 the HDFC had markedly increased surface areas andonly 100,000 cells per 25-cm2 flask were plated in order to min-imize contact-mediated inhibition of growth. After 5 days freshmedium containing BrdUrd and fetal bovine serum at the in-dicated concentrations was added. The medium was replacedevery second day thereafter and the cells were exposed only to580- to 590-nm light (model DUB safelight, Thomas Instru-ment, Charlottesville, VA). At the times described the cells weretreated with trypsin, pelleted, and resuspended in 1 ml of a

Abbreviations: HDFC, human diploid fibroblast-like cells; BrdUrd, 5-bromodeoxyuridine; PDL, population doubling level.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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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) at5.8 ,tg/ml, then Vortex mixed for 5 sec and kept for 2 hr at 40C.Cells were then analyzed immediately, or after addition of 10%(vol/vol) dimethyl sulfoxide (Schwarz/Mann) they were frozenat -700C. Later flow cytometric analysis is unaltered by thisfreezing. Cells analyzed 16 and 24 hr after feeding were, in-stead.of the above, fixed with ethanol, stained with ethidiumbromide and mithramycin, and analyzed as described (23). Alltime points were examined with duplicate culture flasks. Flowcytometry was performed on an ICP-22 cytophotometer (OrthoDiagnostic Systems, Westwood, MA) interfaced to a PDP 11/03 computer (Digital Equipment, Maynard, MA). UG1 excita-tion and GG 435 emission filters were used for-Hoechst 33258fluorescence analysis.

BrdUrd-Hoechst fluorescence histograms were analyzed bycomputerfitting of Gaussian curves with use of the nonlinearleast-squares technique of Marquardt (24). The numbers of Sphase cells were approximated as the unfit portion of the countsbetween G1 and G2 peaks or, at 48 hr and earlier, the S phasecomponent was determined by the method of Dean and Jett(25). Ethidium bromide/mithramycin fluorescence histogramswere also analyzed by the latter technique. The nonlinear least-squares technique was also used to fit the kinetic data to themodel of Smith and Martin, modified to include a noncyclingfraction of cells:a = (1 - f)o1'P(t-TB) + f (for t TB; a = 1 for t < TB),

in which a is the proportion of the initial population remainingin G1 at time t, f is the fraction of absolutely nondividing cells,and TB is the length of the Smith and Martin B-phase lag beforethe onset of S.

Autoradiography. In one experiment 250,000 and 125,000cells per 25-cm2 flask were plated, synchronized, and stimu-lated with serum as described above, and duplicate sets of flaskswere either analyzed with BrdUrd-Hoechst 33258 or contin-uously exposed to [3H]thymidine (40-60 Ci/mmol, New En-gland Nuclear; 1 Ci = 3.7 x 1010 Bq) at 0.05 ,uCi/ml. In bothcases, cultures were refed every second day and were har-vested 16 hr and 1, 2, 4, 7, and 11 days after serum stimulation.For autoradiography the bottoms of flasks were cut into 25 X75 mm slides and processed as described (21). Autoradiographswere exposed in the dark for 21 days. All labeled and unlabeledcells in random x 400 microscopic fields, were enumerated, atleast 400 cells were counted, and the number of fields exam-ined was recorded. The cell density, percent nonlabeled nuclei,and percent nonlabeled nuclei corrected for cell proliferationwere then calculated as described by Matsumura et al (17).

RESULTSBrdUr&Hoechst Assay of Cell Proliferation. Examination

of the kinetics of exit from G1 phase of slowly dividing cellsrequires simultaneous identification of the fraction of cells re-maining noncycling in G1 and a correction for proliferation basedupon the original and final cell number. The correction can beobviated if the cells are arrested in mitosis by Colcemid (26, 27)or vincristine (28); however, in application to HDFC this stath-mokinetic approach was uniformly unsuccessful due to toxicityor leakiness of the block after several days (data not shown). Analternative and successful approach has been devised that yieldsall necessary information and avoids the additional labor anderror introduced by cell counting. Fig. 1 shows representativehistograms obtained by growth of HDFC in the presence of150 AuM BrdUrd followed by staining with Hoechst 33258 andflow cytometric analysis. Cells that have incorporated BrdUrd

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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%fetal bovine serum and 150 ,.tM BrdUrd. The abscissa shows the his-togram channel number, which is proportional to fluorescence inten-sity. The G1 of cells that did not incorporate BrdUrd is indicated as wellas cell cycle phases after one (prefix B) or two (prefix BB) rounds ofDNA synthesis in the presence of BrdUrd.

into their DNA show decreased fluorescence intensity withsuccessive cell divisions, up to and including the second sub-sequent G1. The fluorescence intensity of the G1 cells aftergrowth for one cycle in BrdUrd (BG1) is approximately 35% ofthe original G1 under the conditions shown, with a further de-crease in the G1 peak after the next mitosis (BBG1). The shiftsare sufficiently great so that the five cell cycle phases shown are

well resolved and easily fitted to their Gaussian components byleast-squares techniques. On the basis of the number of divi-sions each of the fitted populations has experienced, the pro-

portion of the original number of cells plated residing in thatpopulation can be calculated, as can the total increase in cellnumber.The degree of "quenching" of Hoechst 33258 fluorescence

increases with increasing concentration of BrdUrd in the cul-ture medium (19, 29). We have found that concentrations ofless than 60 ,uM result in a quenching of BG1 compared to G1of less than 45%, precluding resolution of the G1 peak from theleft-shifted G2 peak (29). Fig. 2A shows the computer-analyzedresults of growth of middle-PDL HDFC in the presence of var-

ious concentrations of BrdUrd. The cells are seen to rapidlyleave the G1 phase between 16 and 48 hr after addition of fetalbovine serum. to 0.1% fetal bovine serum G1/G0 synchronizedcells. The cell cycle kinetics during this period would be in-distinguishable from a probabilistic (Smith and Martin) entryinto S phase. During subsequent days, however, there is a strongdeparture from this behavior as the exit from GC plateaus. Thisresult is suggestive of the existence of two subpopulations ofcells, one dividing with Smith and Martin kinetics, and anotherthat is virtually nondividing. Computer least-squares fitting ofthe data based upon such a model is also shown in Fig. 2A.

In light of previous reports that BrdUrd-induced mutagen-esis and sister chromatid exchanges may result from pertur-bation of deoxycytidine metabolism, independent of incorpo-

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FIG. 2. (A) Effect of different concentrations of BrdUrd on the percent of original cells in G1. Cells of strain 78-18 PDL 28 were grown for theindicated times with 10% fetal bovine serum and the following concentrations of BrdUrd: 60 uM (o); 150 gM, without (A) or with (*) deoxycytidinein equimolar concentration to BrdUrd; 400 AM, without (v) or with (d) deoxycytidine; 800 uM, without (a) or with (x) deoxycytidine. Solid linesindicate the computer least-squares fit of each set of data to the model described in the text. (B) Effect of BrdUrd concentration upon the percentcycling cells (solid lines) and rate of exit from G1 (broken lines) with (m) and without (a) deoxycytidine in equimolar concentration to BrdUrd. Thepercent cycling cells in a similar experiment using strain 79-81 PDL 20 is also shown, with (e) and without (a) deoxycytidine. (C) Increase in cellnumber after the addition of media with 10% fetal bovine serum, using the cells and BrdUrd concentrations indicated inA, and with 30 ,uM BrdUrd(c). Viability, as assessed by trypan blue exclusion, was in all cases greater than 98%, in the same range as controls.

ration of BrdUrd into DNA (30, 31), the possibility that theplateau observed in the exit from G1 is a toxic or inhibitory ef-fect of incubation in BrdUrd must be excluded. As shown inFig. 2 A and B, the results of the kinetic assay are minimallyaltered by the concentration of BrdUrd employed or by thepresence or absence of deoxycytidine. In contrast, there aremarked differences in the growth rate of the cultures, as shownin Fig. 2C. Even at concentrations as low as 30 ,M, BrdUrdresults in a cessation of growth of the culture after approxi-mately one doubling. At concentrations of 400 AM or greaterthere is nearly complete G2 arrest; this arrest is partially re-versible by the addition of deoxycytidine, but this is withouteffect upon the proportion of noncycling cells. The growth ar-rest caused by BrdUrd actually is of benefit in the kinetic anal-ysis because cultures are prevented from becoming density ar-rested during the course of an experiment. As a consequence,decreasing the cell density at the start of an experiment has lit-tle effect upon the calculated noncycling fraction (e.g., see Ta-ble 1). This evidence is very suggestive that while growth ofcells in these concentrations of BrdUrd results in an apprecia-ble G2 arrest, the exit from G1 phase is little affected. To fur-ther examine this question the BrdUrd-Hoechst flow cyto-metric technique was directly compared to conventional auto-radiography and counting of cell numbers by the method ofMatsumura et al. (17). Initial estimates of the proportion of cellsremaining in G1 were very similar, and at 48 hr after stimu-lation the proportion of nondividingcells reached aplateau whosevalue was virtually identical by the two techniques (Table 1).The error associated with the autoradiographic technique was

considerably greater than that with flow cytometry, however,and the human effort expended in the autoradiographic analysisexceeded that required by flow cytometry by more than an or-

der of magnitude.As a final test of the BrdUrd-Hoechst flow cytometric tech-

Table 1. Comparison of autoradiography and BrdUrd-Hoechstflow cytometric assays of percent noncycling cells

% noncycling cellsNo. cells plated BrdUrd-Hoechst Autoradiography

125,000 29.8 ± 0.9 29.6 ± 4.2250,000 27.3 ± 1.6 27.8 ± 6.6

Strain 79-81, PDL 22.5 was used. Number of cells plated was per 25-cm flask. Results are mean ± SD.

nique a serum "shift-up" experiment was conducted, with therationale that toxic effects of BrdUrd on G1 cells would impairthe entry into S phase to a greater extent in cells exposed toBrdUrd for longer times. Analysis of middle-PDL cells grownin 15% fetal bovine serum and 150 p.M BrdUrd showed thatafter 4 days of growth the culture approached a plateau at which39.6 ± 0.5% of the originally plated cells remained noncycling.Cells initially grown in medium with 1% fetal bovine serum and150 A.M BrdUrd, with or without deoxycytidine, demonstrated72.3 ± 2.4% noncycling cells 5 days after serum stimulation.These cultures were then shifted to medium with 15% fetal bo-vine serum and the same concentrations of BrdUrd and de-oxycytidine. By day 9 the exit of cells from G1 approached aplateau at which 38.1 ± 0.8% (without deoxycytidine) and 39.7± 0.8% (with deoxycytidine) of cells were noncycling. Thus,the prior 5-day exposure of the cells to BrdUrd was withoutdeleterious effect upon the ability of the cells to exit the GCphase.

In the remainder of the experiments presented, a concen-tration of BrdUrd of 150 A.M was utilized. With the cell typeand culture medium employed, this value yields optimal BrdUrdquenching of Hoechst 33258 fluorescence and results in a de-gree of growth inhibition sufficient to prevent density arrest ofthe exit of cells from G1.

Effect of Population Doubling Level upon the Fraction ofNoncycling Cells. The results of BrdUrd-Hoechst flow cyto-metric analysis of the cell cycle kinetics of two strains of HDFCas a function of their in vitro PDL are shown in Fig. 3. Fig. 3Ademonstrates very clearly that noncycling cells are observed atall PDLs examined and that the proportion of noncycling cellsincreases progressively with age. The computer-fit curves cor-responding to the revised Smith and Martin model that in-cludes a fraction of noncycling cells are also indicated. The ad-equacy of the model in fitting the data is confirmed by x2goodness-of-fit tests (typical x = 1.8, df = 8, P = 0.986). Fig.3B shows that in both of the HDFC strains examined the in-crease in the proportion of noncycling cells is roughly linearwith population doubling level, although above PDL 50 strain79-81 shows a decline in this rate of increase. These latter cul-tures appear morphologically near-senescent and have a greaterproportion of trypan blue nonexcluding cells than do lower PDLcultures; it seems likely that some noncycling cells are beinglost as the culture approaches senescence. For strain 79-81 thedegree of interculture variability in the proportion of noncy-cling cells is indicated by multiple observations upon cultures

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FIG. 3. (A) Results of BrdUrd-Hoechst analysis of the kinetics of growth of cultures of strain 79-81 at various PDL in media with 16% fetalbovine serum, including the computer fitting of the data to the model described (solid lines). Cells were used at PDL 10 (v), 16 (*), 32 (A), and 62(o). (B) Change in the percent of noncycling cells as a function of PDL for strains 79-81 (c) and 78-18 (o). (C) Changes in the rate of exit from G,(transition probability) as a function of PDL for strain 79-81.

grown independently of each other for 12 population doublingsor greater.The transition probability (rate of exit from G1) of just the

cycling portion of cells is resolved by computer fitting of thedata to the model, and, as shown in Fig. 3C, the transitionprobability decreases linearly with increasing PDL.

Effect of Serum Concentration upon Cell Cycle Kinetics.The cell cycle changes underlying the reduced growth rate ofHDFC in suboptimal concentrations of serum mitogens were

investigated with use of the BrdUrd-Hoechst flow cytometrictechnique. Fig. 4 shows that as the concentration of fetal bo-vine serum in the culture medium is decreased there is a dra-matic decrease both in the percentage of dividing cells and inthe rate of exit of cells from G1 (the transition probability) butthere is no apparent change in the length of the B-phase laguntil the first cells enter S. As shown in Fig. 4B for middle-PDLcells of two strains, the rate of change in both percent dividingcells and transition probability is approximately a logarithmicfunction of the serum concentration, most especially in the rangeof 1% to 16% fetal bovine serum, although at low serum con-

centrations the increase in slope may be more closely linear.Had the kinetic analysis been terminated at 48 hr after mitogenstimulation, the presence of the variable noncycling fractioncould not have been appreciated, and all of the alterations ingrowth rate would have been ascribed to an even greater changein transition probability than actually occurred within the cy-cling population.

DISCUSSIONThis report describes a rapid and quantitative technique thatallows the determination of the noncycling proportion of cells

Days

over an extended period of observation. Application of thistechnique to the kinetic behavior of HDFC has shown that thetransition probability model of cell kinetics must be revised toinclude a proportion of noncycling cells that changes as a func-tion of both mitogen concentration and PDL in vitro.The flow cytometric kinetic analysis is made possible by op-

timization of BrdUrd concentration and staining conditions so

that quenching of Hoechst 33258 fluorescence in BrdUrd-sub-stituted DNA is sufficient to allow separation of each successivephase of the cell cycle (29). This degree of quenching is greaterthan shown in recent years by using flow cytometry (20, 32, 33)but is similar to that reported by Latt et al. (19). It is shown inthis report that concentrations of BrdUrd that are sufficientlyhigh to achieve adequate quenching result in a marked reduc-tion in the growth rate of the HDFC culture (Fig. 2C), but, incontrast, the rate of cell cycle transition out of the initial un-

substituted G1 is unaffected. The subsequent growth arrest ap-pears to be mediated by a block of G2 at highest BrdUrd con-

centrations (19), although at lower concentrations of BrdUrd(e.g., '150 juM) most cells appear to be arrested in the sub-sequent G1 (Fig. 1). The mechanism(s) of these blocks remainsto be elucidated. Thus, although exceedingly well suited to thekinetic assays demonstrated here, the technique is at presentunsuited for the determination of successive cell cycle times inHDFC.The data shown in this report are consistent only with a model

in which the growth response of HDFC to differing concen-

trations of serum is mediated by both an alteration in transitionprobability and, very significantly, by a variation in the pro-portion of cells capable of responding to the particular level ofmitogens. The latter response, we suggest, could result from

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FIG. 4. (A) Kinetics of growth of a middle-passage (PDL 32) culture, strain 79-81, grown in media containing BrdUrd and 0.1% (o), 1% (A), 2%(*), 4% (v), 8% (n), and 16% (o) fetal bovine serum. The solid lines show the computer fitting of the data with the revised model ofSmith and Martin.

(B) Calculated percent noncycling cells (solid lines) and the rate of exit from G1 of the cycling cells (transition probability, dotted lines) for strain79-81 PDL 32 (a) and strain 78-18 PDL 36 (a) as a function of serum concentration.

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a large degree of heterogeneity within the population with re-spect to an inherent threshold level of mitogen concentrationrequired in order to initiate proliferation. We propose that, asan expression of this heterogeneity, for each serum concentra-tion there exist within the population (i) responsive cells, (ii)cells refractory to stimulation at that serum concentration butcapable of being stimulated by an increase in the serum level,and (iii) cells that are refractory to all practical levels of serummitogens. The data, as shown in Figs. 3 and 4, are fit closelyby a revision of the transition probability model that includessuch a feature. This heterogeneity suggests that functional sub-classes of cells exist within the population.

It is apparent from previous reports that examine the kineticsof 3T3, 3T6, simian virus 40-transformed 3T3 (2), and BHK 21cells (3) that, if populations of noncycling cells are present inthese transformed cell lines, then they must be in proportionsvery much smaller than in HDFC. The possibility that thereare, in fact, no noncycling cells in such cell lines can be ex-amined only by a kinetic analysis of sufficient duration suchthat the noncycling "phase" of the kinetic curve is revealed, ifit does in fact exist. In either case, we speculate that the pres-ence of an appreciable population of noncycling cells may bean inherent feature of diploid strains having finite proliferativelife-spans, and that this feature is either absent from or verydiminished in immortal transformed cell lines.

These results support several previous observations that sug-gest that there is a progressive increase in the proportion ofnoncycling cells with in vitro age, although the increase ob-served here is linear, rather than exponential, as previously re-ported (16, 17). Results obtained by 24- to 30-hr [ H]thymidinepulses (16) will, however, underestimate the noncycling frac-tion at low PDL due to division of cycling cells during the pulseperiod and will overestimate the noncycing fraction at higherPDL due to the presence of cells that only cycle "slowly"-i. e.,have a decreased transition probability. The discrepancy withthe data of Matsumura et al. (17) is less easily understood, andit may be related to true differences between HDFC strains orto differences in protocol; it is possible, for example, that dif-ferent mitogenic stimuli may result from trypsin treatment ofthe cells at the onset of the growth period (Matsumura et al.),as compared to the effect of addition of serum alone to pre-viously plated, serum-deprived cultures (this report).We believe that the body of conflicting data regarding the

basis of the increased heterogeneity of interdivisional times withincreasing PDL is resolved by consideration of the limitationsof previous methods and by the observation that both the tran-sition probability and the proportion of nondividing cells changeas a function of increasing PDL. Taking both of these factorsinto account, the proliferative kinetics of HDFC at all PDLscan be explained. In particular, there is no evidence from thekinetic analysis that the cells within the nondividing fractionrevert to the dividing state (greater than a few percent per weekleaving G1/Go would have been discernable). This would beconsistent with the presence of postreplicative or terminallydifferentiated cells at all PDLs.

One may speculate that the increasing fraction of nondivid-ing cells and the decreasing growth rate that accompany agingin vitro may result from a progressive shift upwards with eachcell division in the threshold of mitogen responsiveness hy-pothesized above. A larger fraction of cells would then becomerefractory to stimulation by the usual levels of serum mitogensemployed in culture; eventually this trend would result in thecompletely nondividing culture associated with phase III se-nescence in vitro.The excellent technical support of Patricia C. Otto and Michael Wen-

tang Shen is gratefully acknowledged. This work was supported by Na-tional Institutes of Health Grant AG 01751.

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tochem. 25, 927-934.20. Bohmer, R.-M. (1979) Cell Tissue Kinet. 12, 101-110.21. Rabinovitch, P. S. & Norwood, T. H. (1981) Somatic Cell Genet.

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uest

on

Mar

ch 2

6, 2

021