1992;52:4090-4095. Cancer Res   Harvey D. Preisler, Venu Gopal, Shripad D. Banavali, et al.   Bioactive and Cytotoxic AgentsMultiparameter Assessment of the Cell Cycle Effects of

  Updated version

  http://cancerres.aacrjournals.org/content/52/15/4090

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  .permissions@aacr.orgDepartment at

To request permission to re-use all or part of this article, contact the AACR Publications

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on October 4, 2013. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

[CANCER RESEARCH 52, 4090-4095. August I, 1992]

Multiparameter Assessment of the Cell Cycle Effects of Bioactive and CytotoxicAgents1

Harvey D. Preisler,2 Venu Gopal, Shripad D. Banavali, Daniel Finke, and Syed A. J. Bokari

University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0508

ABSTRACT

This paper describes the use of the bromodeoxyuridine/propidiumiodide method to assess the effects of bioactive and cytotoxic agents onthe kinetic characteristics of acute myelogenous leukemia cells. By careful selection of gates, the following parameters can be measured simultaneously using only 50,000 cells: the proportion of cells in S-phase, thedistribution of cells within the S-phase compartment, the relative rate ofDNA synthesis, the relative distribution of S-phase times, the proportion of S0 cells, and the proportion of cells in GÃŒand G2/M. This methodwas used to demonstrate that while retinole acid, a-interferon, and cy-tosine arabinoside may all "inhibit" DNA synthesis, the actual effects of

these agents differ. Retinole acid appears to arrest cells in C, withoutaffecting the rate of DNA synthesis, while a-interferon and cytosinearabinoside "inhibit" DNA synthesis by reducing the rate of synthesis

per se.

INTRODUCTION

Recognition of the possible role of bioactive agents in potentiating the effects of cytotoxic agents has created a need for thedevelopment of simple methods capable of providing information on complex phenomena. This is especially the case regarding the characterization of agents capable of inhibiting the proliferation of neoplastic cells. The classical methods used toassess the effects of bioactive agents on the proliferation ofhuman cells have serious limitations (1). Measurement of thepHjdThd-1 labeling index (2, 3) provides no information on the

locus in the cell cycle at which a bioactive agent is active, norcan it easily be used to obtain accurate information on possiblealterations in the rate of DNA synthesis. Measurement of [3H]-

dThd incorporation by a cell population by means of liquidscintillation counting provides no information as to whether areduction in [3H]dThd incorporation is due to a reduction in therate of DNA synthesis or whether the reduction in [3H]dThd

incorporation is a reflection of a reduction in the proportion ofcells synthesizing DNA. In vitro cell culture proliferation assaysof more than 24 h are limited by our inability to provide in vitroconditions that accurately reproduce in vivo conditions.

In this paper, we describe how the BrdUrd/PI incorporationmethod (4-9) can be used to avoid the above limitations.

MATERIALS AND METHODS

Cell Acquisition

Bone marrow or peripheral blood specimens were obtained afterinformed consent was obtained from patients with acute or chronicmyelogenous leukemia. Specimens were anticoagulated with heparin

Received 1/2/92; accepted 5/12/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by National Cancer Institute Grant CA 41285.2 To whom requests for reprints should be addressed, at University of Cincinnati

Medical Center, 231 Bethesda Avenue, M.L. 508, Cincinnati, OH 45267-0508.3 The abbreviations used are: dThd, thymidine; PBS, phosphate-buffered saline;

IgG, immunoglobulin G; AMI , acute myelogenous leukemia; RA, retinoic acid;IFN-or, interferon; ara-C, cytosine arabinoside; IL-lra, interleukin-l receptor antagonist; BrdUrd/PI, bromodeoxyuridine/propidium iodide.

and subjected to density cut centrifugation over Ficoll-Hypaque (specific gravity, 1.077; Pharmacia, Piscataway, NJ). The light density cellswere studied.

Exposure to Bioactive and Cytotoxic Agents

Cells were placed in RPM1 1640 supplemented with 10% fetal calfserum at IO6cells/ml. The cells were cultured in the presence or absenceof the bioactive agent for 24 h in complete media (37°C,5% CO2-balance air) or for l h in the presence of ara-C. After these periods ofculture, BrdUrd was added to a concentration of 20 MM.5-Fluoro-2'-

deoxyuridine was also added to the medium (final concentration, 10 MM)to block ini raed Iular thymidylate synthetase activity (6, 7). After 1-hculture, the cells were harvested and washed in PBS; 95% ethanol(precooled to -20°C) was added dropwise with vortexing to a final

concentration of 70%. This mixing procedure prevented the clumpingof cells. The cells were kept at 4°Cfor a minimum of 30 min before they

were processed for BrdUrd staining. These fixed cells may be stored at-20°C for up to 3 months without affecting BrdUrd or PI staining.

BrdUrd/PI Analysis

Staining for BrdUrd

Ethanol-fixed cells were centrifuged, resuspended in 2 N hydrochloric acid (1 ml/5 x IO6cells) containing 0.5% Triton X-100 (Sigma), andincubated for 30 min at room temperature to denature double-strandedDNA. The acid-detergent mixture was prepared fresh just prior to use.After incubation, the cells were pelleted (2000 rpm x 10 min), and theacid supernatant was removed with care being taken to avoid cell lossfrom the pellet (which at this stage was very loose). The pellet was thenresuspended in 1 ml 0. l N sodium tetraborate (Borax; Sigma) to neutralize the acid. The cells were then pelleted and washed once in PBS.The cells could at this stage be kept in ethyl alcohol at -20°Cand the

processing continued at a later date. Fifty M!of PBS containing 1%globulin-free bovine serum albumin and 0.5% Tween-20 were thenadded to the cell pellet. A mouse monoclonal anti-BrdUrd antibodyconjugated with fluorescein isothiocyanate (Becton Dickinson) wasadded to a concentration of 1 Mg/5 x IO6 cells. The incubation wascarried out in the dark at room temperature. Cells processed in the sameway but incubated with mouse IgGl in place of anti-BrdUrd antibodywere used as a negative control. We have shown that prior incubation ofanti-BrdUrd antibody with BrdUrd (twice the amount by weight) for 1h at room temperature neutralizes the antibody. This mixture can alsobe used as a negative control in place of mouse IgGl. After 30 min, thecells were washed twice in PBS and resuspended in 0.5 ml of the same,buffer and single-color BrdUrd data collected by flow cytometry.

DNA Staining Using PI

After collecting data for single color BrdUrd analysis, the cells werepelleted and resuspended in 50 M!of PBS containing 1% globulin-freebovine serum albumin and 0.5% Tween-20. One ml of RNase A (1mg/ml; Sigma) was then added and the cells incubated at 37°Cfor 20

min. After incubation, the cells were pelleted and resuspended in PI (10Mg/ml in PBS; Sigma). Two-color BrdUrd/PI analysis was carried outafter 30 min. The whole procedure, starting from staining for BrdUrdto collection of 2-color BrdUrd/PI data by flow cytometry, shouldpreferably be done on the same day.

Flow Cytometry

Flow-cytometric analysis was carried out using a fluorescence activated cell analyzer (FACScan; Becton Dickinson Immunocytometry

4090

BrdUrd/PI ANALYSIS OF KINETICS OF AML CELLS EXPOSED TO BIOACTIVE AGENTS

Systems, San Jose, CA) interfaced with a Hewlett Packard computer(model 310). A single 15-mW argon-ion laser beam (488 nm) was usedto simultaneously excite the fluorescein and PI dyes. Optimal forwardand side scatter detection was achieved using a solid-state photodiodeand a Brewster angle beam splitter, respectively. Green fluorescencewas collected through a 530-nm band pass filter and red fluorescencethrough a 610-nm long-pass filter.

Single-Color Analysis for BrdUrd. The Consort-30 Program (Bee-ton Dickinson) was used to collect and analyze the single-color BrdUrd

data. CaliBRITE beads (Becton Dickinson) were used to optimize instrument settings and ensure proper electronic compensation: 10,000cells were measured per sample. Green fluorescence from fluoresceinisothiocyanate-labeled cells were collected, and the intensity was amplified logarithmically, stored in list mode, and analyzed.

Histograms created from the green fluorescence of the control cells(IgGl) were used to set a marker so that more than 99% of the negativepopulation fell to the left of the marker. This marker was used todetermine the percent positivity in the corresponding antibody-treatedcells. The ratio of the mean channels of the BrdUrd-positive and -negative populations was calculated by setting markers on these populations using the manual mode of the Cellfit program (Becton Dickinson).

Two-Color Analysis for BrdUrd/PI. The Cellfit program was usedfor bivariate analysis of BrdUrd/PI. Chick erythroid nuclei (DNA-QCparticles; Becton Dickinson) stained with PI were used as a standard tooptimize the instrument settings, check the CV and linearity, and setproper electronic compensation. Following this, the data were collectedfrom the BrdUrd/PI-stained cells. The BrdUrd-associated green fluorescence was amplified logarithmically (every 7 channels representing adoubling of fluorescence). The red fluorescence from PI was amplifiedlinearly; 10.000 cells were measured per tube at a rate of < 150 cells/s;data were stored in list mode and analyzed.

Reagents. The reagents used were: (a) interferon-a 2a (Roferon-A;no. 78210) from Roche Laboratories (Nutley, NJ); (b) RA, a\\-lrans (no.R-2625), BrdUrd (no. B-5002), 5-fluoro-2'-deoxyuridine (no. F-0503),

ribonuclease A (no. R-9009), globulin-free bovine serum albumin (no.A-7638), and cytosine arabinoside (no. C-1768) from Sigma; (c) RPMI1640 (no. 320-1875AJ) and fetal calf serum (no. 230-6140AJ) fromGIBCO Laboratories (Grand Island, NY); and (d) IL-Ira (no. 078-90)(10, 11) from Synergen, Boulder, CO.

RESULTS

Fig. 1 illustrates the types of analyses that can be performedusing the BrdUrd/PI method. The 2-color representation (Fig.\A) permits a general appreciation of the effects of agents andculture conditions on the proportion of cells in S-phase, on thedistribution of cells within the S-phase compartment, on therelative rate of DNA synthesis, on the distribution of S-phasetimes, and on the proportion of S0 cells. Fig. l B (left) illustratesa single color analysis of BrdUrd incorporation into DNA.Assessment of the percent of cells containing BrdUrd providesinformation on the percent of cells in S-phase, while quantita-tion of fluorescence intensity provides information on the relative rate of DNA synthesis. Fig. IB (right) demonstrates thatthe combining of data from cells that have incorporated BrdUrdwith a red fluorescence DNA histogram permits assessment ofthe distribution of cells within S-phase. Fig. 1C illustrates thatby obtaining a mid-S-phase "cut" in the red DNA histogram

one can obtain the distribution of green fluorescence amongthese cells, thereby permitting quantitation of the proportion ofS0 cells as well as of the distribution of S-phase durations.

Fig. 2A provides a BrdUrd/PI analysis of the effects of RA onAML cells in vitro. The 2-color analysis in Fig. 2A illustratesseveral points. Comparison of the preculture and 24-h controlculture demonstrates that overnight culture resulted in theappearance of cells whose DNA content is between 2 and 4 Nbut that failed to incorporate BrdUrd. These cells are putatively

G2/M

red fluorescence

B

Cells which haveI incorporated BrdU

green fluorescence

red fluorescence

red fluorescence green fluorescenceFig. 1. Flow-cytomctric analysis of cell kinetic parameters. A, 2-color display:

green, BrdUrd incorporation; red, distribution of DNA content. All cells in the loprectangle represent cells in S-phase. S0 cells are found in the region where BrdUrdincorporation (green fluorescence) is nil but in which cellular DNA content is >2N but <4 N. B, left panel, single-color analysis of BrdUrd incorporation. Todetermine the distribution of BrdUrd-containing cells within the S-phase compartment, the green fluorescing cells are gated to provide a histogram of their redfluorescence using the RFIT program. The distribution of red fluorescence amongthese cells is then superimposed (cross-hatched area in the right panel) on theDNA histogram of the cell population as a whole. Areas A, B, and C representearly, mid-, and late S-phase, respectively, by DNA content. The distribution ofBrdUrd-containing cells within these 3 areas indicates the distribution of cellswithin the S-phase compartment. C. distribution of green fluorescence (right) ismeasured among the cells whose DNA content is between 2 and 4 N (left). Thelogic behind assessing BrdUrd incorporation among these cells is that, given theusual length of S-phase and the short exposure time to BrdUrd, all cells with thisDNA content and that are synthesizing DNA will be doing so throughout theentire incubation period with BrdUrd. Cells to the left of the dotted vertical line inthe right panel are in S0; those to the right of the line are in S-phase. Given theabove, the distribution of green fluorescence to the right of the dotted line indicatesthe relative distribution of S-phase times among the cells in the population beingstudied.

in S0. Fig. 2B provides a mid S-phase "cut" to quantitate the

proportion of S0 cells. Note that in the t = 0 specimen, 89% ofthe cells in mid-S-phase have incorporated BrdUrd, while this isthe case for only 42-45% of the cells incubated overnight. The

4091

BrdUrd/PI ANALYSIS OF KINETICS OF AML CELLS EXPOSED TO BIOACTIVE AGENTS

Fig. 2. Effect of RA on AML cells in culture. A, 2-color analysis of BrdUrd incorporation and DNA content. Note appearance of S0cells in 24-h cultures and reduction of cells inearly S-phase. The latter is especially the casefor the culture containing RA in which therealso appears to be a relative increase in lateS-phase. Despite these clear-cut differences inthe distribution of cells in S-phase in both cultures, the actual proportion of cells in S-phaseis the same in both cultures. />".distribution of

S0 cells. Note that RA had no effect on theproportion of S0 cells. IgG control representscells incubated with IgG instead of the uniiBrdUrd antibody. C, distribution of cellswithin the S-phase compartment. Note that inthe f = 0 culture. 52% of S-phase cells are inearly S. This falls to 30% after overnight culture and to 6% after overnight culture withRA. D. single color analysis of BrdUrd incorporation. Note that the percent of S-phasecells in both cultures is indistinguishable.

t-0

B

•¿�£.~f,.y.^*...

. «S•uC0)uWo>osTÃŽ4)1^-•¿�' " '•

24 Hr. Culture24 Hr. Culture »FC3 * Fca * RA

IgG Control [t-0]

189%- 42%+.,'4tU]1'II l 45% +•u4% +

R«d Fluorescence Rid Fluorescence Red Fluorescence

8.8%H 3.7%H iiI13.9%H

Qrt«nFluofMCtnc» Qr»«nFluoraiccnc« Qr««nFluor*ic«nc«

Table I Effects of retinole add on AML cells in vitro

SpecimenControl

24 h24 h + RA%

S-phase (DNA)histogram5.4

2.93.1%

S-phase(BrdUrd-positive)8.8

3.73.9%

S-phasedistribution%S015.0

58.055.0Early52.0

30.06.0Mid-27.0

13.025.0Late21.0

57.069.0Fluorescence

intensity"3.7

3.93.8%

Distribution ofS-phasetimes*Minimum''21

2329Maximum'186

200191

" Ratio of median green fluorescence (channel number) of BrdUrd-containing cells to median green fluorescence of the BrdUrd-negative cells.* Percentage of linear mean fluorescence intensity.*'The relative minimum and maximum S-phase durations were calculated by dividing the brightest and lowest green fluorescence values by the peak value (Fig. 1C) and

multiplying by 100. Hence, S-phase durations in the control cells range from 21 to 186% of the median value for the population as a whole. If the median value was 10h. then the times of the cells in the population would range from 2.1 to 18.6 h.

presence of RA had no effect on the proportion of cells in S0(Table 1). Comparison of the 2-color BrdUrd/PI analyses (Fig.2A) for the overnight control culture and the 24-h culture containing RA demonstrates an apparent absence of cells in earlyS-phase in the latter culture. Fig. 2C provides the distributionof cells within the S-phase compartment. These data demonstrate that overnight culture per se resulted in an alteration inthe distribution of cells within the S-phase compartment so thata larger proportion of cells are in late S-phase in the 24-hculture. The presence of RA accentuated this process so thatthere are almost no cells in early S-phase (Table 1). Fig. 2Dprovides a single color histogram analysis of BrdUrd incorporation. These histograms illustrate that the fluorescence intensities of all 3 specimens are indistinguishable but that the percent of cells containing BrdUrd was reduced in the 2 culturedspecimens relative to the / = 0 control. Table 1 provides thenumerical data. Note also that for these AML cells the presenceof retinoic acid did not affect the distribution of S-phase times.

Fig. 3 provides a comparison of the effects of RA, IFN, andan antagonist of the IL-1 receptor on blastic crisis CML cells.In this case, the 24-h culture does not contain S0 cells. Thehigher concentration of RA reduced the overall proportion ofcells synthesizing DNA and the proportion of cells in earlyS-phase, and altered the distribution of cells within the S-phasecompartment (Table 2). Cells incubated with the lower concentration of RA were indistinguishable from control cells. In contrast, IFN and IL-lra dramatically reduced the rate of DNAsynthesis to the point that essentially all cells whose DNAcontent was between 2 and 4 N appeared to enter S0 in the2-color projection (Fig. 3/4). Note that in Fig. 35, however,while the amount of BrdUrd incorporation was markedly reduced (i.e., fluorescence intensity), S-phase cells were still recognizable. Taken together with Fig. 3/4, it is clear that the2-color method as currently used cannot distinguish between S0cells and cells that are synthesizing DNA very slowly. Table 2provides the numerical data. In this study, the data suggest that

4092

Brdllrd/Pl ANALYSIS OF KINETICS OF AML CELLS EXPOSED TO BIOACTIVE AGENTS

24 Hr. Culture•¿�PCS

24 Hr. Culture * PCS•¿�1.6x10-SMoCOímo3•oO•*•»•',•—

J-

Bgreen fluorescence

Hr Culture •¿�PCS

26.3%l:l

A

green fluorescence~— 24 Hr. Culture

l •¿�PCS •¿�RAA 1.6x10'!M

24 Hr. Culture *PCS5x1Û-7MC(Dco

0)o3TJi124

Hr. Culture1 24 Hr. Culture *ILI 0ta*IFN 12500 u/mll Q.tuQ/ml0C01luoresc•a0)IUCiUìi03•o0)1green

fluorescence yreen fluorescence greenfiuorescenc*01or^24Hr Culture r— 2< Hr Culture r__24 Hr.CullunÕ»PCS •¿�RA

| 5 x 10"7M\

30.8%[»\l/\Õo•

IFN[2500A

26.1Au/ml

^%[»!

S,

»IL1<3réA 0.1ug/mlI

\ A26.1%|<•v¡fluorescence intensity

24 Hr Culture •¿�PCS

81.0*1-1

fluorescence intensity

24 Hr. Culture •¿�PCS •¿�RA1.6x10'5M—«

fluorescence intensity fluorescence intensity fluorescence intensity

24 Hr. Culture •¿�PCS•¿�RA

86.0%H

5 x 10 M

95.2%H

green fluorescence green fluorescence green fluorescence

Fig. 3. Effect of RA. IFN. and IL-lra on cell kinetic parameters in myeloid blastic crisis of CML. A, 2-color analysis. B, single color analysis of BrdUrdincorporation. Note that the amount of BrdUrd incorporation as indicated by the fluorescence intensity of S-phase cells is the same as the controls for cells culturedin the presence of RA. but that the percent of cells incorporating BrdUrd is lower in cells exposed to the higher concentration of RA. In contrast, exposure of IFN orthe IL-lra resulted in a decrease in fluorescence intensity, indicating a reduction in the rate of DNA synthesis. C BrdUrd incorporation by cells whose DNA contentwas in mid-S-phasc. Note lack of effect of RA on proportion of S0 cells. In contrast. BrdUrd incorporation by cells exposed to IFN or IL-lra was so low as to makethese cells indistinguishable from S0. See Fig. 2A.

Table 2 Comparison of effects ofretinoic acid, interferon, and IL-lfira on myeloid hlastic crisis CML cells

% S-phasedistribution24

h24

h24h24h24hSpecimen+RA(1.6 x IO-5M)+

RA (5 X IO"7M)+

a-IFN (2500units/ml)+

IL-l-0ra (0.1 Mg/ml)%

S-phaseDNAhistogram19.58.720.513.912.9%

S-phasc(BrdUrd-positive)26.313.030.826.125.1%S019.014.04.8100.0100.0Early56.638.257.344.762.7Mid-27.035.925.522.421.1Late16.425.917.232.916.2Fluorescenceintensity3.52.74.12.72.6%

Distribution ofS-phasetimes"Minimum425457NA*NAMaximum175212215NANA

" Percentage of linear mean fluorescence intensity.* NA, not available.

in this case, in addition to producing a G, arrest, the presenceof RA was associated with a reduction in the proportion of cellswith the shortest S-phase durations and with the appearance ofcells with longer S-phase times than were present in the controlspecimen.

Fig. 4 illustrates the effects of ara-C on DNA synthesis inAML cells. Fig. 4 and the data in Table 3 demonstrate that thiscytotoxic agent reduces the percent of cells in S-phase, reducesthe rate of DNA synthesis, and increases the proportion of "So"

cells. These effects increase when cells are exposed to increasingconcentrations of the agent (Table 3). The data in Table 3suggest that at high concentrations of ara-C, cells are arrestedin G,. This is reflected by the relative decrease in the proportionof cells in early S-phase. Also note that none of these effects isdiscernible if the DNA histogram is used to assess the distribution of cells in S-phase. In fact, if one used this method toevaluate the effects of exposure to ara-C for 1 h, no effect wouldhave been discernible.

DISCUSSION

The methods described here extend the utility of theBrdUrd/Pl technique originally described by Dolbeare et al. (4).The simultaneous assessment of the DNA distribution and BrdUrd incorporation by a cell population permits measurementof the effects of inhibitory (or stimulatory) agents on the percent of cells synthesizing DNA, the rate of DNA synthesis, thedistribution of cells within the cell cycle, the distribution of cellswithin the S-phase compartment, and the proportion of S0cells. The differing effects of RA, IFN, IL-lra, and ara-C havebeen used to illustrate the utility of this method.

While only a limited amount of data are presented here, theyare representative of a larger number of studies that will bereported elsewhere. It is of interest that the mere incubation ofAML cells in suspension culture for as little as 24 h can alter thekinetic characteristics of these cells. As demonstrated here,many AML cells that were synthesizing DNA immediately after

4093

BrdUrd/PI ANALYSIS OF KINETICS OF AML CELLS EXPOSED TO BIOACTIVE AGENTS

Fig. 4. Effect of cytosine arabinoside onDNA synthesis. A. 2-eolor analysis of BrdUrdincorporation and DNA content. The extension of green fluorescence in cells with dDNA content beyond that present in cellswhose DNA content is >2 N may indicate anarrest of cells in very early S-phase, so earlythat the DNA content of these cells is not distinguishable from that of GI cells. B, singlecolor evaluation of BrdUrd incorporation.Note that increasing concentrations of ara-Creduce both the proportion of cells synthesizing DNA and their fluorescence intensity. C,distribution of BrdUrd incorporation by cellswhose DNA content is mid-S-phase. At thehigher ara-C concentrations, BrdUrd incorporation was so low that the weakly positive cellswere not distinguishable from S0 cells. /). lackof effect of 0.03 ¿tg/mlof ara-C on distributionof cells within S-phase.

ControlIncubation x 1 Hr

No araCaraC X 1 Hr0.03 ug/ml

araC0.3X

1Hrug/ml"I araC3.0-JX

1Hrug/ml

green fluorescence green fluor»»c«nc*

¿2.1%1-HVAflreen fluorescence green fluorescence green fluorescence green fluoretcenc«

¡!33.6%[+)

green fluorescence green fluorescence

D

red fluorescence red fluorescence

Table 3 Effects of ara-C on DNA synthesis in AMLcellsGroupIncubationIncubationIncubationIncubationx

1x

1X

1X

1h

(noara-C)h

(ara-C), 0.03xg/mlh

(ara-C), 0.3>¿g/m]h

(ara-C), 3.0 «ig/ml%

S-phase DNAhistogram15.614.913.616.1%

S-phase(BrdUrd-positive)10.88.22.10%

S-phasedistributionSo66.499.498.9100.0Early46.841.250.622.0Mid-24.624.021.322.0Late28.634.828.156.0Fluorescence

intensity2.42.21.71.4%

Distribution ofS-phasetimes"Minimum68NA*NANAMaximum204NANANA

" Percentage of linear mean fluorescence intensity.* NA, not available.

removal from a patient may become arrested in S-phase whenplaced into culture. This was not the case for blastic crisis CMLcells. The presence of RA reduces the percentage of cells inS-phase by apparently arresting the cells in G|. This bioactiveagent does not appear to arrest cells within S-phase.

IFN and IL-Ira, at the concentrations used here, markedly

slowed the rate of DNA synthesis to the point that those cellsremaining in S-phase could not be distinguished from S0 cellswhen the 2-color method described here was used. Unlike RA,these 2 bioactive agents did not arrest cells in G i. In contrast, atlow concentrations, ara-C reduced the rate of DNA synthesiswhile at higher concentrations there appeared to be a G, arrestas well.

Figs. 3 and 4 and the data in Tables 2 and 3 illustrate oneproblem with our current method. As alluded to above, in theIFN and IL-Ira studies and the ara-C 0.3 and 3 Mg/ml studies,it is possible to discern cells that have clearly incorporatedBrdUrd but that are not recognizable in the 2-color displays.

taOoD:r*fi4|!

i—1 1S.Jft?1

Fluorescence IntensityFig. 5. Green fluorescence of mid-S-phase blastic crisis cells exposed to inter-

feron 2500 units/ml and reacted with either IgG or the anti-BrdUrd antibody.Dotted line, dividing line between BrdUrd (+) and BrdUrd (-) cells as estimatedby 2-color analysis. Note that while the distribution of green fluorescence of theIgG control and the antibody-treated cells is to the left of the dotted line, thedistributions are different, demonstrating that small amounts of BrdUrd wereincorporated into DNA. Despite being cultured with IFN, these cells incorporatedBrdUrd as illustrated in Fig. 3Ä.This small amount of incorporation will bedistinguishable from So cells in the mid-S-phase "cut" once a curve subtraction

method is added to our current capabilities. Shown is an analysis of the cellsfocused on in Fig. 3.

4094

BrdUrd/PI ANALYSIS OF KINETICS OF AML CELLS EXPOSED TO BIOACTIVE AGENTS

Furthermore, when the mid-S-phase "cut" is made in these

studies to identify S0 cells, all cells appear to be in S0. It is clearthat the ability to distinguish between cells that have incorporated very small amounts of BrdUrd and those that have incorporated no BrdUrd is much reduced when 2-color analysis isbeing performed. Hence, the method described here for detecting and quantitating the effects of agents on the rate of DNAsynthesis becomes progressively limited when the of DNA synthesis is very slow. Fig. 5 demonstrates that this analysis will bepossible when a "curve subtraction" method is incorporated

into our present studies. While these superimposed curves canbe produced by current FACScan software, the numerical analysis of these data will require the rewriting of the FACScansoftware.

The studies described here demonstrate that multiple measurements can be made in cells exposed to bioactive or cytotoxicagents, measurements that help to define the effects of theseagents on target cells.

REFERENCES

1. Katano, N., Tsurusawa, M., Niwa, M., and Fujimoto, T. Flow cytometricdetermination with bromodeoxyuridine/DNA assay of sensitivity of S-phasecells to cytosine arabinoside in childhood acute lymphoblastic leukemia. Am.J. Paed. Hematol.-Oncol.. 77:411-416, 1989.

2. McDivitt, R. W., Stone, K. R., Craig, R. B., and Meyer, J. S. A comparisonof human breast cancer cell kinetics measured by flow cytometry and thymi-dine labeling. Lab. Invest., 52: 287-291, 1985.

3. Meyer, J. S., and Coplin, M. D. Thymidine labeling index, flow cytometricS-phase measurement, and DNA index in human tumors: comparisons andcorrelations. Am. J. Clin. Pathol., 89: 586-595. 1988.

4. Dolbeare, F.. Gratzner, H.. Pallavicini, M. G., and Gray, J. W. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuri-dine. Proc. Nati. Acad. Sci. USA, 80: 5573-5577, 1983.

5. Dean, P. N., Dolbeare, F., Gratzner, H., Rice, G. C. and Gray, J. W. Cellcycle analysis using a monoclonal antibody to BrdUrd. Cell Tissue Kinet., 77:427-436. 1984.

6. Raza, A., Preisler, H., Meyers. G.. and Bankert. R. Rapid enumeration ofS-phase cells by means of monoclonal antibodies. N. Engl. J. Med., 570:991,1984.

7. Beisker. W., Dolbeare, F., and Gray, J. W. An improved immunocytochem-ical procedure for high-sensitivity detection of incorporated bromodeoxyuri-dine. Cytometry, 8: 235-239, 1987.

8. Gratzner, H. G., and Leif, R. C. An immunofluorescence method for monitoring DNA synthesis by flow cytometry. Cytometry. /: 385-389, 1981.

9. Waldman. F. M., Dolbeare. F., and Gray, J. W. Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNAassay. Cytometry, 6: 657-662, 1985.

10. Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin. F. G.. Dripps, D. J.,Heimdal, P. L., Armes, L. G., Sommer, A., Eisenberg. S. P., and Thompson.R. C. Intri lâ„¢kin 1 receptor antagonist activity of a human interleukin-linhibitor. Nature (Lond.), 343: 336-340. 1990.

11. Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T.,Hannum. C. H., and Thompson. R. C. Primary structure and functionalexpression from complimentary DNA of a human interleukin-l receptorantagonist. Nature (Lond.), 343: 341-346. 1990.

4095