BrdU—Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis

Experimental

Cell Research 174 (1988) 309-318

SPECIAL ARTICLE

A Unique

BrdU-Hoechst Flow Cytometry: Tool for Quantitative Cell Cycle Analysis

P. S. RABINOVITCH*,’ M. KUBBIES,t Y. C. CHEN,” D. SCHINDLER,? and H. HOEHNt *Department of Pathology, University of Washington, Seattle, Washington 98105, and TDepartment of Human Genetics, University of Wiirzburg, Federal Republic of Germany

Unlike other techniques, flow cytometric analysis of BrdU-quenched 33258 Hoechst fluorescence may be used to measure cell activation and the G,, S, and Gr/M compartment distributions in each of three successive cell cycles after growth stimulation of human peripheral blood lymphocytes. Cell cycle kinetic curves can be constructed from the BrdU-Hoechst flow data which allow the simultaneous assessment of growth fraction, lagtime, compartment exit rate, compartment duration, and compartment arrest. Applications of this new versatile technique include the evaluation of drug and growth factor effets, cell aging, and diagnosis in medicine and immunology. 0 1988 Academic PRSS, IIIC.

During the past decade, two different methods of cell cycle analysis have been developed which are based on the fluorometric assessment of nuclear 5-bromodeoxyuridine (BrdU) incorporated during semiconservative DNA replication. Both methods have certain advantages over the traditional methods of assessment of cellular proliferation. One technique utilizes a monoclonal antibody as a probe for detection of BrdU incorporated into DNA [l]. Using flow cytometry, this method has been successfully combined with simultaneous measurement of total DNA content [2] and yields, after pulse labeling with BrdU, excellent estimates of S-phase cell fractions, replication rates, and even cell cycle traverse rates both in synchronous and in asynchronously growing mammalian cell cultures [3, 41. The other method, applied first to cell proliferation by S. Latt, involves the detection of BrdU incorporation into DNA by virtue of the “quenching” of the fluorescence of bisbenzimidazole dyes such as 33258 Hoechst when bound to BrdU-substituted DNA [5, 61. Although the latter technique has been applied to flow cytometry in a number of laboratories to differentiate between cycling and noncycling cells [7-161, the resolution previously achieved was insuffkient for the demonstration of multiple cell cycles after mitogen stimulation. As illustrated in the present report, recent optimizations of the BrdUHoechst technique [15-171 enable a comprehensive analysis of cell activation and cycle progression that may have a variety of applications in cell biology, immunology, and medicine. I To whom reprint requests should be addressed at: Pathology SM-30, University of Washington, Seattle, WA 98195. 309

Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827188 $03.00

310 Rabinovitch et al. MATERIALS

AND METHODS

Cell culture. The BrdU-Hoechst analysis was performed as previously described [15, 161.In brief, peripheral blood lymphocytes (PBL) were obtained by Ficoll-Hypaque gradient separation of whole blood and washed in RPM1 1640 medium and aliquots of 1X1@ lymphocytes/ml were incubated in RPM1 1640 medium with 16% fetal bovine serum (FBS), 180 ug/ml phytohemagglutinin (PHA; Wellcome) and 1.0x 1O-4M BrdU. Addition of deoxycytidine (50 @f) does not appear to be necessary for lymphocyte culture [17], but may be useful during culture of adherent cells 1151.At the indicated times cells were harvested, pelleted, resuspended in DMEM with 10% FBS and 10% DMSO, and stored at -20°C. For experiments with immunofluorescence, PBL were harvested, stained with phycoerythrin-conjugated monoclonal antibody (Becton-Dickinson) following the manufacturer’s directions, washed, fixed in PBS with 30% (v/v) ETCH, and stored at 4°C. Flow cytometry. For flow analysis, cells were thawed, pelleted, and resuspended at 5x lO’/ml in a solution of 0.154 M NaCl, 0.1 M Tris (pH 7.4), 0.5 mit4 MgClr 0.2 % BSA, 0.1% Nonidet-P40 and 1.2 CLg/ml33258 Hoechst. For two-parameter measurements 1181,1.5 &ml ethidium bromide (EB) was added 30 min after prestaining with Hoechst dye, and 15 min later the doubly stained cells were analyzed by an ICP-22 (Ortho Diagnostic Systems) interfaced to a PDP 1I/23 microcomputer (Digital Equipment Corp.). Ultraviolet excitation (UC1 filter) was used with analysis of 33258 Hoechst fluorescence at 450-500 nm and EB fluorescence above 600 nm. The relative proportions of cells in successive cell compartments were calculated as described subsequently. For immunofluorescence/ BrdU-Hoechst analysis, fixed cells were resuspended in the same buffer as above; after 30 min incubation, 5 &ml 7-amino actinomycin D (Calbiochem) was added. Cells were analyzed on a Model 50H cytofluorograf (Ortho Diagnostic Systems) using 100 mW uv excitation (351-364 nm) from an argon ion laser, with detection of 33258 Hoechst fluorescence at 420-500 nm. The spatially separated beam of a krypton ion laser, 150 mW at 531 nm, was used to excite PE and 7AAD, with fluorescence emission detected at 566-586 nm and above 620 nm, respectively. Data were collected and processed in list-mode on the Model 2150 computer (Ortho Diagnostic Systems).

THE BrdU-HOECHST

CELL CYCLE HISTOGRAM

Figure 1 illustrates a temporal sequence of activation of human peripheral blood lymphocytes assayed by the BrdU-Hoechst fluorescence technique. Replication of DNA in the presence of BrdU results in BrdU substitution of thymidine; quenching of the 33258 Hoechst fluorescence of BrdU-substituted S- and GZphase cells (labeled BS and BG2) results in reduced fluorescence of these cells. In contrast, the dye ethidium bromide (EB) does not exhibit BrdU-dependent quenching, and simultaneous staining with EB demonstrates increased fluorescence of S and Gz phases on the EB axis simultaneously with the reduced fluorescence on the Hoechst dye axis [16, 181. Following division of the BG2 cells, the 33258 Hoechst fluorescence of the BrdU-substituted Gi cells (Fig. 1A, labeled BGJ is as little as 30% of that of the original G, cells. The magnitude of this quenching is dependent upon the degree of BrdU substitution, which in turn depends on the concentration of BrdU during cell culture, the AT/GC ratio of the target cell type [19], and the concentration of Hoechst dye [8, 161.Although the relative intensities of 33258 Hoechst and EB fluorescence are altered by energy transfer and quenching, the separation of successive cell cycles and their interpretation is not affected. Figures 1B and 1 C show that a second cell cycle in the presence of BrdU results in increased fluorescence of BBS- and BBGz-phase cells, the increase being greater in EB than in 33258 Hoechst fluorescence emission (B). Division of BBG;! cells results in a separate peak of BBGi cells closer to the origin (C); replication of these cells results in increased Hoechst and

1

128

HOECHST-FLUORESCENCE Fig. 1. 33258 Hoechst fluorescence (abscissa, top and bottom panels) and EB fluorescence (ordinate, top panels) of peripheral blood lymphocytes analyzed at (A) 36 h, (B) 46 h, (C) 60 h, and (D) 72 h after growth in the presence of PHA and BrdU. The bottom panels show histograms of the X-axis (Hoechst dye) projection of the bivariate displays shown above. Nomenclature of the three cell cycles displayed is indicated in (D), where the “B” prefix(es) indicate the number of rounds of replication in the presence of BrdU.

EB fluorescence, distinguishable from the second cycle by the lower Hoechst dye fluorescence of these cells in the third cycle (0).

DATA EXTRACTION Gi, S, and G2/M compartments of each of these three successive cell cycles can be quantitated by analysis of the respective regions of the one- or two-parameter data; Fig. 2 shows that analysis of the two-parameter data is facilitated by collapse of the two-dimensional region corresponding to a single cell cycle onto an axis parallel to that cell cycle distribution, followed by one-parameter cell cycle fitting by conventional methods [20] (the G2/GI ratio is allowed to deviate from 2.0 but this does not affect the method of quantitation). Treated in this fashion, the sequence of panels displayed in Fig. 2 shows that this particular 72-h aliquot of a human peripheral blood lymphocyte culture has, respectively, 21.4, 24.2, and 54.4% of cells in the first, second, and third cycles. Within each cycle, the distribution of cells inthe G,, S, and GdM compartments is derived by the conventional fittings, as shown in the panels of the bottom row. Furthermore, the cell cycle statistics obtained from the actual cells analyzed (“real data”) can be readily converted to numbers which reflect the proliferative history of the cells originally placed in culture (“original data”) by including a correction for division of cells which have divided once, twice, or more times [the number of cells in the second cycle (BG,, BBS, BBGJ is divided by two, the number in the third cycle (BBGi, BBBS, BBBG*) is divided by four, and the number in the fourth cycle, if any, is divided by eight. The proportions of “original” cells in each compartment are then normalized to the new total “original” cell number.] The time

312 Rabinovitch et al. HOE/BRDU

FLUORESCENCE INTENSITY n - 90755 100% REAL DATA: ORIG. DATA :

n. 19465 21,bX (15.0 45 1.91 131.8 9.6 k,Op]

n- 21989 24.2%

n- 69301 54.4%

Fig. 2. Derivation of quantitative cell cycle and cell cycle compartment data from bivariate (Hoechst/ethidium bromide) dot plot displays. The top sequence shows data from the same 72-h lymphocyte culture partitioned into (from left to right) total, first, second, and third cycle cell fractions by indicating the respective regions of the bivariate plot using an interactive computer program. The data within the region were collapsed onto a single axis, after first rotating the axis to be parallel to the axis of the bivariate cell cycle within the region. The resulting one-dimensional histogram is deconvoluted into G ,, S, and Gz compartments by conventional curve-fitting (bottom panels). At the bottom of the figure number-triplets within boxes give the percentages of cells in G,, S, and GI/M for cells in the first, second, and third cell cycle (from left to right), respectively. The data are expressed both as proportions of the cells present at the time of analysis (“real data”) and as “original data” obtained by correction to reflect the proportions of cells originally placed in culture by accounting for the number of cell divisions undergone by cells in each of the enumerated cycles (see text). The latter figure is more easily interpretable in studies of cell cycle kinetics. n=number of cells analyzed.

required for the extraction of all of this information from a single 72-h lymphocyte culture sample is approximately 15 min distributed evenly between flow measurement and computer analysis. BrdU-EFFECTS It is, of course, essential that the method of analysis does not perturb the cell cycle progression. The effects of BrdU incorporation have been examined in human peripheral blood lymphocytes and no cell cycle perturbation is seen when cells are grown (in the dark) in medium containing up to 6.5~ 1O-4M BrdU [ 171. In contrast, in human skin tibroblasts, concentrations above 10m5M lead to cell cycle retardation and partial arrest in BG, and subsequent phases; concentrations below this level with the addition of deoxycytidine can, however, be used for cell cycle kinetic analyses up to the BGi phase, and thus, unlike most techniques, the G2 to BGi transition can still be analyzed [21]. Immortal transformed lines which

BrdU-Hoechst YOUNG DONOR

FANCONI ANEMIA

cell cycle analysis

OLD DONOR

313

CYCLOSPORIN 1

! 36 HRS

48 HRS

60 HRS

72 HRS

t FLUORESCENCE INTENSITY Fig. 3. Univariate 33258 Hoechst fluorescence histograms of peripheral blood lymphocytes from (left to right) a 3-month-old healthy donor, a 17-year-old patient with Fanconi anemia, and a go-yearold donor, and cells from a 30-year-old donor treated with 0.1 pg/ml cyclosporin. Cells were analyzed at 36, 48, 60 and 72 h after growth in PHA and BrdU.

have been examined (e.g., Chinese Hamster ovary cells, murine spontaneously transformed cell lines, and human tumor lines) are more similar to lymphocytes in tolerating high BrdU concentrations with minimal effects on cell cycle progression (unpublished data).

THE KINETICS

OF CELL CYCLE PROGRESSION

Discrimination of three sequential cell cycles over a serial time course provides a unique view of cell cycle kinetics. Figure 3 shows a comparison of the cell cycle progression of PBL cultured from a young donor (A), cells from a patient with Fanconi anemia (B), an elderly donor (C) and cells from a young donor treated with cyclosporin (D). Comparison of (A) and (C) readily demonstrates the qualitative differences between these representative donors of different ages: the lagtime before cells begin to enter S phase is longer in cells from donors of an older age, the rate of cell cycle progression of these cells is slower, and at later times a greater fraction of cells remains noncycling in GdG, [17]. Figure 3 B demonstrates that cells from patients with Fanconi anemia show a substantial arrest in both first and second cycle Gz phases; thus the presumptive abnormality in DNA repair of these cells yields an observable cell cycle aberration even in the absence of DNA damaging agents [22]. In young cells treated with cyclosporin the principal feature is an extended delay before cell proliferation begins.

314 Rabinovitch et al. CELL CYCLE PHASE TRANSITION

CURVES

The kinetic features of serial cell cycle studies are best visualized by quantitation of the cell cycle compartment distributions and graphic analysis of these data. Choice of a suitable model of cell cycle behavior together with computer fitting then permits extraction of the fundamental cell cycle parameters. We have previously demonstrated that the BrdU-Hoechst data can be analyzed in a manner analogous to the data derived by the BrdU-labeled mitosis method; plots of the proportion of cells in the first, second, and third cell cycle versus time are virtually identical by the two techniques [23]. Analysis of these curves can be used to derive interdivisional times [24, 251. In contrast to the labeled-mitoses method, however, BrdU-Hoechst flow cytometry yields a greater quantity of information: this technique identifies not just the numerical order of the cell cycle of metaphase cells, but instead quantitates the Gr, S, and G2 compartment distributions of each of three successive cycles of all interphase cells. Direct measurement of cell cycle compartment specific parameters and of information pertaining to progression-arrested cells is thus possible. We have chosen to analyze the BrdU-Hoechst kinetic data in a fashion which recognizes the heterogeneity of cell cycle times within the population; such heterogeneity implies that estimates of only average cycle times can be incomplete or even misleading. Several models have been proposed to describe this heterogeneity, many of which functionally yield very similar distributions. One of the most widely referenced is that of Smith and Martin [26] who showed that the heterogeneity in time of onset of S phase and of interdivisional times can be explained as a consequence of a first order or exponential process in G,. This model can be applied to BrdU-Hoechst kinetic data by making several modifications: addition of a normally distributed heterogeneity in the lag-time before the onset of the G1 to S transition [26], provision for noncycling cells [ 151, and allowing for cell cycle compartment arrest [ 171.Figure 4 shows data derived from experiments such as those shown in Fig. 3 plotted according to the above model. The percentage of cells remaining within a particular cell cycle phase or earlier is plotted semilogarithmically versus the time after stimulation with mitogen. These percentages were obtained by curve-fitting of the BrdU-Hoechst histograms as described earlier, and are corrected for cells having divided once, twice, or more times in order to obtain growth-independent values for each compartment. As shown by the computer fitted solid lines in Fig. 4, the above model provides an excellent correspondence to the data. Each such line is a distribution curve for the times of transition from one compartment (as labeled) to the next. The similarity in transition rates (slopes) of the sequential exit curves in these panels may be explained if a single first order process which occurs in GdG, is the major source of intercellular variability in cell cycle times. Alternative models which also fit these data have been discussed [17]. Analysis of these data provide the following information: (1) GdGi lag phase and minimum compartment durations (determined by the time between intercepts with the time axis of successive compartment exit curves): (2) the mean duration of the cell cycle phase, given by the sum of the fixed minimum compartment length and the mean duration of the

BrdU-Hoechst

20

B 20

:

: : 40

cell cycle analysis

315

Cl

: :. : " 10023 k0 60 80 HOURS

60

80

100

Fig. 4. Cell cycle compartment exit curves derived from BrdU-Hoechst analysis of (A) young (3month-old donor); (B) old (80-year-old donor); (C) Fanconi anemia (17-year-old); and (D) cyclosporintreated (0.1 pg/ml) cells. In each panel, the exit from G,JG, (O), BS (A), BG2 (*), BG, (V), BBS (0) and BBG2 (0) phases are shown. The solid lines indicate the least-squares fit according to the modified Smith and Martin model.

additional variable component (given by the reciprocal of the transition rate); (3) transition rates from one compartment to the next for cells which are not arrested in the compartment (slope of the exit curve); and (4) compartment-specific cell cycle arrest (determined by the difference of the plateau phase levels between successive exit curves, as extrapolated by the computed fit).

CELL CYCLE PARAMETERS The cell cycle parameters derived from the data shown in Fig. 4 are listed in Table 1. Comparison of these parameters of PBL from representative young and old donors shows several of the features recently described [17]; cells from older donors have a substantial delay in the onset of G, phase exit, and the rate of the G,- to S-phase transition is lower. In addition, while PBL from the young donors show little or no arrest in subsequent cell cycle phases (the transition curves have plateaus which differ by at most several percent), cells from elderly donors show S and G2 and subsequent G, (BGJ phase arrests. The mechanism(s) of such arrest may relate to increased sensitivity of PBL from older donors to DNA damage, or defective DNA repair, as previously postulated on the basis of sensitivity to effects of [3H]thymidine [27]. A representative result of analysis of PHA-stimulated PBL from a patient with Fanconi anemia is shown in Fig. 4 C and compared with the results from the other donors in Table 1. The distinctive feature of the cell cycle in this genetic disease, even in these cells not treated with DNA

316 Rabinovitch et al.

1

20

40

00

80

100

1

20

40

00

80

100

HOECHST

Fig. 5. Analysis of 7-amino actinomycin D (7AAD) and 33258 Hoechst fluorescence of cells simultaneously labeled with PE-anti-CD4 or PE-anti-CD8 antibody. The analysis is gated on PE fluorescence so that the cell cycle of only CD4+ “helper/inducer” (A) or CD8’ “cytotoxicisuppresso?’ cells (I?) is displayed. Cells were grown in the presence of PHA and BrdU for 60 h.

damaging agents, is a large arrest of cells in the G2 phase of the first cell cycle, and to some degree in the second cycle as well [22]. There is, in addition, a lengthening of the minimum length of the G2 phase and a lower transition rate out of S and G2 phases (compared to the G,jGi exit rate), and thus, a lengthening of the mean S and Gz compartment durations. This provides an example of how this technique distinguishes, and quantitates separately, cell cycle prolonging and arresting effects; information which is not derived by alternative techniques. Fig. 40 and the corresponding section of Table 1 demonstrate that the principal effects of cyclosporin upon PBL cell kinetics are a delay of GdGi exit (16 h longer than the young control and 5 h longer even than PBL from the elderly donor). Other effects noted are an increased proportion of noncycling (G,,/G, blocked) cells, reduced GdG, transition rates, and an increase in the minimum S-phase compartment length. It is evident from this single example that studies of drugrelated cell cycle perturbations could benefit from the use of the BrdU-Hoechst technique. APPLICATIONS

TO IMMUNOLOGY

The BrdU-Hoechst technique is amenable to an analysis which combines identification of immunophenotypes (by use of immunofluorescent monoclonal antibodies) with the measurement of cell activation. Growth of lymphocytes in BrdU, followed by immunofluorescent labeling, then cell permeabilization by ethanol fixation allows the simultaneous detection of the immunofluorescent label and BrdU-Hoechst-quenched cell cycle analysis. Figure 5 shows the analysis of cells labeled with phycoerythrin (PE)-labeled anti-CD4 antibody or PE anti-CD8 antibody simultaneously with 33258 Hoechst fluorescence (BrdU-quenched) and 7-amino actinomycin D (7AAD) fluorescence (non-BrdU-quenched DNA fluorescence). As with the use of ethidium bromide, the 7AAD fluorescence aids in separation of Gi, S, and Gz phases of each cell cycle. This dye has fluorescence

BrdU-Hoechst

cell cycle analysis

317

TABLE 1 Compartment durations, from x-axis intercepts, Condition Age

transition rates, and compartment arrest computed slopes, and extrapolated plateau regions of Fig. 4 Young 3 months

Elderly 80 years

Fanconi 17 years

Cyclosporin 30 years

27.5+0.8 6.4.kO.4 4.5f0.4 6.4f0.6

38.5f0.9 8.8f0.5 3.7f0.2 3.750.2

34.lil.O 6.2f0.4 10.7+0.5 4.4kO.2

42.2f1.5 10.3+0.6 3.8kO.l 7.520.2

5.8f0.7 4.3kO.5 4.4+1.0 5.0fl.2

4.250.6 3.7*0.8 4.2k1.3 I .9*0.3

22.5kO.6 0.0 1.3fO.l 3.4f0.2

55.4f0.8 2.5f0.3 3.920.5 5.6f0.8

Minimum compartment duration (h)

GdG, s G2 BG, Transition rates (percent/h)

GdG, s G BG,

6.5+1.0 3.3kO.3 2.8f0.4 3.7zkO.6

2.1kO.37 1.6kO.32 l.lkO.25 0.5f0.02

Compartment arrest (%o)

GdG, s G2 BG,

29.1kO.6 2.720.1 19.5fl.O 9.620.6

43.8f2.8 1.0+0.1 7.0f 1.6 0.0

emission characteristics which allow simultaneous quantitation of 7AAD and PE fluorescence when excited by a single laser [28]. The immunofluorescence intensity was used for a gated analysis of the cell cycle: the cell cycle distribution of only CD4+ cells is shown in Fig. 5A, and the cell cycle distribution of CD8+ cells is shown in Fig. 5B. This example demonstrates that a greater proportion of CD4+ cells leave the G,,/Gr phase when stimulated with PHA. This is consistent with results obtained previously with sorted populations of CD4+ and CD4- cells [29]. The figure also demonstrates, however, that of those CD8+ which do cycle, some have progressed through the cycle (to BBS, BBG*, and BBG, phases) more rapidly than CD4+ cells. Use of simultaneous immunofluorescence analysis with the BrdU-Hoechst technique should open a fruitful area of investigation by allowing the sophistication of this cell cycle approach to be applied to analysis of antibody-defined functional subsets.

CONCLUSION The above descriptions demonstrate that BrdU-Hoechst flow cytometry offers a unique view of the quantitation of cell proliferation. This analysis is rapidly performed and provides considerably greater information than radioactive thymidine uptake or even conventional flow cytometry. Since these studies can be performed on any flow cytometer having uv excitation capabilities, it is hoped

318 Rabinovitch et al. that this presentation will stimulate the more widespread use of this new approach to the studies of mitogen response and the cell cycle. Supported by NIH Grant AGO1751 and DFG Grant 849/2-l.

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