Characterization of arsenic-induced cytogenetic alterations in acute ...

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Gain or loss of genes plays important roles in leukemogenesis of APL via cooperation ... Genetic alterationAcute promyelocytic leukemiaNB4 cell lineArsenic ...
Med Oncol (2012) 29:1209–1216 DOI 10.1007/s12032-011-9946-4

ORIGINAL PAPER

Characterization of arsenic-induced cytogenetic alterations in acute promyelocytic leukemia cell line, NB4 Marjan Yaghmaie • Hossein Mozdarani • Kamran Alimoghaddam • Seyed Hamidullah Ghaffari Ardeshir Ghavamzadeh • Marjan Hajhashemi



Received: 24 February 2011 / Accepted: 4 April 2011 / Published online: 5 May 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Gain or loss of genes plays important roles in leukemogenesis of APL via cooperation with PML-RARA. Fluorescence in situ hybridization (FISH) was applied to investigate the DNA copy number changes of hTERT, ERG, CDKN1B (P27), CDKN2A (P16), and TP53 genes in an acute promyelocytic leukemia (APL) cell line (NB4). Five bacterial artificial chromosome probes (BAC) for 9p21.3, 17p13.1, 12p13.2, 5p15.33, 21q22.2 regions were prepared using sequence independent amplification (SIA) and were hybridized to NB4 cells treated with different doses of arsenic trioxide (As2O3; ATO) at various time intervals. NB4 cells were also karyotyped by G-banded chromosome analysis 24 h after culture initiation. FISH analysis prior to treatment showed CDKN1B, CDKN2A, and TP53 gene deletion but ERG and hTERT gene amplification. After treatment with ATO, the number of the NB4 cells with deleted CDKN1B and CDKN2A as well as the counts of the cells with hTERT amplification was significantly reduced in time- and does-dependent manners. In addition, we observed expressive increase in signal patterns of CDKN1B and CDKN2A along with significant decline in hTERT signal patterns in ATO-treated cells as compared with the control group (in time- and dose-dependent manners). On the other hand, no difference in signal patterns for Erg and p53 was observed in response to ATO

M. Yaghmaie  H. Mozdarani (&) Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, P.O.Box 14115-111, Tehran, Iran e-mail: [email protected] K. Alimoghaddam  S. H. Ghaffari  A. Ghavamzadeh  M. Hajhashemi Haematology, Oncology and Stem cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

exposure. The results of the present study show the cytogenetic alteration in hTERT, CDKN1B, and CDKN2A in NB4 cells after treatment with ATO might introduce a new mechanism of antitumor activities of ATO in APL cell line, NB4. Keywords Genetic alteration  Acute promyelocytic leukemia  NB4 cell line  Arsenic trioxide  Fluorescence in situ hybridization

Introduction Acute promyelocytic leukemia (APL) is a subset of acute myeloid leukemia genetically characterized by the reciprocal translocation between chromosomes 15 and 17 that fuses the promyelocytic leukemia (PML) gene and the retinoic acid receptor alpha (RARA) gene. As a result of this translocation, the PML-RARA fusion protein is generated, which inhibits differentiation of myeloid cells [1]. Studies with transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL. APL occurred in these mice only after a long latency (8.5–12 months) and penetrance was 15–30% [2]. It has been well-accepted that defining minimal common regions of chromosomal loss in particular cancers is a popular strategy in searching for tumor suppressor genes. Amplification is also considered to be a late or secondary event in cancer and is one of the mechanisms that can cause the overexpression of oncogenes [3]. The array comparative genomic hybridization (aCGH) profile has shown gain of 5p13–pter and loss of 9p21.1 and 17p in NB4 cell line [4]; hTERT, CDKN2A, and TP53 are located in these regions, respectively. The cyclin-dependent kinase inhibitors (CDKIs), like CDKN1B and CDKN2A, play critical roles in check-point functions during the cell

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cycle and inhibitors of cell proliferation. Loss of their activities can impair development and differentiation and contribute to the uncontrolled proliferation characteristic of cancer cells [5, 6]. Continued proliferation in tumor cells also requires telomerase to maintain telomere-related chromosomal stability and to prolong the telomere lengthrestricted replicative life span of cells. Telomerase is constitutively expressed in germ line cells and in most malignant tumor cells [7]. Lack of functional TP53 due to gene deletion and considerable overexpression of ERG transcription also contribute to leukemogenesis [8, 9]. Therefore, we investigated the loss of the FISH signals consistent with deletion of CDKN1B, CDKN2A, and TP53 tumor suppressors at 12p, 9p, and 17p and the gain of FISH signals consistent with amplification of ERG and hTERT at 21q and 5p in order to identify specific groups of genetic changes that are present in NB4 cell line and altered by ATO treatment. ATO has shown substantial efficacy in treating both newly diagnosed and relapsed patients with APL, even in patients resistant to conventional chemotherapeutic agents or all-trans retinoic acid [10]. ATO was shown to exert concentration-dependent dual effects in APL cell. ATO at high-concentrations (0.5–2.0 lmol/l) could induce in vitro growth inhibition and apoptosis of APL cells; however, at lower concentrations (0.1–0.5 lmol/l), it induces differentiation [10]. Such effects have been observed in cultured cell lines and animal models, as well as clinical studies. Although the exact mechanism of ATO efficacy remains unknown, induction of differentiation, cell cycle arrest, and apoptosis are the principal modalities involved in the antitumor effect of ATO [11, 12]. Some reports attributed its effect to induction of reactive oxygen species. ATO can also induce degradation of the PML-RARA fusion protein, and this effect was thought to underlie ATO anti-APL activity [11]. However, the presence of PML-RARA fusion protein is neither necessary nor sufficient for the efficacy of ATO [13–15]. This work may help to identify specific groups of genetic changes that are present in NB4 cell line and are altered by ATO treatment.

Materials and methods Cell culture The NB4 cells, a permanent cell line originally derived from the marrow of a patient with acute promyelocytic leukemia (APL) (M3) in relapse, [16], obtained from National Cell Bank of Iran, Pasteur Institute of Iran, were cultured in RPMI-1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Auckland, USA), with 2 mM L-glutamine (Sigma, St Louis, MO, USA), 25 mM HEPES (N-2-

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hydroxyethylpiperazine-N-2-ethanesulfonic acid) (Sigma, St Louis, MO, USA), and penicillin–streptomycin (100 U/ml–100 lg/ml) (Sigma, St Louis, MO, USA) in a humidified atmosphere of 95% air/5% CO2 at 37°C. Cells were maintained at less than 5 9 108 cells/l with daily adjusting of cell density through adding fresh medium and corresponding concentrations of compounds. For G-banded chromosome analysis of the changes, colcemid (Sigma Chemical, St Louis, MO, USA) was added at the final concentration of 0.1 lg/ml to the NB4 cells, and the cells were harvested and fixed according to standard cytogenetic protocols. The fixed cell suspension was then dropped (4–6 drops) onto a clean-chilled-wet slide. G-banded chromosome analysis was used to karyotype sixteen well-spread metaphases of untreated NB4 cells, and the remaining cells were used for FISH. Arsenic treatment The cells were synchronized before ATO treatment by adding fresh medium without FBS. ATO (Sigma Chemical, St Louis, MO, USA) was prepared as a 0.1 mmol/l stock solution in RPMI-1640 medium. The cells treated with 0.5, 1, and 2 lmol/l ATO were collected at 24 and 48 h. After the incubation time, colcemid (stock: 10 lg/ml) was added to the cultures at a final concentration of 0.1 lg/ml, and the cells were harvested after 30 min according to routine cytogenetic protocols. Viability tests The effect of different concentrations of ATO on viability of NB4 cells was assessed by using trypan blue exclusion assay and also by microculture tetrazolium test (MTT assay). Percent viability was normalized to the untreated cells. To do trypan blue exclusion assay, samples of cells were mixed with an equal volume of 0.4% trypan blue, and then counted on a hemocytometer slide (improved Neubauer) under a light microscope (Zeiss, Germany) with 109 objective lens to determine the number of viable cells. MTT assay was done based on the uptake of thiazolyl blue tetrazolium bromide (MTT, Sigma) by viable cells as described by Momeny et al. (2009) [17]. A total of 5,000 cells were plated onto 96-well plates (SPL Life sciences, Pocheon, Korea) and treated with different concentrations of ATO for 24 and 48 h. The cells were incubated with 200 ll of MTT (0.5 mg/ml) at 37°C for 2 h. Untreated cells were used as the control. Precipitated formazan was solubilized with the use of 100 ll of dimethyl sulfoxide (DMSO), the optical densitometry was done at a wavelength of 578 nm. The inhibition rate (IR) of ATO was evaluated using the equation: IR (%) = 1 - ODexp/ODcon 9 100, where ODexp and ODcon are the optical densitometries of treated and untreated

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Table 1 Viability of NB4 cells treated with various concentrations of ATO at different time intervals assessed by trypan blue exclusion and MTT assays Time after treatment (h)

ATO concentration (lM)

Viability

24

0.5

48

Trypan blue assay

MTT assay

100

90

1

92

80

2

72

65

0.5

95

85

1

74

65

2

59

48

cells, respectively. The results of viability tests are shown in Table 1. As seen in Table 1, low dose of ATO did not affect viability of cells significantly, and even after treatment of cells with 2 lM, more than 60% at 24 h and about 50% after 48 h were viable. Probe preparation The BAC clones were obtained from the BACPAC Resource Center (Children’s Hospital of Oakland, Research Center) of RP11 human BAC clone library and mapped to chromosomal band of interest (UCSC data base, http://www.genome.ucsc.edu/) to validate DNA copy number changes of different genes of hTERT, ERG, CDKN1B, CDKN2A, and TP53. BAC DNA was isolated from bacteria using the alkaline lysis method [18].We used RP11-89D11 BAC clone for TP53, RP11-70N14 for CDKN1B, RP11-95I21 for ERG, RP11-149I2 for CDKN2A, and RP11-990A6 and RP11-117B23 for hTERT gene location. To generate sufficient quantities of BAC DNA for the preparation of FISH probes, we used sequence-independent amplification (SIA) [19]. After the initial SIA from a BAC, the DNA fragments were fluorescently labeled in a second PCR reaction by the incorporation of nucleotides that were chemically modified with fluorochromes. In order to achieve the right size range (300–500 bp) of DNA fragments for optimum hybridization kinetics and results, the labeled SIA products were mildly treated with DNase I. After labeling and DNase treatment (in the case of the SIA products), Cot-1-DNA was added to reduce non-specific background hybridization signals and also used as a coprecipitent in order to remove unincorporated labeled nucleotides. The TP53, hTERT, CDKN2A, and ERG were labeled with Texas Red and CDKN1B with fluorecein (Perklin Elmer, USA). Commercially available probes were mixed with the BAC probes as a positive control for hybridization; TP53 probe was mixed with CEP17 (Spectrum green), CDKN1B with

CEP12 (Spectrum Orange), CDKN2A with CEP9 (Spectrum green; all Abbott Molecular, USA), and hTERT with PDGFRB (Spectrum Gold; Kreatech, Netherland) before hybridization. The commercially available centromeric probes of CEP17, CEP9, and CEP12 (Abbott Molecular, USA) were used to make sure that there was no hybridization failure with the genes the loss has been observed. Fluorescence in situ hybridization The slides of untreated and treated NB4 cells were air dried, and 1–1.5 ll of the probe mix (has detailed above) was applied for a 9 9 9 mm2 area, covered with a coverslip and sealed with rubber cement. The slides were subjected to denaturation at 76°C for 5 min followed by hybridization at 37°C for 12–16 h using an automated FISH hybridization chamber (HybriteTM, Vysis, Germany). After hybridization, the slides were washed and counterstained with DAPI according to routine FISH protocols. The fluorescent signals were visualized with an epi-fluorescence microscope (Olympus, BX51, Japan), equipped with a CCD camera. A total of 100 cells were analyzed in two different areas for each treatment. The images were captured and processed using the Applied imaging software (Cytovision, USA). Statistical analysis The two-sample proportion test was used to compare DNA copy number changes at different time courses and the chisquare test to evaluate DNA copy number changes at different concentrations of ATO by using the R package version 2.10.1 [20].

Results NB4 karyotype Cytogenetic analysis on 16 metaphases of NB4 cells showed a complex karyotype and variation from cell to cell. Chromosomal numbers ranged from 79 to 88 with a modal number of 79. However, all the hypertriploid cells exhibited the t(15;17). Representative karyotype could be shown as follows: human hypertriploid karyotype—79 nb4 (79*88) \ 3n [ XX, -X, ?2, ?6, ?7, ?7, der(8)t(8;?) (q24;?),-9, ? 10,t(10;19)(q21.1;p13.3) 9 2, ? 11, der(11) add(11)(q25), ? 12, der(12)t(12;?)(p11;?), ?13, ?14, t(15;17)(q22;q11–12.1), ?17, i(17)(q10), ? 18, i(18)(q10), -19, ?20, ?mar (Fig. 1). The karyotype analysis was only done in NB4 cell line before treatment with ATO. However, if there was any cytogenetic aberration in the chromosomes of interest, it would be detectable by FISH analysis by the

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Fig. 1 Typical NB4 cell karyotype; hypertriploid cell line with complex chromosomal aberrations. Human hypertriploid karyotype— 79 nb4 (79–88) \ 3n [ XX, -X, ?2, ?6, ?7, ?7, der(8)t(8;?) (q24;?), - 9, ?10, t(10;19)(q21.1;p13.3) 9 2, ? 11, der(11)t(11;?)

(? ? ::11p15 ? 11q22.1::11q13 ? 22.1:), ? 12, der(12)t(12;?)(p11;?), ?13, ?14, 14p ? , t(15;17)(q22;q11–12.1), ?17, i(17q), ? 18,i(18q), -19, ?20, ?1mar

use of centromeric probe that we combined with p53, CDKN2A, CDKN1B, and hTERT probes in our analysis.

cells with hTERT amplification were reduced after ATO treatment. This reduction in cells was significant for CDKN1B and CDKN2A after 48 h at different concentrations of ATO (1 and 2 lmol) (P \ 0.001) and in different time courses (24 and 48 h) (P \ 0.0001) (Fig. 3a, b). hTERT DNA copy numbers were also declined significantly at different concentrations of ATO (P \ 0.0001) and at different time courses (P \ 0.001) (Fig. 3c). This reduction was not significant for Tp53 and ERG after 24and 48-h treatment with different concentration of ATO.

Molecular cytogenetic findings The FISH experiment was performed on NB4 cells using 5 probes generated from BAC clones. The hypertriploid karyotype has three copies of chromosomes 21, four copies of chromosomes 17, four copies of chromosomes 12, two copies of chromosomes 9, and three copies of chromosomes 5. We therefore expected to see three signals for ERG on 21q, three signals for TP53 on 17p, four signals for CDKN1B on 12p, two signals for CDKN2A on 9p, and three signals for hTERT on 5p in untreated NB4 cells. Unexpectedly, however, we observed more than 3 signals for hTERT and less than four signals for CDKN1B, no signal for CDKN2A and more than three signals for TERT (Fig. 2). Furthermore, two CDKN2A alleles, but only one CDKN1B and TP53 allele, were lost. We then performed FISH using NB4 cells treated with different concentrations of ATO (0.5, 1, 2 lmol) after 24 and 48 h to identify the hTERT, TP53, ERG, CDKN2A, and CDKN1B copy number status (Fig. 3). Our results show that the numbers of the cells with deleted CDKN1B and CDKN2A as well as the counts of the

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Discussion In this study, through monitoring the DNA copy number changes by FISH analysis, we have shown an increase in DNA copy numbers of CDKN2A and CDKN1B along with a significant decrease in hTERT DNA copy numbers in NB4 cells upon treatment with ATO. Loss of tumor suppressor genes and sustained activation of proto-oncogenes are considered as the common causes for deregulated cell proliferation and apoptosis. The discoveries of these genomic changes have had a profound influence on our understanding of the molecular mechanisms underlying leukemogenesis. We selected five tumor suppressors and

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Fig. 2 FISH assays performed on the NB4 cells which were analyzed for chromosomal imbalances in the ERG, CDKN1B, hTERT, CDKN2A, and TP53 regions. Amplification of ERG and hTERT region and loss of the TP53, CDKN1B, and CDKN2A region are evident. a Hybridization of the ERG FISH probes. Orange (gray) signals corresponding to the ERG loci. b Hybridization of the hTERT FISH probes. Orange (gray) signals corresponding to the hTERT loci and yellow (bright) signals for PDGFRb as control. c Hybridization of the CDKN1B/CEP12 FISH probes. Four orange (gray) signals

corresponding to centromeric region of chromosome 12; three and two green (bright) signals corresponding to the CDKN1B loci. d Hybridization of the p53/CEP17 FISH probes. Four orange (gray) signals corresponding to centromeric region of chromosome 17, two green (bright) signals corresponding to the TP53 loci. e Hybridization of the CDKN2A/CEP9 FISH probes. Two green (bright) signals corresponding to centromeric region of chromosome 9, one orange (gray) signal or cells without orange (gray) signal corresponding to CDKN2A deletion

oncogenes which are important in leukemogenesis to evaluate the response to ATO in NB4 cells using FISH, which allows the assessment of larger number of cells than can be examined by standard cytogenetic techniques, and gives chromosome specific information [21]. Copy number changes of our candidate genes have been reported previously in SNP array analysis of APL patients [2, 22, 23]. NB4 cells are cytogenetically very complex, with multiple chromosomal alterations [16, 24]; the SNP-chip analysis of NB4 cells also showed ploidy = 3.67, indicating that the karyotype is hypertriploid. Loss of the chromosomal region of TP53 was evident in the karyotype of NB4 cell line. Deletion of TP53 region has been detected by using aCGH in NB4 cells and also confirmed by FISH in previous study [4]. TP53 at 17p13 is a tumor suppressor gene encoding nuclear phosphoproteins involved negatively in the cell cycle control of transition from G1 to S phase. Lack of

functional TP53 expression due to deletions or mutations leads to insufficient gene dosage, and suboptimal TP53 function contributes to malignancy [25]. CDKN2A is an important cyclin-dependent kinase inhibitor that allows cells to pass through the G1 check point; we found loss of two alleles of this gene despite the presence of two copies of chromosomes 9 in untreated and treated NB4 cell with ATO. Deletions of CDKN2A and/or CDKN2B have been described at frequencies of up to 80% in cell lines of hematologic malignancies [26]. We also found allelic loss of CDKN1B (Fig. 2). A decrease in CDKN1B induces quiescent cells to proliferate, whereas an exit from the cell cycle is associated with up-regulation of CDKN1B [27]. Deletion of 12p13 is a recognized finding in leukemia, suggesting that the CDKN1B gene may act as a tumor suppressor gene in leukemia. After ATO treatment, we observed that the

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Med Oncol (2012) 29:1209–1216 b Fig. 3 Significant reduction in cells with CDKN1B (a) CDKN2A

(b) allelic loss after 48 h at 1 and 2 lM concentration of ATO (P \ 0.001) and hTERT amplification (c) at different concentration of ATO (P \ 0.0001)

number of cells with deleted CDKN1B and CDKN2A chromosomal regions were declined (Fig. 3a, b). It has been suggested that ATO induces growth arrest in the G1 phase of the cell cycle by enhancement of cyclin-dependent protein kinase inhibitors, CDKN2A and CDKN1B [11]. ERG proto-oncogene is a member of the ETS family of transcription factors [2]. Extra signals hybridizing to the ERG region were the result of trisomy 21 as identified during G-banded chromosome analysis rather than chromosomal amplification, such as a double minute. Alterations of this gene, which is critical for the control of proliferation, differentiation, and apoptosis, might have a substantial impact on cellular processes. ERG transcription factor is altered by as yet unknown molecular mechanisms involved in leukemogenesis [28]. It has been reported that massive telomere loss is an early event of DNA damage-induced apoptosis. Telomerase activity is important for maintenance of telomere ends. Inhibition of telomerase activity suppresses the growth of human cancer cells and leads to chromosomal damage and apoptosis [29]. Chou et al. reported that ATO at concentrations of 0.75–1.0 lM inhibited telomerase transcription and resulted in increased chromosomal end lesions, which promote either genomic instability and thus carcinogenesis or cancer cell death in NB4 cells [30]. We observed hTERT amplification in NB4 cells. Telomerase activity is important for maintenance of telomere ends. However, both deficiency and overabundance of telomerase contribute to formation of mammalian malignancies [31–33], and normal mammalian growth and development are rigidly dependent on optimal levels of telomerase activity [34]. The number of cells with hTERT amplification was reduced after ATO treatment in our study (Fig. 3c). In addition, ATO reduced the signals for hTERT in time- and dosedependent manners (Fig. 3c). It has been reported that As2O3, at concentrations of 0.5–2 lM, could significantly induce apoptosis in the NB4 cell line [12, 35, 36] and at concentrations higher than 1 lM induces programmed cell death through suppression of telomerase activity and telomere length [37]. We assume the cells with TERT amplification grow faster and have higher rate of apoptosis, so we observed reduced amount of cells with hTERT gene amplification after ATO treatment. Our results might also indicate that ATO is able to inhibit growth in vitro through reduction in DNA copy numbers of hTERT in NB4 cells.

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Taken together, the results of the present study indicate that antiproliferation activity of ATO on NB4 cells might be through cytogenetic modifications in hTERT, CDKN1B, and CDKN2A (Fig. 3). These outcomes might introduce a new mechanism of antitumor effects of ATO in APL cells. Acknowledgments This work was supported by the Haematology, Oncology, and Stem cell Transplantation Research Center of Shariati Hospital, Tehran University of Medical Sciences. We acknowledge Prof Stefan K Bohlander and Dr Purvi Kakadia for providing outstanding technical assistance for generating FISH probes. We especially thank Dr Arash Jalali for statistical analysis and Philippa May and Majid Momeny for editing this paper. Conflict of interest of interest.

The authors declare that there are no conflicts

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