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 ( 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 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] (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. well resolved and easily fitted to their Gaussian components by For autoradiography the bottoms of flasks were cut into 25 X least-squares techniques. On the basis of the number of divi- 75 mm slides and processed as described (21). Autoradiographs sions each of the fitted populations has experienced, the pro- were exposed in the dark for 21 days. All labeled and unlabeled portion of the original number of cells plated residing in that cells in random x400 microscopic fields, were enumerated, at population can be calculated, as can the total increase in cell least 400 cells were counted, and the number of fields exam- number. ined was recorded. The cell density, percent nonlabeled nuclei, The degree of "quenching" of Hoechst 33258 fluorescence and percent nonlabeled nuclei corrected for cell proliferation increases with increasing concentration of BrdUrd in the cul- were then calculated as described by Matsumura et al (17). ture medium (19, 29). We have found that concentrations of less than 60 ,uM result in a quenching of BG1 compared to G1 of less than 45%, precluding resolution of the G1 peak from the RESULTS left-shifted G2 peak (29). Fig. 2A shows the computer-analyzed BrdUr&Hoechst Assay of Cell Proliferation. Examination results of growth of middle-PDL HDFC in the presence of var- of the kinetics of exit from G1 phase of slowly dividing cells ious concentrations of BrdUrd. The cells are seen to rapidly requires simultaneous identification of the fraction of cells re- leave the G1 phase between 16 and 48 hr after addition of fetal maining noncycling in G1 and a correction for proliferation based bovine serum. to 0. 1% fetal bovine serum G1/G0 synchronized upon the original and final cell number. The correction can be cells. The cell cycle kinetics during this period would be in- obviated if the cells are arrested in mitosis by Colcemid (26, 27) distinguishable from a probabilistic (Smith and Martin) entry or vincristine (28); however, in application to HDFC this stath- into S phase. During subsequent days, however, there is a strong mokinetic approach was uniformly unsuccessful due to toxicity departure from this behavior as the exit from GC plateaus. This or leakiness of the block after several days (data not shown). An result is suggestive of the existence of two subpopulations of alternative and successful approach has been devised that yields cells, one dividing with Smith and Martin kinetics, and another all necessary information and avoids the additional labor and that is virtually nondividing. Computer least-squares fitting of error introduced by cell counting. Fig. 1 shows representative the data based upon such a model is also shown in Fig. 2A. histograms obtained by growth of HDFC in the presence of In light of previous reports that BrdUrd-induced mutagen- 150 AuM BrdUrd followed by staining with Hoechst 33258 and esis and sister chromatid exchanges may result from pertur- flow cytometric analysis. Cells that have incorporated BrdUrd bation of deoxycytidine metabolism, independent of incorpo- Downloaded by guest on September 27, 2021 Cell Biology: Rabinovitch Proc. Natl. Acad. Sci. USA 80 (1983) 2953

B 90 0.7 0 co C .4 80 a-. C 0.5

c)0

e<,70- u=_ ! 0.3 C>%

0 .5 1.0 2.0 4.0 80 0. Days BrdU concentration (moles/liter x 10 4) ,

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 the indicated times with 10% fetal bovine serum and the following concentrations of BrdUrd: 60 uM (o); 150 gM, without (A) or with (*) deoxycytidine in equimolar concentration to BrdUrd; 400 AM, without (v) or with (d) deoxycytidine; 800 uM, without (a) or with (x) deoxycytidine. Solid lines indicate the computer least-squares fit of each set of data to the model described in the text. (B) Effect of BrdUrd concentration upon the percent cycling cells (solid lines) and rate of exit from G1 (broken lines) with (m) and without (a) deoxycytidine in equimolar concentration to BrdUrd. The percent cycling cells in a similar experiment using strain 79-81 PDL 20 is also shown, with (e) and without (a) deoxycytidine. (C) Increase in cell number after the addition ofmedia with 10% fetal bovine serum, using the cells and BrdUrd concentrations indicated inA, and with 30 ,uM BrdUrd (c). Viability, as assessed by exclusion, was in all cases greater than 98%, in the same range as controls.

ration of BrdUrd into DNA (30, 31), the possibility that the nique a serum "shift-up" experiment was conducted, with the plateau observed in the exit from G1 is a toxic or inhibitory ef- rationale that toxic effects of BrdUrd on G1 cells would impair fect of incubation in BrdUrd must be excluded. As shown in the entry into S phase to a greater extent in cells exposed to Fig. 2 A and B, the results of the kinetic assay are minimally BrdUrd for longer times. Analysis of middle-PDL cells grown altered by the concentration of BrdUrd employed or by the in 15% fetal bovine serum and 150 p.M BrdUrd showed that presence or absence of deoxycytidine. In contrast, there are after 4 days of growth the culture approached a plateau at which marked differences in the growth rate of the cultures, as shown 39.6 ± 0.5% of the originally plated cells remained noncycling. in Fig. 2C. Even at concentrations as low as 30 ,M, BrdUrd Cells initially grown in medium with 1% fetal bovine serum and results in a cessation of growth of the culture after approxi- 150 A.M BrdUrd, with or without deoxycytidine, demonstrated mately one doubling. At concentrations of 400 AM or greater 72.3 ± 2.4% noncycling cells 5 days after serum stimulation. there is nearly complete G2 arrest; this arrest is partially re- These cultures were then shifted to medium with 15% fetal bo- versible by the addition of deoxycytidine, but this is without vine serum and the same concentrations of BrdUrd and de- effect upon the proportion of noncycling cells. The growth ar- oxycytidine. By day 9 the exit of cells from G1 approached a rest caused by BrdUrd actually is of benefit in the kinetic anal- plateau at which 38.1 ± 0.8% (without deoxycytidine) and 39.7 ysis because cultures are prevented from becoming density ar- ± 0.8% (with deoxycytidine) of cells were noncycling. Thus, rested during the course of an experiment. As a consequence, the prior 5-day exposure of the cells to BrdUrd was without decreasing the cell density at the start of an experiment has lit- deleterious effect upon the ability of the cells to exit the GC tle effect upon the calculated noncycling fraction (e.g., see Ta- phase. ble 1). This evidence is very suggestive that while growth of In the remainder of the experiments presented, a concen- cells in these concentrations of BrdUrd results in an apprecia- tration of BrdUrd of 150 A.M was utilized. With the cell type ble G2 arrest, the exit from G1 phase is little affected. To fur- and culture medium employed, this value yields optimal BrdUrd ther examine this question the BrdUrd-Hoechst flow cyto- quenching of Hoechst 33258 fluorescence and results in a de- metric technique was directly compared to conventional auto- gree of growth inhibition sufficient to prevent density arrest of radiography and counting of cell numbers by the method of the exit of cells from G1. Matsumura et al. (17). Initial estimates of the proportion of cells Effect of Population Doubling Level upon the Fraction of remaining in G1 were very similar, and at 48 hr after stimu- Noncycling Cells. The results of BrdUrd-Hoechst flow cyto- lation the proportion ofnondividingcells reachedaplateau whose metric analysis of the cell cycle kinetics of two strains of HDFC value was virtually identical by the two techniques (Table 1). as a function of their in vitro PDL are shown in Fig. 3. Fig. 3A The error associated with the autoradiographic technique was demonstrates very clearly that noncycling cells are observed at considerably greater than that with flow cytometry, however, all PDLs examined and that the proportion of noncycling cells and the human effort expended in the autoradiographic analysis increases progressively with age. The computer-fit curves cor- exceeded that required by flow cytometry by more than an or- responding to the revised Smith and Martin model that in- der of magnitude. cludes a fraction of noncycling cells are also indicated. The ad- As a final test of the BrdUrd-Hoechst flow cytometric tech- equacy of the model in fitting the data is confirmed by x2 goodness-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- Table 1. Comparison of autoradiography and BrdUrd-Hoechst crease in proportion flow the of noncycling cells is roughly linear cytometric assays of percent noncycling cells with population doubling level, although above PDL 50 strain % noncycling cells 79-81 shows a decline in this rate of increase. These latter cul- No. cells plated BrdUrd-Hoechst Autoradiography tures appear morphologically near-senescent and have a greater proportion of trypan blue nonexcluding cells than do lower PDL 125,000 29.8 ± 0.9 29.6 ± 4.2 cultures; it seems likely that some noncycling cells are being 250,000 27.3 ± 1.6 27.8 6.6 ± lost as the culture approaches senescence. For strain 79-81 the Strain 79-81, PDL 22.5 was used. Number of cells plated was per 25- degree of interculture variability in the proportion of noncy- cm flask. Results are mean ± SD. cling cells is indicated by multiple observations upon cultures Downloaded by guest on September 27, 2021 200 Proc. Natl. Acad. Sci. USA 80 (1983)

UV 1.4 80- A oB 70 1.2 "0 "Id ,a....~ -~60 010? .40 E 1.0 ' ° 'va, c ^^OA I W50 c 40 -20.6x0 0..,,"s-o" 6 V >l.. u . 40 r r x ,,o ," 0.6 - 30 0 .r0 c 0 -0.4 o.. I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 20 IP 00 10 Ig 0.2

Uo 2 4 6 8 I0 1I2 14 0 10 20 30 40 50 60 70 10 20 30 40 50 60 70 Days Population doubling level Population doubling level

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% fetal bovine 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 doublings over an extended period of observation. Application of this or greater. technique to the kinetic behavior of HDFC has shown that the The transition probability (rate of exit from G1) of just the transition probability model of cell kinetics must be revised to cycling portion of cells is resolved by computer fitting of the include a proportion of noncycling cells that changes as a func- data to the model, and, as shown in Fig. 3C, the transition tion of both mitogen concentration and PDL in vitro. probability decreases linearly with increasing PDL. The flow cytometric kinetic analysis is made possible by op- Effect of Serum Concentration upon Cell Cycle Kinetics. timization of BrdUrd concentration and staining conditions so The cell cycle changes underlying the reduced growth rate of that quenching of Hoechst 33258 fluorescence in BrdUrd-sub- HDFC in suboptimal concentrations of serum mitogens were stituted DNA is sufficient to allow separation of each successive investigated with use of the BrdUrd-Hoechst flow cytometric phase of the cell cycle (29). This degree of quenching is greater technique. Fig. 4 shows that as the concentration of fetal bo- than shown in recent years by using flow cytometry (20, 32, 33) vine serum in the culture medium is decreased there is a dra- but is similar to that reported by Latt et al. (19). It is shown in matic decrease both in the percentage of dividing cells and in this report that concentrations of BrdUrd that are sufficiently the rate of exit of cells from G1 (the transition probability) but high to achieve adequate quenching result in a marked reduc- there is no apparent change in the length of the B-phase lag tion in the growth rate of the HDFC culture (Fig. 2C), but, in until the first cells enter S. As shown in Fig. 4B for middle-PDL contrast, the rate of cell cycle transition out of the initial un- cells of two strains, the rate of change in both percent dividing substituted G1 is unaffected. The subsequent growth arrest ap- cells and transition probability is approximately a logarithmic pears to be mediated by a block of G2 at highest BrdUrd con- function of the serum concentration, most especially in the range centrations (19), although at lower concentrations of BrdUrd of 1% to 16% fetal bovine serum, although at low serum con- (e.g., '150 juM) most cells appear to be arrested in the sub- centrations the increase in slope may be more closely linear. sequent G1 (Fig. 1). The mechanism(s) of these blocks remains Had the kinetic analysis been terminated at 48 hr after mitogen to be elucidated. Thus, although exceedingly well suited to the stimulation, the presence of the variable noncycling fraction kinetic assays demonstrated here, the technique is at present could not have been appreciated, and all of the alterations in unsuited for the determination of successive cell cycle times in growth rate would have been ascribed to an even greater change HDFC. in transition probability than actually occurred within the cy- The data shown in this report are consistent only with a model cling population. in which the growth response of HDFC to differing concen- trations of serum is mediated by both an alteration in transition DISCUSSION probability and, very significantly, by a variation in the pro- This report describes a rapid and quantitative technique that portion of cells capable of responding to the particular level of allows the determination of the noncycling proportion of cells mitogens. The latter response, we suggest, could result from

8( 0-~~~~~~~~~ 4)6 60~~~~~~~~~~. 50 ~ l 2 4 Days % fetal bovine serum

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 of Smith 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 strain 79-81 PDL 32 (a) and strain 78-18 PDL 36 (a) as a function of serum concentration. Downloaded by guest on September 27, 2021 Cell Biology: Rabinovitch Proc. Natl. Acad. Sci. USA 80 (1983) 2955 a large degree of heterogeneity within the population with re- One may speculate that the increasing fraction of nondivid- spect to an inherent threshold level of mitogen concentration ing cells and the decreasing growth rate that accompany aging required in order to initiate proliferation. We propose that, as in vitro may result from a progressive shift upwards with each an expression of this heterogeneity, for each serum concentra- cell division in the threshold of mitogen responsiveness hy- tion there exist within the population (i) responsive cells, (ii) pothesized above. A larger fraction of cells would then become cells refractory to stimulation at that serum concentration but refractory to stimulation by the usual levels of serum mitogens capable of being stimulated by an increase in the serum level, employed in culture; eventually this trend would result in the and (iii) cells that are refractory to all practical levels of serum completely nondividing culture associated with phase III se- mitogens. The data, as shown in Figs. 3 and 4, are fit closely nescence in vitro. by a revision of the transition probability model that includes The excellent technical support of Patricia C. Otto and Michael Wen- such a feature. This heterogeneity suggests that functional sub- tang Shen is gratefully acknowledged. This work was supported by Na- classes of cells exist within the population. tional Institutes of Health Grant AG 01751. It is apparent from previous reports that examine the kinetics of 3T3, 3T6, simian virus 40-transformed 3T3 (2), and BHK 21 1. Smith, J. A. & Martin, L. (1973) Proc. Natl. Acad. Sci. USA 70, cells (3) that, if populations of noncycling cells are present in 1263-1267. 2. Shields, R. & Smith, J. A. (1977) J. Cell Physiol 91, 345-355. these transformed cell lines, then they must be in proportions 3. Brooks, R. F. (1975)J. Cell Physiol 86, 369-372. very much smaller than in HDFC. The possibility that there 4. Absher, P. M., Absher, R. G. & Barnes, W. D. (1974) Exp. Cell are, in fact, no noncycling cells in such cell lines can be ex- Res. 88, 95-104. amined only by a kinetic analysis of sufficient duration such 5. Absher, P. M. & Absher, R. G. (1976) Exp. Cell Res. 103, 247- that the noncycling "phase" of the kinetic curve is revealed, if 255. it does in fact exist. In either case, we speculate that the pres- 6. Smith, J. A. (1977) Cell Biol lnt. Rep. 1, 283-298. 7. Shields, R. (1979) Cell Biol lnt. Rep. 3, 659-662. ence of an appreciable population of noncycling cells may be 8. Grove, G. L. & Cristofalo, V. J. (1976) Cell Tissue Kinet. 9, 395- an inherent feature of diploid strains having finite proliferative 399. life-spans, and that this feature is either absent from or very 9. Grove, G. L. & Cristofalo, V. J. (1978) Cell Biol lnt. Rep. 2, 185- diminished in immortal transformed cell lines. 188. These results support several previous observations that sug- 10. Mets, T. & Verdonk, G. (1978) Cell Biol Int. Rep. 2, 561-564. gest that there is a progressive increase in the proportion of 11. Merz, G. S. & Ross, J. D. (1969)J. Cell Physiol 74, 219-222. 12. Smith, J. R., Pereira-Smith, 0. M. & Schneider, E. L. (1978) Proc. noncycling cells with in vitro age, although the increase ob- Natl Acad. Sci. USA 75, 1353-1356. served here is linear, rather than exponential, as previously re- 13. Maciera-Coelho, A. & Taboury, F. (1982) Cell Tissue Kinet. 15, ported (16, 17). Results obtained by 24- to 30-hr [ H]thymidine 213-224. pulses (16) will, however, underestimate the noncycling frac- 14. Maciera-Coelho (1974) Nature (London) 248, 421-422. tion at low PDL due to division of cycling cells during the pulse 15. Vincent, R. A. & Huang, P. C. (1976) Exp. Cell Res. 102, 31-42. period and will overestimate the noncycing fraction at higher 16. Cristofalo, V. J. & Sharf, B. B. (1973) Exp. Cell Res. 76, 419-427. 17. Matsumura, T., Pfendt, E. A. & Hayflick, L. (1979) J. Gerontol. PDL due to the presence of cells that only cycle "slowly"-i. e., 34, 323-327. have a decreased transition probability. The discrepancy with 18. Latt, S. A. (1973) Proc. Natl Acad. Sci. USA 70, 3395-3399. the data of Matsumura et al. (17) is less easily understood, and 19. Latt, S. A., George, Y. S. & Gray, J. W. (1977)J. Histochem. Cy- it may be related to true differences between HDFC strains or tochem. 25, 927-934. to differences in protocol; it is possible, for example, that dif- 20. Bohmer, R.-M. (1979) Cell Tissue Kinet. 12, 101-110. ferent mitogenic stimuli may result from trypsin treatment of 21. Rabinovitch, P. S. & Norwood, T. H. (1981) Somatic Cell Genet. 7, 281-287. the cells at the onset of the growth period (Matsumura et al.), 22. Russell, W. C., Newman, C. & Williamson, D. H. (1975) Nature as compared to the effect of addition of serum alone to pre- (London) 253, 461-462. viously plated, serum-deprived cultures (this report). 23. Rabinovitch, P. S., O'Brien, K., Simpson, M., Callis, J. B. & We believe that the body of conflicting data regarding the Hoehn, H. (1981) Cytogenet. Cell Genet. 29, 65-76. basis of the increased heterogeneity of interdivisional times with 24. Marquardt, D. W. (1963)J. Soc. Ind. Appl. Math. 11, 431-441. increasing PDL is resolved by consideration of the limitations 25. Dean, P. N. & Jett, J. H. (1974)J. Cell Biol 60, 523-527. of 26. Bohmer, R.-M. (1980) Cell Tissue Kinet. 13, 497-503. previous methods and by the observation that both the tran- 27. Dosik, G. M., Barlogie, B., White, R. A., Gohde, W. & Dre- sition probability and the proportion of nondividing cells change winko, B. (1981) Cell Tissue Kinet 14, 121-134. as a function of increasing PDL. Taking both of these factors 28. Barfod, I. H. & Barfod, N. M. (1980) Cell Tissue Kinet. 13, 1-8. into account, the proliferative kinetics of HDFC at all PDLs 29. Kubbies, M. & Rabinovitch, P. S. (1983) Cytometry 3, 276-281. can be explained. In particular, there is no evidence from the 30. Davidson, R. L. & Kaufman, E. R. (1979) Somatic Cell Genet. 5, kinetic analysis that the cells within the nondividing fraction 873-885. 31. Davidson, R. L., Kaufman, E. R., Dougherty, C. P., Ovellette, revert to the dividing state (greater than a few percent per week A. M., DiFolco, C. M. & Latt, S. A. (1980) Nature (London) 284, leaving G1/Go would have been discernable). This would be 74-76. consistent with the presence of postreplicative or terminally 32. Nuisse, M. (1981) Cytometry 2, 70-79. differentiated cells at all PDLs. 33. Beck, H.-P. (1981) Cytometry 2, 170-174. Downloaded by guest on September 27, 2021