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Leukemia (1997) 11, 1253–1257  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Induction of apoptosis by 2-chlorodeoxyadenosine in B cell chronic lymphocytic leukemia R Castejo´n1, JA Vargas1, M Briz2, E Berrocal1, Y Romero1, JC Gea-Banacloche1, MN Ferna´ndez2 and A Dura´ntez1 Service of Internal Medicine I and 2Service of Hematology, Clı´nica Puerta de Hierro, Universidad Auto´noma, Madrid, Spain

1

We investigated whether 2-chlorodexoyadenosine could induce apoptosis in B cell chronic lymphocytic leukemia (BCLL) cells in vitro using clinically achievable drug doses, measuring apoptosis ratio by flow cytometry. B cells were isolated from previously untreated patients and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with 2-chlorodeoxyadenosine at different concentrations, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of drug, but the level of apoptosis was greater in cells treated with 2-chlorodeoxyadenosine after the second day of culture. The present in vitro study of B-CLL cells from previously untreated patients suggests this chemotherapeutic agent activates a program of cell death by apoptosis using a drug dose equivalent to the physiological concentration used in patients in vivo. These data reveal an interesting possibility in the 2-chlorodeoxyadenosine treatment of untreated patients by neoplastic B cell apoptosis induction. Keywords: apoptosis; 2-chlorodeoxyadenosine; B-CLL

most cells are in the resting phase.6,7 In vitro studies have shown the appearance of DNA strand breaks and other features of cell death by apoptosis in resting lymphocytes incubated with 2-CdA.8 The death of cells undergoing apoptosis is preceded by chromatin cleavage at the linker regions between nucleosomes by specific endonucleases, which results in extensive fragmentation of the DNA into oligonucleosomal subunits.9 The DNA fragments can be demonstrated by agarose gel electrophoresis giving rise to a ‘ladder’ of nucleosomal-sized multimers. This process is accompanied by morphologic features of chromatin condensation, nuclear margination, and fragmentation of the nucleus into membrane-bound bodies. Flow cytometry has recently become a chosen technique for quantitative analysis of apoptosis.10,11 It is a rapid, simple and reproducible method for measuring the percentage of apoptotic cells. Moreover, flow cytometry data correlate excellently with the classical DNA fragmentation assays.12,13 Thus, we investigated whether 2-CdA could induce apoptosis in B-CLL cells in vitro using clinically achievable drug doses, measuring apoptosis ratio by flow cytometry.

Introduction B cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in the Western world. B-CLL is characterized by the slow and progressive accumulation of monoclonal, apparently mature, CD5+B lymphocytes.1,2 The majority of circulating cells appears to be nondividing and it has been suggested that a prolonged life span is mainly responsible for the accumulation of the leukemic cells. However, spontaneous programmed death by apoptosis appears when B-CLL cells were cultured in vitro.3 The initial management of this disorder is not complicated since a high proportion of patients have an asymptomatic disease with slow progression, and those with symptomatic disease often respond (up to 60% of cases) to low doses of oral alkylating agents and steroids.2 However, the disease remains incurable, since resistance to alkylating agents frequently develops. The vast majority of patients have a reduced life expectancy because of their leukemia. Recently, purine analogues such as 2-chlorodeoxyadenosine (2-CdA) and fludarabine have been synthesized, which have chemotherapeutic activity in refractory or untreated B-CLL.4 2-Chlorodeoxyadenosine is a deoxyadenosine analog, consisting of substitution of a chlorine atom at the 2-position of the purine ring.5 It is unique compared with traditional antimetabolite drugs in that it is equally active against dividing and resting lymphocytes which may be especially important in indolent lymphoid malignancies such as B-CLL, because

Correspondence: R Castejo´n, Laboratorio de Medicina Interna I, Clı´nica Puerta de Hierro, C/ San Martı´n de Porres, 4, 28035 Madrid, Spain Received 28 October 1996; accepted 28 March 1997

Materials and methods

Patients Twenty-one patients with chronic lymphocytic leukemia were entered on this study, nine were male and 12 female. The median age was 70 years with a range of 31–89 years. Five of them had received chemotherapy for at least 4 months before the samples were drawn and the other 16 patients were previously untreated. Diagnosis of B-CLL was unequivocal according to clinical, morphologic and immunological criteria. The patients were classified according to Rai et al14,15 in stages 0–IV: seven patients were at clinical stage Rai 0, four at Rai I, eight at Rai II and two at Rai IV (Table 1).

Cell separation Peripheral blood mononuclear cells (PBMC) were obtained from sterile heparinized venous blood from patients by differential centrifugation on Ficoll–Hypaque (Lymphoprep; Nyegard, Oslo, Norway). B cells were isolated after rosetting out T cells with 2-aminoethylisothiouronium bromide-treated sheep red blood cells. This procedure gave a population of T celldepleted B-CLL cells containing less than 2% T cells and with a B cell purification higher than 95%. Cell viability was always found to be .95% as judged by trypan blue dye exclusion.

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Culture conditions B-CLL separated cells were cultured at a concentration of 2 × 106 cells/ml in complete medium (RPMI 1640, BioWhittaker (Walkersville, MD, USA), supplemented with 10% heat-inactivated FCS Bio-Whittaker and antibiotics), with or without 2-chlorodeoxyadenosine (Leustatin; Ortho Biotech, Raritan, NJ, USA) for 24 and 48 h, at the final concentrations of 1.75 × 10−6 M, 1.75 × 10−7 M and 1.75 × 10−8 M (500, 50 and 5 ng/ml, respectively) in a fully humidified atmosphere of 5% CO2 at 37°C. For in vivo treatment each patient received 0.12 mg/kg daily as 2-h intravenous infusions for 5 consecutive days (0.6 mg/kg total).16 The mean end of infusion plasma 2-CdA concentration is 48 ± 19 ng/ml, that is 1.75 × 10−7 M in our in vitro assay.17

Analysis of apoptosis Apoptosis was measured by flow cytometry and the level of DNA fragmentation. The distribution of cellular DNA content was determined by standard propidium iodide staining and flow cytometry using a FACScan flow cytometer with argon laser excitation at 488 nm (Becton Dickinson, San Jose´, CA, USA). The 200 g centrifugated cell pellet was resuspended in 70% ethanol and placed on ice for 15 min. The cells were centrifuged, washed in PBS and resuspended in 0.5 ml PBS containing propidium iodide (5 mg/ml) and RNAse (50 mg/ml). Tubes were incubated in the dark at 4°C for 30 min prior to flow cytometry analysis. The staining was stable for at least 24 h when cells were kept refrigerated.12 Flow cytometric apoptosis quantification was made with ‘Lysys II’ and ‘Paint-a-gate’ analysis software. Necrotic cells, nuclear fragments or cell debris, with minimal DNA content, were easily distinguishable from apoptotic cells and were excluded from the analysis to avoid overestimation of apoptotic cell quantification. We used a two parameter dot-plot of a fluorescence peak width vs fluorescence peak area signals to gate out cell aggregates (doublets, triplets, etc) and necrotic cells from cell suspensions. DNA fragmentation was assessed on 2% agarose gels to identify the typical ladder pattern of apoptosis.3

apoptosis in vitro. No apoptotic cell death was found on freshly collected peripheral blood (Table 1) but untreated BCLL cells rapidly initiated apoptosis when placed in culture (Table 2). After 24 h, 14.5 ± 7.1% (mean ± s.d.) of cells showed evidence of apoptosis when DNA from cells were analyzed by flow cytometry. By the second day of culture, 19.2 ± 13.8% of the untreated cells were affected (Table 2 and Figure 1). 2-CdA-treated cells were incubated for 24 and 48 h with three different levels of drug (Table 2). The percentage of apoptotic cells after 24 h cultured with the lowest 2-CdA level (1.75 × 10−8 M) was similar to the spontaneous apoptosis percentage shown in untreated cells (16.0 ± 10.3). Apoptotic cell numbers were increased when drug concentration in culture was 1.75 × 10−7 M, with 19.2 ± 21.5% of apoptotic cells in the first 24 h. Moreover, leukemic lymphocytes treated with 1.75 × 10−6 M 2-CdA showed apoptosis levels over 24.7 ± 11.1% after the first day of culture. These data have no statistical significance (Table 2 and Figure 1). When cells were incubated for 48 h with 2-CdA (1.75 × 10−8 M), there were no differences with respect to the apoptosis shown in untreated cells culture (22.3 ± 19.1%). However, a significant increase of apoptosis was observed with respect to the basal (P = 0.014) when the 2-CdA level in the culture was 1.7 × 10−7 M. After 48 h we obtained an apoptotic cell ratio about 3.7 ± 6.6 higher than the spontaneous one. The highest drug dose (1.75 × 10−6 M) produced an elevated percentage of apoptotic cells 55.1 ± 26.0% by 48 h. The last results are significant with respect to the basal (P = 0.000) and the ratio between apoptosis by 1.75 × 10−6 M 2-CdA and spontaneous cell death is about 5.0 ± 6.6 (Table 2 and Figure 1). All these data demonstrate that apoptosis was dosedependent, and occurred after 48 h in cultured cells. DNA fragmentation assay was shown in Figure 2. Incubation of lymphocytes with 2-CdA obtained from patients with Rai stage 0 or I disease in comparison to more advanced disease did not show significant differences in druginduced apoptosis (data not shown). Pronounced interpatient

Table 1 Characteristics of the patients and percentage of apoptotic B cells on freshly collected peripheral blood

Patient

Statistical methods ANOVA and Newman–Keuls test for repeated measurements were made to analyze the results. The data from the groups were compared using the Student’s t-test for paired samples. A P-value of less than 0.05 was considered significant. Results Flow cytometry has proved to be able to identify apoptotic cells, after staining with DNA-specific fluorescent dyes, as a subdiploid peak. Since the morphology of an apoptotic cell shows a highly condensed chromatin in a fragmented nucleus, it is conceivable that both a DNA loss and a decrease of DNA accessibility to the dye are responsible for the lower fluorescence of apoptotic cells. In our cell cultures, CLL lymphocytes were exposed to different concentrations of 2-CdA for various times. Untreated lymphocytes were also analyzed for the extent of spontaneous

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Age

Sex M/F

Rai stage

Apoptotic cells %

63 73 70 75 74 74 68 72 31 82 89 87 68 73 58 48 77 69 78 65 74

M F M F M F F M F F F F F M F M F M F M M

0 0 0 0 0 0 0 I I I I II II II II II II II II IV IV

0.7 3.9 0.0 1.0 0.0 0.0 6.1 0.0 0.0 0.0 3.3 0.0 0.0 2.0 4.3 3.5 0.0 4.9 0.0 5.1 0.0

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Figure 1 Percentage of apoptotic cells after 24 and 48 h culture of patient 2. (a) Freshly isolated cells and spontaneous apoptosis after incubation; (b) 1.75 × 10−6 M 2-chlorodeoxyadenosine-treated cells; (c) 1.75 × 10−7 M 2-chlorodeoxyadenosine-treated cells; (d) 1.75 × 10−8 M 2chlorodeoxyadenosine-treated cells.

variation was evident in spontaneous and drug-induced apoptosis (Table 2). Discussion The aim of the present study was to investigate the induction of apoptosis in human neoplastic B cells using 2-chlorodeoxyadenosine. In fact, in vitro exposure of CLL lymphocytes to 2CdA triggered cell death characteristic apoptosis as we have shown by flow cytometry. Flow cytometric analysis of cells after propidium iodide staining is widely utilized for evaluating cell death, determining ploidy in tumor samples and measuring cell cycle parameters of cultured cells.18 The apoptotic cells are identified as an unequivocal hypodiploid DNA peak in the red fluorescence channels as a result of the reduced DNA content of apoptotic cells.11 This method permits simple, quantitative and reproducible measurement of apoptosis.12 Chronic lymphocytic leukemia does not result directly from enhanced proliferation, but rather is caused by the progressive accumulation of long-lived lymphocytes with enhanced viability. Paradoxically, short-term culture renders many of these cells susceptible to apoptosis.3 Increasing the cell density or changing the source of serum did not affect this spon-

taneous rate of cell death.3,19 One assumes this arises because of the lack of an unidentified essential cytokine.20 Thus, apoptosis was observed during culture in the absence of drug in most cases. In contrast to drug-induced apoptosis, the degree of spontaneous cell death was lower and seemed to increase on prolongation of incubation. The diagnosis of CLL is not consonant with the need for therapy.21 Patients with early stage disease are usually not treated unless symptoms develop or disease progresses, and have a low median survival. Patients with advanced disease have a much shorter survival. Chlorambucil should be still considered the standard treatment.22,23 Response rates range from 40 to 70%, but complete responses are rare. For patients with no response to standard therapy or a relapse after such therapy, purine analogues (2′-deoxycoformycin, fludarabine and 2-chlorodeoxyadenosine) are the treatment of choice.24 2′-Deoxycoformycin is a potent inhibitor of adenosine deaminase.25 Fludarabine is approved for the treatment of patients with chronic lymphocytic leukemia who are refractory to alkylating agents,26 and 2-CdA for patients with untreated or interferon-refractory hairy cell leukemia. The effectiveness of single-agent 2-CdA in patients with CLL who fail to respond to alkylator therapy was first reported in 1988.27 In subsequent studies high complete remission rates were obtained with 2-

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Table 2 Percentage of apoptotic cells after incubation for 24 and 48 h without (spontaneous cell death) and with three different levels of 2 chlorodeoxyadenosine

Patients

24 h No drug

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Mean ± s.d.

48 h

2-CdA 2-CdA 2-CdA 1.75 × 10−6 M 1.75 × 10−7 M 1.75 × 10−8 M

No drug

2-CdA 1.75 × 10−6 M

2-CdA 1.75 × 10−7 M

2-CdA 1.75 × 10−8 M

16.7 13.2 3.5 7.1 10.4 10.2 10.1 15.6 16.3 13.8 22.5 13.3 30.0 20.7 6.4 26.1 9.2 9.4 21.1 9.8 34.1

ND 23.6 12.8 28.7 15.2 27.3 20.6 38.3 40.7 21.9 34.3 44.8 24.3 6.0 20.7 30.6 9.2 11.6 31.6 38.6 52.3

33.9 13.3 13.7 7.6 2.3 3.0 94.2 7.4 34.5 13.8 25.2 26.0 14.8 2.0 9.2 ND 0.6 19.6 24.1 13.9 40.9

9.3 13.0 9.3 11.2 ND ND 23.1 29.9 ND 14.1 31.7 26.9 2.2 1.9 7.0 ND 4.8 8.9 32.2 18.5 28.4

30.0 18.2 14.7 54.1 9.0 2.6 14.8 45.6 25.3 13.4 ND 19.8 33.0 4.6 7.9 9.0 20.4 11.5 9.9 13.1 30.0

ND 47.1 73.9 74.8 33.3 75.9 68.8 84.1 89.5 63.8 ND 68.7 69.1 36.3 9.9 23.9 7.8 34.5 73.5 92.5 54.5

49.8 29.6 40.9 76.8 19.3 75.6 8.9 58.1 49.9 38.4 ND 62.2 70.7 11.0 11.9 ND 10.3 34.8 79.5 14.2 49.5

7.4 12.2 23.8 25.4 ND ND 14.1 28.8 ND 20.5 ND 66.2 9.6 9.2 1.6 ND 20.7 11.1 61.0 25.9 21.0

14.5 ± 7.1

24.7 ± 11.1

19.2 ± 21.5

16.0 ± 10.3

19.2 ± 13.8

55.1* ± 26.0

42.9** ± 25.2

22.3 ± 19.1

ND, not done. *P = 0.000; **P = 0.014.

Figure 2 DNA fragmentation identificated as typical ladder pattern of apoptosis on 2% agarose gel. 1, molecular weight marker; lane 2 necrotic cells after high temperature treatment; lane 3, B-CLL cells after incubation for 48 h; and lane 4, B-CLL cells after incubation with 1.75 × 10−7 M 2-chlorodeoxyadenosine for 48 h.

CdA in previously treated patients.28,29 The response in patients with B cell chronic lymphocytic leukemia resistant to fludarabine was also measured.30–33 All these observations suggested that 2-CdA treatment of patients who had not been exposed to prior therapy might result in higher response rates.34,35 In the first report to document the response rates of 2-CdA in patients with previously untreated CLL, the response rate seen is nearly double that obtained in previously treated patients.36 All data use the same response criteria as recommended by the NCI.37 The purine nucleoside analogue mechanisms of action are

complex but include the induction of apoptosis.5,8 Recent studies have demonstrated that programmed cell death by apoptosis is the mechanism of action of the most common treatment for CLL: the alkylating agent chlorambucil.38,39 2-Chlorodeoxyadenosine is unique compared with traditional antimetabolite drugs in that it is equally active against dividing and resting lymphocytes6,7 which may be especially important in indolent lymphoid malignancies, such as chronic lymphocytic leukemia, because most cells in these disorders are in the resting phase. Although subject to controversy, it is thought that quiescent lymphocytes contain DNA strand breaks that are continuously being formed and repaired. It is hypothesized that 2-CdA treatment of normal resting lymphocytes causes an increase in the amount of DNA strand breaks and thus leads to activation of poly (ADP-ribose) polymerase, which results in a lethal depletion of cellular NAD and ATP and consequential cell death.5 The present in vitro study of chronic lymphocytic leukemia B cells from previously untreated patients shows that this chemotherapeutic agent activates a program of cell death by apoptosis. These results are very important because the 2-CdA physiological concentration used with patients in vivo is equivalent to 1.75 × 10−7 M dose used in our in vitro assays, which cause a significant induction of apoptosis. Although the percentage of apoptosis is higher with the most elevated dose, this drug level is not suitable for using in vivo because the dose is limited by toxic effects in patients. These data suggest an interesting possibility in the 2-CdA treatment of previously untreated patients by chronic lymphocytic leukemia B cell apoptosis induction. Flow cytometric analysis of apoptotic cells is a rapid and simple method that may be a reliable indicator of the effectiveness of 2-chlorodeoxyadenosine patients’ in vivo treatment.

Apoptosis by 2-chlorodeoxyadenosine in B-CLL R Castejo´n et al

Acknowledgements This work has been partially supported by grants No. 96/2173 from the Fondo de Investigaciones Sanitarias de la Seguridad Social No. 029/96 from the Comunidad Autonoma de Madrid and LAIR Foundation, Spain. We thank Ms C Lorences and Ms G Peraile for their skilful technical assistance, and Dr I Milla´n, MS, for the statistical analysis.

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