continued cell cycling ability of hl-60 cells following 3harac incorporation: a combination of...

Continued Ceil Cycling Ability of HL-60 Cells Following 3HaraC Incorporation. A Combination of "Double-Labeling" and Sister Chromatid Analysis

Michael Sailer, Sheila N. Jani Sait, and Azra Raza

ABSTRACT: This study was designed to determine if HL-60 cells could undergo one or more cycles of DNA synthesis despite containing 3H-cytosine arabinoside (3HaraC) in their genome. HL- 60 cells were incubated with 3HaroC for 2 hours, washed and maintained in a medium containing bromodeoxyuridine (BrdU). At fixed time points, cells were arrested in metaphase and prepared for chromosomal analysis. Treatment of the sample by an immunofluorescent monoclonal anti-BrdU antibody allowed us to determine the differential fluorescent pattern of sister chromatids in metaphase cells that had undergone two or more rounds of DNA synthe- s•s in the presence of BrdU. Processing the samples by automdiography demonstrated the presence of black grains (3HaraC) overlying the chromosomes. Thus, we were able to examine each metaphase for the presence of 3HaraC as well us the number of cycles it had completed in the presence of BrdU. We showed that despite the presence of aHaraC in their DNA, some HL-60 cells were able to undergo two or more complete rounds of DNA replication.

INTRODUCTION

Cytosine arabinoside (araC) is wide ly used for the t reatment of acute non lymphocytic leukemia (ANLL) in varying doses and schedules [1, 2]. Fol lowing incorpora- t ion into the DNA of S phase cells, araC is thought to exert it 's lethal effect by arresting further DNA synthesis [3, 4]. One possible mode of resistance to araC could be the abi l i ty of a cell to cont inue DNA synthesis despi te the presence of the drug in it 's genome. In a previous s tudy [5] we showed that HL-60 cells that had incorpora ted a radio iso tope- labeled araC (ZHaraC) dur ing S phase, were able to complete DNA synthesis , pass through G2, and enter mitosis as ev idenced by the presence of 3HaraC grains in metaphase cells. In the present s tudy we a t tempted to define whether or not HL-60 cells could undergo more than one comple te cycle of DNA repl icat ion despi te the presence of 3HaraC in their genome.

In order to de termine the number of cycles a cell has undergone fol lowing incorporat ion of 3HaraC, we took advantage of the semiconservat ive nature of DNA synthesis. The chromosomes that repl icate in the presence of b romodeoxyur id ine (BrdU) for one round of DNA synthesis wil l contain two unif i l iar ly subst i tuted sis-

From the Department of Hematologic Oncology (M. S., A. R.), and the Department of Genetics and Endocrinology (S. N. J. S.), Roswell Park Memorial Institute, Buffalo, NY.

Address requests for reprints to Dr. Azra Roza, Deputy Chief, Dept. of Hematologic Oncol- ogy, Bosewell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263.

Received July 29, 1986; accepted November 23, 1986.

3 1 1

© 1987 Elsevier Science Publishing Co., Inc. Cancer Genet Cytogenet 27:311-318 (1987) 52 Vanderbilt Ave., New York, NY 10017 0165-4608/87/$03.50

312 M. Sailer, S. N. J. Sait, and A. Raza

ter chromatids. On the other hand, a cell that has undergone two rounds of DNA repl icat ion in the presence of BrdU wil l eventual ly have one unifi l iar and one bifi- l iarly subst i tuted chromat id [6, 7]. The use of a monoclonal anti-BrdU ant ibody has been shown to be effective in d is t inguishing between metaphase cells that have bifi l iarly subst i tuted DNA from those that do not [8].

Finally, the use of our previously descr ibed double l ab~ techmque [9, 10] pro- v ided us wi th the oppor tuni ty of assessing 3HaraC incorporat ion into the DNA si- mul taneous ly wi th determinat ion of the number of cycles that ind iv idua l cellg had comple ted in the presence of BrdU.

MATERIALS AND METHODS

HL-60 ceils were main ta ined in RPMI 1640 med ium with 10% fetal calf serum, and passed every seventh day. Ten mil l i l i ters of the cell suspens ion at a concentrat ion of 1 x 1 0 7 cells/ml was incubated with 10 Ci/3HaraC/ml (specific acit ivity 30 Ci/ mmol) for 2 hours at 37°C in a humidi f ied a tmosphere containing 5% CO2. Fol lowing incubation, cells were washed twice wi th cold araC and once with RPMI 1640 medium containing 10% fetal calf serum (FCS). Cell counts were performed using an electronic counter (Coulter Electronics Inc., Hialeah, FL) and the viabi l i ty was checked using Trypan blue dye exclusion method. The cells were resuspended in RPMI 1640 med ium with 10% FCS at a concentrat ion of 6 x 10 ~ cells/m|. Bromo- deoxyur id ine (5 ~g/ml) was added to the m e d i u m and the spec imen was d iv ided into six groups. The cells were then incubated in a humidi f ied a tmosphere at 37°C with 5% CO2, and chromosome preparat ions were obtained at 24, 36, 48, 60, 72, and 96 hours, as follows.

Chromosome Preparations

Colcemid was added at a concentrat ion of 0.01 Izg/ml 2 hours before harvest ing the cells. The specimen was then resuspended in a hypotonic solut ion [11] and incubated for 20 minutes. After fixation with Carnoy's solut ion (glacial acetic acid/meth- anol, 1:3) chromosomal preparat ions were obtained on Alcian blue coated coversl ips in the s tandard fashion [12].

Double Label Technique

Fol lowing fixation in 70% ethanol, the Alcian blue coated coversl ips were processed with the monoclonal anti-BrdU ant ibody by the previous ly descr ibed Raza, Preisler, Mayers, and Bankert (RPMB) technique [13, 14]. The secondary ant ibody used was conjugated to f luorescein isothiocyanate (FITC) so that the BrdU labeling was reflected by the presence of fluorescence. These RPMB processed coversl ips were then coated with NTB2 nuclear track emuls ion (Eastern Kodak Co., Rochester, NY) and exposed in the dark for 1 week. The radioautographs were deve loped with a D-19 developer, fixed, and the coversl ips were then moun ted onto glass sl ides with Elvanol 51-50 and examined under a fluorescence microscope. The metaphase cells were analyzed for incorporat ion of 3HaraC and the fluorescence pat tern of the sister chromatids, as follows.

Assessment of Metaphase Cells

For each spec imen s tudied at 24, 36, 48, 60, 72, and 96 hours, chromosomal preparations were obtained and processod by the double label technique. A metaphase cell was considered posi t ive for 3HaraC incorpora t ion if it demonst ra ted the presence of 5 grains or more overlying the chromosomes. The number of cycles a meta-

"Double-Labeling" and SCE 313

phase had completed in the presence of BrdU was determined by dividing the fluorescence pattern of the mitotic cells into three groups.

Group I (Dark). If the metaphase cell showed no fluorescence in either sister chromatid of the individual chromosomes, it was considered to be "dark." This appearance indicated that the chromosomes contained two unifiliarly substituted strands.

Group II (Dark-Bright). If the metaphase cell manifested one sister chromatid to be dark and the other was brightly fluorescent, it indicated the presence of one unifi- liar and one bifiliarly substituted chromatid. This would signify the completion of two rounds of DNA synthesis in the presence of BrdU.

Group III (Bright). If the metaphase cell showed that both sister chromatids of individual chromosomes were brightly fluorescent, then the cell must have undergone at least three rounds of DNA replication in the presence of BrdU.

RESULTS

The mitotic cells from samples at 24, 36, 48, 60, 72, and 96 hours were examined for the presence of grains as well as fluorescence in each sister chromatid. Figure 1A demonstrates a mitotic cell from the 48-hour sample with differential staining of sister chromatids, indicating that the cell has undergone two complete cycles of DNA replication in the presence of BrdU. Figure 1B illustrates the same mitotic cell as Figure 1A demonstrating the presence of black grains (3HaraC) overlying the

Figure 1A A mitotic cell from the 48-hour sample distinctly shows the differential staining of sister chromatids indicating that the cell has undergone two complete cycles of DNA replication in the presence of BrdU.

314 M. Sailer, S. N. J. Salt, and A. Raza

Figure 1B The same mitotic cell as in Figure 1A demonstrating the presence of black grains (3HaraC) overlying the chromosomes.

chromosomes. Table 1 provides the details of cell counts, cell viability, number of metaphases counted at each time point, and the classification of metaphase cells into the three groups described in the Methods section. The mitotic index at each time point varied between 0% to 2%.

Table 1 shows that, although the cell counts increased slightly from a baseline of 5 x 105 cells/ml to 7.8 x 105 cells/ml at 96 hours, the viability decreased from 88% before addition of 3HaraC to 68% at 96 hours. A substantial number of cells showed clear morphologic evidence of severe damage in that nuclei were often fragmented, the cytoplasm was vacuolated, and the phenomenon of karyorrhexis was seen frequently. These changes were most striking in the 24-hour sample, where we were unable to obtain any satisfactory metaphase cells for analysis. For the remaining time points, we examined a minimum of 37 and a maximum of 60 mitotic cells from each sample. All of these metaphase cells revealed 3HaraC incorporation, even though the amount was considerably decreased by 96 hours.

At 36 hours, 43% of the metaphases examined showed only dark sister chromatids, whereas 57% had already undergone two rounds of DNA replication in the presence of BrdU and demonstrated a dark-bright or differential fluorescence pattern in the sister chromatids. None of the metaphases examined had only bright chromatids at the 36-hour time point. The 48, 60, 72, and 96 hour samples showed a progressive decrease (from 27% to 5% of the metaphases examined) in group I type chromatids and an increase in group III type pattern (from 10% to 56%). Of note is the fact that, even in the 96-hour sample, 5% of the metaphases examined (three of 56 cells analyzed) continued to show only dark ehromatids indicating a marked prolongation of the cycling time induced by araC in these cells. However,

"Double-Labeling" and SCE 315

Table 1 Cell count, viability and pattern of fluorescence in sister chromatids in HL-60 cells following a 2-hour exposure to 3H-cytosine arabinoside

0 hr 24 hr 36 hr 48 hr 60 hr 72 hr 96 hr

Cell count/ml Control 5.5 x 105 4.4 x 106 - - 5.6 × 108 - - 9.4 X 10 8 4.2 x 10 6

Experiment 5 x 105 6.2 x 105 6.3 x 105 6.6 x 105 7.1 x 105 7.8 x 105

Viability (trypan blue) Control 98% 96% - - 96% - - 97% 87% Experiment 98% 84% 82% 78% 76% 69% 68%

Number of metaphases counted (% of total)

Control Group I (dark) Group II

(dark/bright) Group III

(bright) Experiment

Group I (dark) Group II

(dark/bright) Group III

(bright)

9 (36%) 4 (9%) - - 0 15 (60%) 35 (80%) - - 9 (33%)

1 (4%) 5 (11%) 5 (190%) 18 (67%)

20 (43%) 13 (27%} 5 (14%} 10 (17%) 3 (5%) 26 (57%) 30 (65%) 12 (32%} 22 (39%)

0 5 (10%) 20 (54%) 32 (53%) 31 (56%}

the majority of the mitotic cells at 96 hours (56%) revealed brightly fluorescent sister chromatids reflecting the completion of at least three rounds of DNA replication.

DISCUSSION

Cytosine arabinoside has been increasingly util ized in the treatment of acute nonlymphocytic leukemia (ANLL) since the introduct ion of high-dose regimens (HDaraC) [15, 16]. This use of single agent therapy has made the interpretation of laboratory data infinitely simpler. Although the administrat ion of HDaraC for 6 days [17] often provides adequate therapy for many individuals, some patients in our experience were noted to show evidence of persistent leukemia [18, 19]. The majority of these individuals had cell cycle times ranging between 80 and 100 hours [20]. Because araC was administered for 6 days, all leukemic cells should have undergone one round of DNA replication during treatment, thereby assuring exposure to araC during at least one S phase. Persistence of leukemic cells in this setting reflects the presence of metabolic rather than kinetic resistance [21]. For example, the leukemic cells of one individual may be so exquisitely sensitive that exposure to araC during one S phase proves to be sufficiently lethal, whereas, another patient may have cells that require exposure to araC during at least two rounds of DNA synthesis. One way of answering this question would be to determine the ability of cells to cont inue cycling despite the incorporation of araC into their DNA.

In a previous study [5], we were successful in demonstrating the ability of HL- 60 cells to complete S phase, pass through G2, and enter mitosis despite containing 3HaraC in their genome. Whether such cells were eventually committed to die or could cont inue into the next generation is the question we have addressed. In the

316 M. Sailer, S. N. J. Salt, and A. Raza

present study, following exposure to 3HaraC for 2 hours, we allowed HL-60 cells to grow in tissue culture medium containing low doses of BrdU. Because of the semi- conservative nature of DNA replication, we were able to differentiate between metaphase cells that had completed only one round of DNA synthesis in the presence of BrdU versus those that had gone through two or more complete cycles. Simulta- neous detection of black grains by autoradiography allowed us to demonstrate the presence of 3HaraC on individual mitotic cells. The results indicate clearly that at least some cells were able to continue cycling for several rounds of DNA replication despite the presence of 3HaraC in their genome. It must be remembered, however, that the majority of cells were severely damaged and undergoing nuclear fragmen- tation as evidenced by the morphologic appearance of the 24-hour sample (Table 1). In addition, no metaphase cells could be obtained from this sample indicating at least a marked prolongation of the S phase in the surviving cells.

Why did some ceils survive while others were lethally damaged in a relatively homogeneous population of tissue culture cells? There can be two possible expla- nations for this. Firstly, assuming that araC can only be incoporated into the DNA of an S phase cell, it is possible that some ceils were engaged actively in DNA synthesis for a very brief period of the 2-hour exposure to 3HaraC either because they were in early or late S phase. In this setting, relatively small amounts of 3HaraC would be incoporated, compared with other S phase cells that were actively syn- thesizing DNA during the entire length of 3HaraC exposure. Survival of such cells could be accounted for on the basis of kinetic resistance to araC and would be akin to slowly cycling cells in ANLL patients noted above. On the other hand, it is possible that some cells even though kinetically sensitive to araC by virtue of being in S phase were metabolically resistant to the drug. Such cells despite being in S phase either did not incoporate 3HaraC into their DNA or were able to continue DNA synthesis regardless of the presence of 3HaraC in their DNA. Both mechanisms may be active in the studies reported here.

Our data is also in agreement with the recent report by Crowther et al. [22] who followed chinese hamster ovary (CHO) cells pulse labeled with araC by time-lapse microcinematography through several divisions. They showed that cells were able to undergo several divisions following a pulse exposure to araC, but even though 81% cells from the third division entered mitosis, only 50% of the divisions were successful and none was normal. They concluded from these observations that araC can induce a delayed form of cell killing. Other investigators have also demonstrated the occurrence of chromatid breaks [23], aberrant replication [24], and inter- phase death [25] occurring in ceils after araC exposure. The advantage of our double label [26, 27] method is the ability to demonstrate 3HaraC incorporation in cells whose sister chromatids were differentially recognized by the anti-BrdU antibody.

This form of delayed cell death following exposure to araC could account for the presence of persistent leukemia in ANLL patients who continue to demonstrate the presence of myeloblasts after treatment with HDaraC. Whether these individuals enter remission or not must be partly dependent on the proportion of cells that are lethally damaged versus those that can recover rapidly from the damage caused by araC. We hope that by further measuring the cell cycle times of these cells we will be able to obtain a more comprehensive view of the mechanism of araC action. For example, patients with long cell cycle times may benefit more by therapy with low doses of araC, which is administered over several weeks, as opposed to a short course of HDaraC. We now plan to administer continuous infusions of BrdU to patients simultaneously with araC therapy to determine the number of cycles that myeloblasts complete in vivo despite the presence of araC. The present study con- ducted in HL-60 cells in vitro hopefully will serve as a model for future design of in vivo trials in ANLL patients.

"Doub le -Labe l ing" and SCE 317

Supported by National Cancer Institute Grants CA-28734 and CA-41285. The authors thank Mr. Anthony Navone for his expert technical assistance, Grace Kuwik

for her excellent secretarial help.

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continued cell cycling ability of hl-60 cells following 3harac incorporation: a combination of...

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