[CANCER RESEARCH 54, 4289-42M. August 15. 1994]

Advances in Brief

Simultaneous Analysis of DNA Replication and Apoptosis during Treatmentof HL-60 Cells with Camptothecin and Hyperthermia andMitogen Stimulation of Human Lymphocytes1

Xun Li, Frank Tráganos, and Zbigniew Darzynkiewicz2

The Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595

Abstract

A new method is described which combines the identification of DNAreplicating and apoptotic cells in a single measurement by flow cytometry.The detection of DNA replicating cells is based on incorporation of5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine, followed by selective

photolysis at the site of incorporation of the halogenated DNA precursors.Single-strand breaks in DNA resulting from the photolysis are subsequently labeled with digoxygenin or biotin-conjugated dUTP in a reaction

catalyzed by exogenous terminal deoxynucleotidyl transferase. The double-stranded DNA breaks in apoptotic cells resulting from activation of

the endonuclease can be labeled in this reaction as well. However, incontrast to the photolysed DNA, the low molecular weight fraction of DNAof apoptotic cells is extractable from the cells, and the degree of DNAelution can be modulated by cross-linking with formaldehyde. Thus,

apoptotic cells can be distinguished and quantified by virtue of theirfractional DNA content. Replication of less than I % of a genome of a cellin the presence of 5-bromo-2'-deoxyuridine (equivalent of a 5-inin 10 JUM5-l)i iiiii"-2'-(lrci\v in idiiie pulse) can be detected by the selective photolysis

method. The method was applied to study apoptosis and proliferation ofhuman leukemic HL-60 cells and normal, mitogen-stimulated lymphocytes. Whereas apoptosis of HL-60 cells induced by the DNA topoisomer-

ase I inhibitor camptothecin was selective to DNA replicating cells, apoptosis induced by hyperthermia showed no such selectivity. Lymphocytesthat preferentially underwent apoptosis in cultures stimulated by phyto-

hemagglutinin did not initiate DNA replication. By offering the possibilityfor identification of both DNA replicating and apoptotic cells in a singlemeasurement, the method may find an application in studies of theprognostic value of both cell proliferation and death in human tumors andthe apoptotic response of DNA replicating is. nonreplicating cells todifferent treatments.

Introduction

Tumor growth rate is regulated by the frequency of cell proliferation and the rate of cell death. Cell death in tumors, whether spontaneous or treatment-induced, occurs predominantly by the mode of

apoptosis (reviewed in Refs. 1 and 2). A plethora of methods toanalyze the rate of cell proliferation, either directly by measuring theproportion of cells synthesizing DNA or indirectly by estimating theexpression of one or more of the proliferation-associated proteins,

have been developed (3, 4). These methods have become common inexperimental and clinical studies, and the prognostic value of prolif-

erative markers is now widely recognized. In contrast, the analysis ofcell death in tumors has been neglected thus far, and until recently,relatively few methods have been available to identify apoptotic cells(reviewed in Ref. 5). Recent studies, however, indicate that the

Received 5/5/94; accepted 6/22/94.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by National Cancer Institute Grant CA 28704.2 To whom requests for reprints should be addressed, at The Cancer Research Institute,

New York Medical College, ITO Grassland Road, Elmsford. NY 10523.

intrinsic propensity of tumor cells to undergo either spontaneous ordrug-induced apoptosis is an important cellular feature that may berelated to overall prognosis (6-9). This propensity is modulated by theexpression of several oncogenes such as bcl-2 (10), c-myc (11), and

ras (9) or the tumor suppressor gene, p53 (12, 13). The availability ofan assay which combines the analysis of cell proliferation and celldeath in a single measurement, therefore, is desirable. The conventional procedure for analysis of DNA proliferation by detecting incorporation of the thymidine analogue BrdUrd' with antibody re

quires DNA denaturation by heat or acid to make the epitope(BrdUrd) accessible to the antibody (3,14). We observed that this stepis incompatible with the detection of apoptotic cells, the DNA ofwhich is totally extracted during heat or acid treatment.

The analysis of cell proliferation presented in this paper also isbased on incorporation of BrdUrd or IdUrd, which, however, isfollowed by the DNA SBIP. This approach, which does not requireDNA denaturation, stems from the observation that DNA containingincorporated BrdUrd undergoes degradation when exposed to UVlight (15). The DNA strand breaks generated by photolysis are thenlabeled with b-dUTP or d-dUTP in a reaction using exogenous TdT.

The latter step, previously used to label DNA strand breaks in cellsundergoing apoptosis (16, 17), has been designed in the presentmethod to label DNA containing the halogenated DNA precusors.Apoptotic cells, in turn, are identified based on their reduced DNAcontent, following extraction of the degraded, low molecular weightDNA. Thus, the method allows one to discriminate between cellscontaining DNA strand breaks due to apoptosis and cells which haveDNA strand breaks induced by photolysis as a result of BrdUrd orIdUrd incorporation. A preliminary report describing the principle ofSBIP and its application to detection of DNA replication in tissuesections of tumors labeled with BrdUrd in vitro has been published(18).

Materials and Methods

Cells. All experiments were carried out on the human leukemic HL-60 cell

line or on normal human peripheral blood lymphocytes stimulated to proliferate in vitro by the mitogen PHA which was administered 48 h prior toincubations with BrdUrd. The HL-60 cell line, the cell generation time (Tc) of

which is approximately 22 h, was maintained in RPMI 1640 (GIBCO/BRLLife Technologies, Inc., Grand Island, NY) supplemented with 10% fetal calfserum, 100 units/ml penicillin, 100 fig/ml streptomycin, and 2 mM L-glutamine

and was periodically tested for Mycoplasma infection. The cells were growingexponentially at densities below 5 x 10s cells/ml. BrdUrd (Sigma Chemical

Co., St. Louis, MO) or IdUrd (Sigma) were added to cultures (in completemedium) at 10-50 fiM for various periods of time as indicated in the legendsto the figures. To induce apoptosis, HL-60 cells were either treated with 0.15

3 The abbreviations used are: BrdUrd, 5-bromo-2'-deoxyuridine; IdUrd, 5-iodo-2'-

deoxyuridine; SBIP, strand breaks induced by photolysis labeling method; b-dUTP,biotin-conjugated dUTP; d-dUTP, digoxygenin-conjugated dUTP: TdT, terminal deoxynucleotidyl transferase; PHA, phytohemagglutinin; CAM, camplothecin; FITC, fluo-

rescein isothiocyanate.

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Fig. 1. DNA replication in HL-6() cells delectedby the SBIP method following incubation with Br-dUrd or IdUrd with or without exposure to UV light.HL-60 cells were incubated with 10 UM BrdUrd(left) or IdUrd (righi) for 1 h, washed, and eitherilluminated with UV light (top) or left in the dark(bottom). The samples were then fixed and exposedto d-dUTP in the presence of TdT; DNA was coun-terstained with propidium iodide. Green (FITC-anti-

digoxygenin, representing DNA strand breaks) andred (DNA) fluorescence was recorded for each of1 X IO4 cells. Contour maps of the data, presenting

bivariate analysis of DNA strand breaks versus DNAcontent, were produced with the LYSYS II softwareof the FACScan flow cytometer. Note: only in cultures exposed to UV light were DNA strand breaksgenerated, and they were limited to S cells, as identified by DNA content. The cells incubated in theabsence of BrdUrd or IdUrd but exposed to UV lighthad similar fluorescence as the cells in bottom panels. Le., showed no evidence of DNA strand breaks(not shown). The single-parameter DNA histograms

for each culture are inset in the lop panels.

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DNA ContentFig. 2. The sensitivity of detection of BrdUrd incorporation by the SBIP method as a function of length of incubation with the halogenated DNA precursor. Exponentially growing

HL-60 cells were cultured in the presence of 10 (¿MBrdUrd for 5, 10, 20, or 30 min; each aliquot was split, and one-half of the cells were exposed to UV light for 5 min while theremainder were kept in the dark. After fixation, the cells were rehydrated and incubated with d-dUTP and TdT. Incubation with BrdUrd for 10-30 min provided sufficient strand breaksto unequivocally identify the DNA-replicating cells from those in G, and G2 + M which did not incorporate the precursor: the limits of d-dUTP-associated green fluorescence of theunirradiated cell aliquot (bottom panels) is indicated by the Irapezoid in each upper panel. Reducing incubation lime to 5 min still allowed identification of the DNA-replicating cells,although there was some overlap with the unirradiated control distribution (lower panel). The green fluorescence photomultiplicr gain was adjusted to give maximum fluorescence ofthe S population in the upper panel but was the same for each pair of the measurement (UV-exposed and nonexposed sample). The DNA content distribution, which was the samefor all variations in BrdUrd exposure, is inset in the top leftmost panel.

JAMCAM for 3 or 4 h or heated at 43°Cfor 40 min and then grown at 37°C

for 3 or 4 h as described before (16, 17). In other experiments, the cells werefirst preincubated with 10 ¡J.MBrdUrd for 30 min and then rinsed free of drugand treated with CAM or heat, as above.

Following incubation with the halogenated DNA precursors, the cells weresuspended in cold phosphate-buffered saline and exposed to UV light. It has

been empirically determined by testing a variety of light sources and illumination conditions that exposure of the cell suspension in Petri dishes positioned

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SBIP METHODOLOGY

directly on the glass surface of the Foto UV 300 analytical DNA transillumi-nator containing four 15-watt 300-nm bulbs (Fotodyne, Inc., New Berlin, WI)for 5 min was optimal in terms of induction of strand breaks in BrdUrd-labeledcells, while 15 min was required for IdUrd-labeled cells. The cells were thenfixed in suspension in 0.3% methanol-free formaldehyde (pH 7.4) at 0-4°C for15 min, then rinsed in Hanks' balanced salt solution, suspended, and stored in

70% cold (4°C)ethanol. In some experiments, different concentrations of

formaldehyde (0.1-1.0%) were tested, while cells were also fixed directly in

70% ethanol, bypassing the formaldehyde treatment step (see legend to Fig. 3).DNA Strand Break Labeling. In most experiments reported in this study,

the strand breaks generated by photolysis of DNA were labeled with d-dUTP

followed by staining with a fluoresceinated antibody to digoxygenin. This wasaccomplished using the commercial kit for DNA strand break labeling, developed to identify apoptotic cells (ONCOR, Inc., Gaithersburg, MD; ApopTagkit), according to the protocol included with the kit by the vendor. In someinstances, b-dUTP was used according to the procedure described by us before

(15, 16). Both methods provided essentially the same results.Cytometry. Green (dUTP) and red (DNA) fluorescence of individual cells

was measured on a FACScan flow cytometer (Becton Dickinson, San Jose,CA). The data from IO4 cells/sample were collected and stored using LYSYS

II software. Further details were presented before (16, 17). The experimentswere repeated at least three times, yielding essentially identical results.

Results

Detection of Incorporated BrdUrd or IdUrd. Fig. 1 illustratesincorporation of BrdUrd and IdUrd by exponentially growing HL-60

cells that were incubated in the presence of a 10-/AMconcentration of

these halogenated DNA precursors for 1 h. The data clearly show thatthe cells identified by their DNA content as residing in G, and inG2+M phases of the cell cycle failed to incorporate BrdUrd or IdUrdduring pulse labeling; their green (FITC) fluorescence was identical tothat of cells that were not illuminated with UV light. In contrast, cellshaving a DNA content intermediate between 2C and 4C, i.e. in Sphase, exhibited strong green fluorescence. HL-60 cells that were

incubated in the absence of BrdUrd or with BrdUrd but in thepresence of the DNA polymerase inhibitor aphidicolin (2 /XM)or theantimetabolite ara-C (25-100 /AM)remained unlabeled (not shown).

The SBIP method was sensitive enough to detect BrdUrd incorporation following short exposure times of HL-60 cells to the precursor(Fig. 2). There was virtually no overlap of BrdUrd-labeled S cellsbetween unirradiated and irradiated cultures after a 10-min incubation,while the overlap was minimal after as little as 5-min incubation with

BrdUrd. Since the S phase duration of this cell line is about 10 h,approximately 0.8% of the genome is replicated in 5 min.

Detection of Apoptotic Cells. Following cell fixation in ethanol, alarge portion of DNA from apoptotic cells can be extracted if the cellsare repeatedly washed in aqueous media or even briefly treated withphosphate buffer at somewhat elevated pH (5, 19). This observation isthe basis for identification of apoptotic cells by flow cytometry ascells with a fractional DNA content (19). DNA in necrotic cells, in

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DNA ContentFig. 3. DNA replication and apoptosis in cultures of HL-60 cells treated with DNA topoisomerase I inhibitor CAM or subjected to hyperthermia. A, exponentially growing cells

were maintained in (he presence of BrdUrd for 30 min and then were fixed and incubated with TdT and d-dUTP lo label DNA strand breaks. Note the lack of cells with DNA strandbreaks. B, the cells were treated identically as in (A), except thai following incubation with BrdUrd, the cells were exposed to UV light for 5 min. Noie the extensive number of DNAstrand breaks in S cells. C, the cells were treated as in (B) but after a 30-min exposure to BrdUrd were incubated in the absence of this precursor for an additional 4 h. Note the cellprogression through the cycle; compared (B), the cohort of cells with DNA strand breaks have moved from early and mid-S to late S. G2 + M, and some even to G,. D, the cells were

preincubalcd for 30 min with IO JÌMBrdUrd and then were grown in the presence of 0.15 p.MCAM for4h. Note the loss of S cells and the presence of apoptotic cells (Ap) characterizedby a large number of DNA strand breaks; the Ap cells are well separated from the nonapoptotic cell population. E, the cells were processed as in (D), except for treatment withformaldehyde (in all other panels, the cells were treated with 0.3% formaldehyde for 15 min). In the absence of formaldehyde, apoplotic cells have minimal DNA content and are lessdistinguishable from the background. F, the cells were preincubaled with 10 /¿MBrdUrd for 30 min, then washed free of the precursor, heated al 43°Cfor 30 min, and then grownat 37°Cfor an additional 3.5 h. Compared with CAM (D). most apoptotic cells have a low number of DNA strand breaks; the S cells remain within the nonapoptotic cell population.

Insets, DNA frequency distribution histograms of the respective populations. Broken lines, the maximal level of FITC fluorescence of the respective control cells, identically treatedexcept for the exposure lo UV lighl.

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SBIP METHODOLOGY

contrast, appears to be more stable (5). Initially, we observed that, inthe course of the SBIP procedure, the loss of DNA from apoptoticcells that were prefixed in ethanol was extensive, resulting in difficulties in distinguishing such cells from subcellular debris (18). Attempts, therefore, have been made to partially stabilize DNA, allowing cells undergoing apoptosis to both be distinguished from live cellsand yet be effectively separated from the locus of subcellular debris.Empirically, by testing different concentrations, we have observedthat cell prefixation for 15 min in 0.25-0.3% formaldehyde, followed

by storage of the sample in 70% ethanol, was optimal for bothdetection of apoptotic cells as well as identification of cells thatincorporated BrdUrd or IdUrd (Figs. 3 and 4).

Apoptosis of HL-60 cells was induced, in the present experiments,

by the DNA topoisomerase I inhibitor CAM or by hyperthermia. Asis evident from the data shown in Fig. 3, live cells could easily bediscriminated from apoptotic cells in both the CAM- and heat-treated

cultures based on the lowered DNA stainability of the latter. Identification of cells incorporating BrdUrd among the nonapoptotic cellswas also possible, in as much as these cells had a full complement ofDNA and a distinct, d-dUTP-associated fluorescence (Fig. 3, compare

B with A).The extent of d-dUTP-associated fluorescence of apoptotic cells

provided information about their BrdUrd labeling and thus their DNAreplication status vis-à-vis their sensitivity to treatment. Thus, forexample, in the case of CAM-treated cells, it was apparent thatapoptotic cells had very high d-dUTP fluorescence (Fig. 3D). This

fact, combined with the observed loss of BrdUrd incorporating cellsfrom the "live" cell population (Fig. 3, compare D and B), provided

direct evidence that the BrdUrd-incorporating cells selectively underwent apoptosis in these cultures. Their strong d-dUTP fluorescence is

the result of extensive DNA breakage due to both the action of theapoptosis-specific endonuclease as well as photolysis.

When HL-60 cells were subjected to hyperthermia (Fig. 3F), mostapoptotic cells had lower d-dUTP fluorescence than did CAM-treated

cells (Fig. 3D). Furthermore, among the nonapoptotic cells, a distinctS cell population was still apparent. Thus, unlike CAM, apoptosiscaused by hyperthermia was not selective to S cells.

These results confirm our earlier findings on the cell cycle specificity of these agents, obtained by the identification of S cells based ontheir DNA content (17). The present data, however, are direct anddefinite in terms of identifying both the cells undergoing apoptosisand the cells replicating DNA. In addition, the method providesinformation regarding the cell cycle kinetics of the population. Thus,for example, by comparison of the cell populations shown in Fig. 3,B and C, it is quite evident that the cells which incorporated BrdUrdduring S had moved to G2+M, and some had even divided, during the4 h of incubation following exposure to the precursor. In contrast toFig. 3C, the nonapoptotic cells in heat-treated cultures had hardly

moved through the cell cycle during this time interval (Fig. 3F).Hyperthermia, thus, in addition to inducing apoptosis, significantlyslowed cell cycle progression through S.

Fig. 4 illustrates apoptosis and BrdUrd incorporation in a culture ofhuman peripheral blood lymphocytes stimulated to proliferate byPHA. The data clearly indicate that the apoptotic cells did not incorporate BrdUrd.

Discussion

Halogenated DNA precursors incorporated into DNA can be detected with the SBIP method by photolytic induction of strand breaks,followed by labeling 3'-OH ends at the break points. The latter step is

identical to the detection of DNA strand breaks in apoptotic cells, asdescribed before (16, 17). The strategy for discrimination between the

1 000

DNA ContentFig. 4. Detection of S cells in mitogen-stimulated human lymphocyte cultures using the

SBIP method. The mononuclear cell fraction was isolated from human peripheral bloodand washed and incubated for 48 h with 10 iig/m\ PHA. The culture was treated for 30min with 10 JAMBrdUrd; the cells were then harvested and divided into two aliquots, oneof which was exposed to UV light (upper panel), while the other remained in the dark(bottom panel). Note that the labeling of DNA strand breaks in the apoptotic cellpopulation (Ap) is minimal. The DNA histogram is displayed as an inset in the upperpanel.

BrdUrd-incorporating and apoptotic cells, presented in the present

study, is based on the differential extractability of DNA in apoptoticcells compared to that of the photolysed DNA in live cells. Specifically, in contrast to DNA in apoptotic cells, which has numerousdouble-strand breaks resulting in a low molecular weight fraction that

can easily be eluted from cells (5, 19), the breaks in photolysed DNAare primarily of the single-strand type (15); such DNA resists elution.After testing different concentrations of formaldehyde (0.1-1.0%), itwas found that a 15-min exposure of cells to 0.25-0.3% of this

fixative was optimal for the retention of a fraction of DNA in apoptotic cells that was sufficient to allow their discrimination fromcellular debris. Photolysed DNA remained stable under these conditions. Additional identification of apoptotic cells was obtained byprocessing parallel samples that were not illuminated with UV light;apoptotic cells, prefixed in 1% formaldehyde, retained the bulk oftheir DNA after cross-linking and were intensely labeled with d-dUTP

in the absence of UV illumination (16, 17).Simultanous identification of DNA-replicating and apoptotic cells,

as offered by the present method, is expected to find an application asa prognostic assay which combines an analysis of both the marker ofcell proliferation and cell death. The method will also be helpful instudies of drug cytotoxicity, when the identification of apoptotic cellsand the cell cycle phase specificity of a drug are of interest. Mostimportantly, however, because detection of cells undergoing DNAreplication does not require prior DNA denaturation, as is the casewith the conventional BrdUrd antibody-based methodology (14), this

approach may be used in combination with labeling of cell surfacemarkers or other cell features that are generally destroyed during theharsh conditions (2.0 or 4.0 M HC1 or >90°C)required to denature

DNA. The sensitivity of the method, which detects a 5-min pulse of

BrdUrd, appears to be comparable to the sensitivity of the conventional, BrdUrd-antibody assay (14). However, because numerous

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d-dUTP molecules can be incorporated per single DNA break, as a

function of TdT activity, one may expect that by optimizing the UVillumination (e.g., by selecting the most optimal wavelength of UVlight, the dose, and geometry of cell exposure to induce maximalphotolysis) and incubation with TdT, further increases in sensitivitycan be achieved.

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12. Slinchenmyer, W. J., Nelson, W. G., Slebos, R. J., and Kastan, M. B. Loss ap53-associated G, checkpoint does not decrease cell survival following DNA damage. Cancer Res., 53: 4164-4168, 1993.

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14. Gratzner, H. G. Monoclonal antibody to 5-bromodeoxyuridine. A new reagent fordetection of DNA replication. Science (Washington DC), 218: 474-475, 1982.

15. Hutchinson, F. The lesions produced by ultraviolet light in DNA containing 5-bro-mouracil. Quart. Rev. Biophys., 6: 201-246, 1973.

16. Gorczyca, W., Gong, J., and Darzynkiewicz, Z. Detection of DNA strand breaks inindividual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nicktranslation assays. Cancer Res., 53: 1945-1951, 1993.

17. Gorczyca, W., Gong, J., Ardelt, B., Tráganos, F., and Darzynkiewicz, Z. The cellcycle related differences in susceptibility of HL-60 cells to apoptosis induced byvarious antitumor agents. Cancer Res., 53: 3186-3192, 1993.

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19. Gong, J., Tráganos, F., and Darzynkiewicz, Z. A selective procedure for DNAextraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.Anal. Biochem., 218: 314-319, 1994.

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1994;54:4289-4293. Cancer Res   Xun Li, Frank Traganos and Zbigniew Darzynkiewicz  Hyperthermia and Mitogen Stimulation of Human Lymphocytesduring Treatment of HL-60 Cells with Camptothecin and Simultaneous Analysis of DNA Replication and Apoptosis

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