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Teratogenesis, Carcinogenesis, and Mutagenesis Supplement 1:171–186 (2003

)

Chromosomal Aberrations Induced by
5-Azacytidine Combined With VP-16
(Etoposide) in CHO-K1 and XRS-5
Cell Lines

A.P.A. Guimarães,1 F.L. Dias,2 R.S. Cardoso,1 S.N. Kronka,3 and
E.T. Sakamoto-Hojo1,2n
1
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto,
Universidade de São Paulo, SP, Brasil
2
Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão
Preto, Universidade de São Paulo, SP, Brasil
3
Departamento de Ciências Exatas, Faculdade de Ciências Agrárias e Medicina
Veterinária da UNESP-Campus de Jaboticabal, SP, Brasil

A cytogenetic study was carried out with 5-azacytidine (5-azaC) and etoposide
(VP-16) in CHO-K1 and XRS-5 (mutant cells deficient for double-strand break
rejoining) cell lines to verify the interaction effects of the drugs in terms of
induction of chromosomal aberrations. 5-azaC is incorporated into DNA causing
DNA hypomethylation, and VP-16 (inhibitor of topoisomerase II enzyme) is a
potent clastogenic agent. Cells in exponential growth were treated with 5-azaC for
1 h, following incubation for 7 h, and posttreatment with VP16 for the last 3 h. In
K1 cells, the combined treatments induced a significant reduction in the
aberrations induced in the X and ‘‘A’’ (autosome) chromosomes, which are the
main target for 5-azaC. However, in XRS-5 cells, the drug combination caused a
significant increase in the aberrations induced in those chromosomes, but with a
concomitant reduction in the randomly induced-aberrations. In addition, each
cell line presented characteristic cell cycle kinetics; while the combined treatment
induced an S-arrest in K1 cells, alterations in cell cycle progression were not
found for XRS-5, although each drug alone caused a G2-arrest. The different cell
responses presented by the cell lines may be explained on the basis of the evidence
that alterations in chromatin structure caused by 5-aza-C probably occur to a
different extent in K1 and XRS-5 cells, since the mutant cells present a typical

Contract grant sponsor: FAPESP; Contract grant sponsor: CAPES; Contract grant sponsor: CNPq.

n
Correspondence to: Elza T. Sakamoto-Hojo, Departamento de Biologia, Faculdade de Filosofia Ciências
e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901 Ribeirão Preto,
SP, Brasil. E-mail: etshojo@usp.br

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/tcm.10072

r 2003 Wiley-Liss, Inc.

Mutagen. resulting in a hypomethylated state. Suppl. and also generate large insertions and deletions. Key words: 5-azacytidine. and a complete remission rate of 45–60% was achieved [12. 13]. which are represented at lower frequency than that expected throughout the eukaryotic genome [7. These breaks become the target for recombination and sister chromatid exchange. 5-azaC is widely used in many fields of biological research. 3]. When 5-azaC is incorporated into the DNA (instead of cytidine). There is considerable evidence that methylated sites contribute to the silencing of gene activity in various biological contexts [4]. In general. except for those located at the inactive X-chromosome and parentally imprinted genes [9. drug combinations are commonly prescribed for different types of malignant neoplasias with the purpose to improve the chemotherapeutic regimens.172 Guimarães et al. 8]. a negative interaction effect between 5-azaC and inhibitors of topo II enzyme (ellipticine and teniposide) was observed in CHO cells. inhibitors of topo II are widely used in anticancer chemotherapy. consequently producing chromosomal aberrations [17]. In contrast. before the re-ligation step [15]. the nitrogen atom is not methylated. This intermediate is termed cleavable complex and consists of a topo II monomer that is bound covalently to the 50 termini of a DNA double- strand break (DSB) [16].  c 2003 Wiley-Liss. on the basis of its property to cause DNA hypomethylation. regions of the genome defined as CpG islands contain the predicted frequency of CpG dinucleotides that are usually unmethylated. In an earlier study. thus introducing high levels of transient protein-associated DNA breaks in treated cells. alternatively. particularly . the mechanism by which the cytotoxic effect of a certain antitumoral drug can be modulated by another compound is poorly understood. Etoposide is a commercially available antineoplastic drug classified as an inhibitor of DNA-topoisomerase II enzyme. and the cytotoxic and antitumoral effect of these inhibitors resides in their ability to inhibit the topo II activity at an intermediate step following DNA scission. chromosomal aberrations. in most cases. Bender et al.6]. which catalyses the breakage and rejoining of both DNA strands during replication [14]. but. etoposide. The anti-leukemic drug 5-Azacytidine (5-azaC) is a cytidine analogue with a nitrogen replacing the carbon atom at 5-position of the pyrimidine ring [1]. 10]. However. hyper-condensed chromosome structure (especially the X. [11] demonstrated that 5-azaC is able to slow the growth of tumor cells during subsequent cell passages by reactivating growth-regulatory genes (p16) silenced by de novo methylation. Besides its application in anticancer therapy. It has been reported that an intensive regimen of etoposide (VP16) combined with 5-azacytidine was efficient to treat patients with refractory or relapsed acute nonlymphocytic leukemia (ANLL). 1:171–186. 5-aza-C could induce reactivation of DNA repair genes in XRS-5 cells. which has been correlated with gene activity and cellular differentiation [2. Teratogenesis Carcinog.and ‘‘A’’ chromo- somes). and genes silenced by hypermethylation can be reactivated by 5-aza-C [5. DNA methylation normally occurs at cytosine residues within the CpG dinucleotides. 2003. as a useful tool to study the role of DNA methylation at different cellular processes. DNA methylation INTRODUCTION In chemotherapy. Inc.

Before establishing such levels of concentrations. or treatment with inhibitors of DNA topoisomerase II enzyme [21].005 mg/ml penicillin). many preliminary .02. and 4 mM.38 mg/ml Hepes (Sigma). MATERIALS AND METHODS Cell Lines CHO-K1 and XRS-5 were kindly provided by Pof. The aim of the present work was to study the interaction effects of 5-azaC (added to cell cultures during the S-phase) with VP16 posttreatment at the G2-phase of the cell cycle. Since 5-aza-C is unstable in aqueous solution.1. 2. the concentrations were 1. 10. antibiotics (0. and 8 mM. 5-azaC was added to CHO-K1 cell cultures at the concentrations of 2. 8 h before fixation. Chemicals Etoposide (VP-16) was obtained from Bristol-Myers Squibb.. and 5-aza-C from Sigma.0 mg/ml VP-16. VP-16 was prepared in deionized distilled water at the concentrations of 0. 4. lower concentrations of 0. 0. it was stored at 201C and prepared in phosphate-buffered saline (PBS) immediately before use. which was added to the cultures of CHO-K1 cells at the G2-phase (last 3 h).01. The cells were maintained as monolayers growing at 371C in 25-cm2 flasks containing HAM-F10 þ DEM (Sigma. 5-azaC treatments (in a 60-min pulse) were performed in serum-free medium. and 2. Louis. St.05 mg/ml were chosen on the basis of the results from preliminary experiments. A. a significant reduction (35–68%) in the frequencies of chromosome aberrations was observed in both cell lines. it can be expected to obtain additional information on the mechanism by which the DNA damage induced by 5-azaC would interact (during one cell cycle) with the subsequent effects induced by etoposide. excluding those located on the X-chromosome [19]. followed by three washes in PBS solution and incubation in complete medium at 371C. Natarajan (Leiden University Medical Center. and 0. followed by 0.5 or 1. VP-16 treatment was performed in a 30-min pulse. while for XRS-5 cells. concerning the induction of chromosome aberrations in Chinese hamster ovary fibroblast cell lines: CHO-K1 (wild-type) and XRS-5 (mutant cells). By comparing the cell response between both cell lines. Cell Treatment With 5-aza-C and/or VP-16 Cells in exponential growth were seeded (1  106 cells per flask) and allowed to grow for 20 h. and 100 mg/ml. MO) culture medium supplemented with 15% fetal calf serum (Cultilab. All the experiments were carried out with CHO-K1 and XRS-5 cells at the 5–10th culture passage after thawing. an inhibitor of topo II enzyme. For the mutant XRS-5 cells.01 mg/ml streptomycin and 0. In both cases.T. i. The effects of combined treatments with 5-azaC and the antitumoral compound cytosine arabinoside (Ara-C) were also studied in CHO-K1 compared with XRS-5 cells. XRS-5 cells were described to be deficient for DNA double-strand break rejoining and more sensitive to the induction of chromosome aberrations following irradiation [20]. 5-Azacytidine Combined With Etoposide 173 a significant reduction in the production of chromosomal aberrations [18]. The Netherlands).e. Brazil). but only for the aberrations that were randomly distributed.

as well as chromosome exchanges. The air-dried chromosome preparations were stained with 2% Giemsa diluted in phosphate buffer. [23]. The Z-Test for comparison of proportions was applied to compare the distribution of cells at different phases of the cell cycle. Chromosome Spread Preparations and Cytogenetic Analysis Cells were harvested by standard cytogenetic methods (hypotonic shock with 1% sodium citrate. RESULTS Clastogenic Effects of 5-aza-C In the experiments performed with CHO-K1 cells.000. by determining the mitotic indexes in addition to the frequencies of chromosomal aberrations. San Jose. The probability of error considered was r0. these values were used to calculate the magnitude of reduction.174 Guimarães et al. Additional experiments were carried out using different fixation time points: 3. and 8 mM 5-azaC treatments induced an increase in the frequencies of chromosomal aberrations as a . experiments were carried out to evaluate the level of drug cytotoxicity. Statistical Analysis Multi-factorial analysis of variance with repeated measures was applied to the data. 2. In the combined treatments. CA). followed by the Tukey test in order to verify the interaction effects of 5-azaC treatment combined with VP16 concerning the induction of chromosomal aberrations. Following the criteria established in the literature [22]. respectively. and the cell cycle analysis was performed by staining the cells with propidium iodide and applying the detergent-trypsin method described by Vindelov et al. The cultures were incubated with colcemid (1 mg/ml) during the last 90 and 120 min (before fixation) for XRS-5 and CHO-K1 cells.05.000 cells per treatment. The mitotic indexes were obtained by counting the number of mitotic cells in a total of 2. and cells were distributed along the different phases of the cell cycle according to the DNA content.000 cells was considered to minimize the sensitivity of the test and accept only the strictly significant differences. One hundred metaphases were analyzed per culture to score the chromatid or isochromatid breaks. and 5 h. 4. Instead of 10. 4. methanol/acetic acid (3:1) as fixative. an ‘‘n’’ ¼ to 5. and metaphase block with colcemid. which was performed on gated nuclei using the software program (CellFit) provided by Becton Dickinson. Cell Cycle Analysis Similar experiments (as described for cytogenetic analysis) were carried out with both cell lines. the expected values for the CA frequencies were calculated as the sum of the effects caused by each drug alone minus the control value. Ten thousand cells were analyzed using a FACSCAN flow cytometer (Becton Dickinson. The gap lesions were also included in the analysis. metaphases with an acceptable chromosome number (2172) were analyzed to determine the frequencies of chromosomal aberrations.

Similar experiments were carried out with XRS-5 cells by treating them with 1. excluding X and ‘‘A.05) increase in the aberrations induced in those chromosomes (18 to 37%).05 mg/ml). The mitotic indexes suffered a significant (Po0. and also cell death. In XRS-5 cells.. In both cell lines. varying from 28 to 76% and 23 to 60%. the combined treatments induced a significant (Po0. and also in a specific autosome called ‘‘A’’ chromosome. but the differences were not statistically significant (P40. In spite of this. lower VP-16 concentrations were tested (0.’’ In K1 cells. A lack of interaction effect occurred in the frequency of total CAs (considering the aberrations in the whole genome). independently of VP-16 concentration.01.05) increase in the CA frequencies. Considering the toxicity caused by each drug and the possibility of a delay in the cell cycle. but different fixation times (3. 5-azaC induced a high incidence of breaks in the X-chromosome. and compared with the control value. Po0. except the treatment with 5-azaC 4 mM (Table II.05) reduction in the aberrations induced in the X and ‘‘A’’ chromosomes. and they induced a significant (Po0. additional experiments were performed using the same protocol of cell treatment. respectively. Although VP-16 decreased the mitotic indexes. Interaction Effects of the Combined (5-Azac þ VP16) Treatments Comparison between the cell responses of both cell lines to the combined treatments indicated that each one presented a characteristic pattern. but the differences were significant (Po0. and 0.05) reduction in the aberrations randomly-induced in the genome. the differences observed in the CA frequencies were significant.01) increase in the CA frequencies as compared with the control levels.02. except in the case of XRS-5 treated with the highest concentration of two drugs in combination.01) (Table I). Clastogenic Effects of VP-16 Experiments with CHO-K1 cells showed that 0. respectively.01) increase in the CA frequencies in relation to the control level. or 5 h) after drug . 1B).05) in relation to the control value (Table I). the differences with the control values were statistically significant (Po0. 5-Azacytidine Combined With Etoposide 175 function of drug concentration. Fig. 1A). however.5 and 1. concomitantly. Only 5-azaC 8 mM induced a significant (Po0.01. CHO-K1 and XRS-5. the mutant XRS-5 cells presented a significant (Po0. in the wild-type K1 cells. A significant reduction (Po0. 4. ranging from 35 to 55% in relation to the expected values (Fig. 0. and by the Tukey test analysis. which induced a significant (Po0.05 and Po0. the differences were much higher (Po0. those scored in all chromosomes.0 mg/ml VP-16 induced a statistically significant (Po0.05) reduction in the mitotic index (Table I). for each cell line (Table II). and 4 mM of 5-aza-C. 2.05) decrease for the highest 5-azaC treatments (2 and 4 mM) (Table I). The analysis of the total number of abnormal cells also showed an increase as a function of drug concentration (Table I).e. the mitotic indexes for the combined treatments did not differ among the different cultures. for the treatments with 4 and 8 mM. i.05) only for K1 cells.05) among the different treatments.01) in the mitotic indexes (compared with the control) was found for XRS-5 cells submitted to the combined treatments with 2 and 4 mM 5-aza-C þ VP16 (Table II). while in XRS-5 cells there was a significant (Po0.

7 4771.0 n nn n nn 8 F 4.774.772.4 1 0.4 4174.371.2 49.772.370.5 w Three hundred cells were analyzed in three independent experiments.770.3 nn 2 F 6.1 48.1 2 F 3.870.470.2 2 0.372.373.05 3.770.2 67.7 n nn F F F F F 4 0.470.7 F 0. n Po0.3 n57.3 1 0.371.9 7.0 18.570.370.05 4.7 3.7 n nn nn 8 0.8 23.02 4.0 5672.6 3074.4 n n nn 4 F 4.2 2175.3 53.7 4072.3 20. MI=mitotic index.6 3872.2 23. Considering the Total Aberrations in the Genomew Treatments CHO-K1 Treatments XRS-5 5-aza-C VP-16 MI(%)7SE Abnormal cells7SE CAs/100cells7SE 5-aza-C VP-16 (mg/ml) MI(%)7SE Abnormal cells7SE CAs/100 cells7 SE (mM) (mg/ml) (mM) F F 7.1 3373.372.0 F F 7.02 3.670. SE=standard error of the mean.2 1170.5 3.3 n n nn F 0.374.773.170.2 6471.2 41.5 n nn 4 1.774.8 47.4 n27.7 72.2 3172.6 11.01 3.470.371.3 2 0.05 3.93 73.573.270.070.7 1 F 5.01 4.378.5 44.770.170.374.777.5 n51. CAs=chromosomal aberrations.01.0 2572.5 5576.470. Significant difference aanalyzed by the Tukey test.8 5074.570.5 2972.270.672.370.7 57.4 +63.4 F 0.TABLE I.7 n4176.7 4973.770.5 3.870.02 4.5 n nn nn 8 1.01 4.770.670.05. nnPo0.9 F 0.170.379.771.0 2. Chromosomal Aberrations Obtained in the Combined Treatments With 5-azaC and VP16 in CKO-K1 and XRS-5 Cell Lines.7 70.270.5 2.870.9 41.373.8 26.7 36.02 3.070.2 n nn nn 4 0.373.0 3973.9 4 F 3.9 10.6710.22 n n nn 2 0.1 4178.372.4 32.070.5 n nn F F F F F 4 0.070.0 2.6 n nn nn F 1.5 3.5 22.8 41.0 2.570.8 n nn nn 2 1.374.7 5171.778.5 n nn F F F F F 4 0/05 2.870.2 1771.8 5574.7 1 0.6715.670.1 43.5 n nn F F F F F 2 0.3 33.770.370. .01 3.0 1.773.4 41.5 48.7 471.

7) 8 0.9 (49.0) 8 1.777.9) 0.2 (67.2) 9.375.071.2) 4 1.4) F F F F 2 0.4) 2.7 (85.671.474.1 (32.7) 14.375.5) 23.075.6 (28.5) F 0.5 24.6 (9.02 45.05 60.370.05 52.378.071.671.9) 0.670.471.770.4) 2 0.772.9) 3.371.3) 2 F 15.6) 1.6 (0.5 (76.0 55.0 (99.0 (65.8 (71.370.772.0 57.4 (63.7) 7.4 (15.071.7 (90.05 66.7 (4.371.5 (100) 0.5) 18.5 18.8) 3.0) 5.5 32.1 (40.0 (0.675.0 (0.3 (75.0 (92.4 (36.675.7 (30.0 ( 0.6 (53.01 23.076.372.2 (35.3 74. .1 (95.7 (100) 0.1) F 1.3 (57.3 (85.1) 33.0) 15.7710.0) 4 0.5 (100) 0.7 ( 59.470.6) F 0.3 (92.6) 1 0.0711.0) 2 0.0 49.8 (71.172.0) F F F F 4 0.5 (28.6 (38.5 (71.6) 8 F 10.0) 21.01 28.7) 24.2 (24.9) 4 F 31.1) 1 F 23.075.476.0 (63.078.01 25.371.078.8 ( 14.6 (0.0) F F F F 4 0.1) 6.6) 15.374.5 (23.4) 21.1 (0.01 28.5 22.6 (42.0) 2 0.8) 1 0.4711.0 54.075.471.9 (50.0728.4715.9) 1 0.5) n Percentage shown is in relation to the total number of CAs.771.02 39.8 (70.0 (0.773.3) F F F F 4 0.02 34.672.570.070.05 40.070.6 (99.3) 4 F 11.0) 21.4) 2 1.0) 23.4 ( 99.3 (7.6) 22.7 (46.TABLE II.02 25.370.6 (29.4) 2.0) F 0.070.370.6 (100) 0.3721.37 2. Distribution of Chromosomal Aberrations at the Preferential Sites (X+‘‘A’’) for 5-azaC and in the Rest of the Genome [Total-(X+‘‘A’’)] in CHO-K1 CHO-K1 and XRS-5 Cells Submitted to the Combined Treatments With 5-azaC and VP-16 CHO-K1 XRS-5 Treatments Chromosomal aberrationsa Treatments Chromosomal aberrationsa 5-aza-C (mM) VP-16 (mg/ml) Total–(X+‘‘A’’)7SE (%) X+‘‘A’’7SE (%) 5-aza-C (mM) VP-16 (mg/ml) Total–(X+‘‘A’’)7SE (%) X+‘‘A’’7SE (%) F F 471.2 (7.1) 2 F 7.3) 10.7 (36.0) F 0.7 (62.0) F F 11.

and those which were randomly-induced in the total genome. The expected values for CA frequencies were calculated as the sum of the effect caused by each drug alone minus the control value.178 Guimarães et al. . Po0. (*Statistically significant. Frequencies of chromosomal aberrations in CHO-K1 (A) and XRS-5 (B) cells submitted to the combined treatments with 5-azaC and VP16.05). taking into consideration those induced in the X and ‘‘A’’ chromosomes. Fig. 1. Three hundred cells were analyzed for each cell line.

2). and a statistically significant increase in the percentage of cells (B46%) undergoing the S-phase was observed in K1 cells submitted to the combined treatments (5-aza-C þ VP-16) in comparison to the control value (27. 19. an increase in the mitotic index was also observed. and the results were similar to those obtained in the previous experiments. we observed a significant decrease (Po0. Therefore. The interaction effect was not significant for the combined treatments (5-aza-C þ VP-16).e.5 to 39. 2).8% of cells at G0/G1. and cytotoxicity. The results obtained for both cell lines indicated an increase in the mitotic indexes for the cultures harvested at later times. and VP-16 was added at G2-phase (3 h before fixation). S. 5-aza-C þ VP-16 combined treatments induced an interac- tion effect in XRS-5 cells (32. 27. Similarly. These results indicated that the reduction in the CA frequencies is not a consequence of drug cytotoxicity leading to a blockade in the cell cycle. on the border of statistical significance (P = 0.0 mg/ml presented similar CA frequencies. On the other hand.05) in the incidence of K1 cells at G0/G1 and G2/M phases for the combined treatment (8 mM 5-aza-C þ 1. 5-Azacytidine Combined With Etoposide 179 removal (Table III). and the magnitude of reduction in the CA frequencies did not suffer a significant difference among the different harvesting times. as a post-treatment. and G2/M cell cycle phases. While the treatment with 0.4. Cell Cycle Kinetics Cell cycle analysis was performed by flow cytometry in order to study the alterations in the cell cycle progression in response to the treatment with 5-aza-C and VP-16. and 31. and this increase was considerable for 4 mM 5-azaC.3.3%).01 mM VP-16 alone induced a significant increase (Po0.0%. and the mitotic indexes were recovered at later times. being statistically significant (Po0. Concomitantly. XRS-5 untreated cells showed 49. cells in exponential growth were treated with 5-aza-C 8 h before fixation. In . but without variation in the CA frequencies.05) in the incidence of cells undergoing the G2/M phase.0 VP-16) (Fig.6. XRS-5 cells treated with 5-aza-C suffered an increase in the percentage of cells at G2/M transition. and 13. While CHO-K1 cell line without any treatment presented 58.05). but independent of the time of cell harvesting (Table III). Each cell line presented characteristic cell cycle kinetics. i. 24]. DISCUSSION Although many authors emphasize the importance of 5-aza-C as a hypomethy- lating agent. the combined treatments (5-aza-C 4mM þ VP-16) did not induce significant variations in the distribution of XRS-5 cells at different phases of the cell cycle (Fig.. when the majority of cells were undergoing the S-phase of the cell cycle. but without variation in the CA frequencies. Small variations on the cell cycle progression were observed in response to the treatment with 5-aza-C or VP-16 alone. respectively.0512). being higher at later times.6% of reduction in CA frequencies). probably due to DNA hypomethylation as a consequence of inhibition of DNA methyltransferase activity [25].9. In vitro studies demonstrated that this compound has efficient cytotoxic and clastogenic activity during the S-phase [18. K1 cells treated with VP-16 1. in XRS-5 cells. there are few reports in the literature describing its activity in the chromosome structure. In the present study.

CAs = chromosomal aberrations.6 1 F F F 5 7.05 4 2.0 1.0 56 F F 0.4 4 0.0 F 5 4.0 1.2 37 F 4 F 4 4.0 1.7 50 F 8.05 5 4.4 4 0.7 59 F F 1.1 39 F 8. Chromosomal Aberrations and Mitotic Indexes Observed for Different Fixation Times in CHO-K1 and XRS-5 Cells Treated With 5-azaC at S-Phase and Posttreated With VP-16 at G2-Phase of the Cell Cyclen CHO-K1 cells XRS-5 cells Treatments Treatments 5-aza-C VP-16 Fixation Time MI Total Reduction 5-aza-C VP-16 Fixation Time MI Total Reduction (mM) (mg/ml) (h) (%) (%) (%) (mM) (mg/ml) (h) (%) (%) (%) F F 3 6.05 3 3.7 8.05 5 6.4 47 F 8.2 58 39.05 4 6.9 55 F F 0.0 3 1.8 4 F F F 5 8.0 F 3 3.0 F 4 4.2 10 F F F 4 8.1 53 F F 0.5 67 26.5 n The magnitude of reduction in the frequencies of chromosomal aberrations was calculated using the expected values (sum of the effects caused by each drug alone minus the control value) MI= mitotic index.8 57 36.9 32 F 4 F 5 6.9 52 32.1 65 22.6 0 F F F 3 6.1 69 27.2 55 F F 1.7 35 F 8.0 5 5.05 3 3.0 2 F F F 4 7.0 4 4.6 8.0 4 3.7 4 0.TABLE III.3 40 F 4 F 3 2.0 5 4.0 3 2.5 8 F F 1. .

5-azaC treatments (in a 60-min pulse) were performed in serum-free medium. which are the main target for 5-azaC. 2. This effect probably occurs due to the presence of heterochromatic regions. since there is evidence indicating that incorporation of 5-azaC into DNA during its replication and the consequent induced . which was added to the cultures at the G2-phase (3 h before the harvesting time). FACS profiles showing the percentage of K1 and XRS-5 cells at different phases of the cell cycle in cultures treated with 5-azaC and VP-16. and 5-azaC induced high frequencies of chromosome breaks at preferential sites. as previously demonstrated at similar conditions [18]. In both cell lines. which showed a characteristic uncoiled and elongated shape. following by VP-16. both cell lines. 5-Azacytidine Combined With Etoposide 181 Fig. Ten thousand cells/treatment were scored by flow cytometry. in the long arm of the X-chromosome and also in one of the autosomes (called ‘‘A’’ chromosome). 8 h before fixation. the increase in the CA frequencies caused by 5-azaC alone was proportional to drug concentration.

e. However. the differences in the cell response observed between the cell lines can be a consequence of alterations in chromatin structure and in the DNA-nuclear matrix.27]. VP-16 does not bind directly to DNA. in spite of the fact that 5-azaC can induce chromatin decondensation. leading to increased frequencies of aberrations in those chromosomes. This information gives support to suggest that in cells submitted to the combined treatment at high drug concentrations (4 mM aza- C þ 0. i.’’ It has been reported that DNA methylation can alter the distribution of the binding sites for topo II enzyme [32]. making gene transcription and repair activity in XRS-5 cells difficult. but it interferes with topoisomerase II enzyme activity by stabilizing the cleavable complex [28. while in K1 cells 5aza- C treatment may facilitate the binding of DNA repair enzymes. and the induction of aberrations occurred proportionally to drug concentration. This aspect can influence the binding of transcription factors. Comparison between the cell responses of both cell lines to the combined treatments indicated that each one presented a characteristic pattern. as suggested by Slijepcevic and Natarajan [33]. in contrast.05 mg/ml VP-16). however. leading to the reduction in the aberrations induced by the combined treatments in K1 and XRS-5 cells. This is supported by the evidence that the sensitivity of XRS-5 cells to chemicals has been attributed to their super-condensed chromosomes. which presents the highest extent of methylation [26. mainly in the heterochromatic regions. 5-azaC incorporation into DNA could change the cleavage sites for the topo II inhibitor. Experiments carried out with VP-16 tested alone at G2-phase demonstrated its clastogenic activity in both cell lines. In spite of this. as observed only in XRS-5 cells. which are characteristics of XRS-5 cells [36. since their chromosomes are normally super-condensed. this effect could occur at less extension in XRS-5 cells. 5-azaC administered to CHO cells during the S-phase of the cell cycle caused a significant reduction in the frequency of chromosomal aberrations induced by the DNA . A lack of interaction effect occurred in the frequency of total CAs (considering the aberrations in the whole genome). but they also depend on the chemical structure of the compounds. while in XRS-5 cells there was a significant increase in the aberrations induced in those chromosomes. Furthermore. alterations in the methylation pattern caused by 5-aza-C can lead to changes in chromatin structure. thus reducing the induction of chromosome damage. In a previous work. and the extent by which this drug incorporates into DNA in CHO-K1 and XRS-5 cells can be different. considering the typical chromosome morphology of the mutant cells [35]. with consequent difficulty accessing DNA repair enzymes [34]. those scored in all chromosomes.182 Guimarães et al. except in the case of XRS-5 treated with the highest concentration of two drugs in combination. in the wild-type K1 cells the combined treatments induced a significant reduction in the aberrations induced in the X and ‘‘A’’ chromosomes. The topo II enzyme participates in cell proliferation [30] and its maximum activity occurs during the G2-phase of the cell cycle [31]. excluding X and ‘‘A. especially in the heterochromatic X and ‘‘A’‘ chromosomes. DNA hypomethylation may affect the chromosome condensation pattern. protecting the DNA for the induction of damage by VP-16. 37]. Therefore. may make access to the elements of DNA repair machinery difficult. in the mutant cells the hyper-condensation status of the chromosomes.. the mutant XRS-5 cells presented a significant reduction in the aberrations induced randomly in the genome. 29].

in this study VP-16 showed a synergistic effect in the induced-aberrations at the preferential sites for 5-azaC. without any treatment. leading to reduced frequencies of randomly induced chromosomal aberrations. and a statistically significant increase in the percentage of cells (B46%) undergoing the S-phase (S-phase arrest) was observed in K1 cells treated with 5-aza-C þ VP-16. the cell type. who demonstrated that 5-aza-C leads . indicating that the cell response to chemicals in combination with 5-azaC should be influenced by the kind of drug. In contrast. it can be suggested that in XRS-5 cells. Sakamoto-Hojo et al. during the G2 phase of the cell cycle [18]. Jeggo and Holliday [21] described the isolation and the partial characterization of 6 XRS strains sensitive to X-rays and deficient to DSB repair. Similarly. 19.6.3. on the other hand. as well as cytotoxicity [40]. and also to the reduced accessibility to DNA repair enzymes.3%). showing that XRS cells reverted to the wild type after 5-aza-C treatment. silenced by hypermethylation. K1 cells presented 58. which is active in the DNA repair process. and this can be attributed to the different interaction of the topo II inhibitor with the DNA in those super-condensed chromosomes. However. and 31.e. which can influence its interaction with DNA. which can be reactivated by 5-azaC [42]. teniposide (VM26) and ellipticine (EPC). 27. but the same effect was not observed for those located on the X-chromosome. 41].4. becoming more resistant to X-rays. the wild-type CHO-K1 cells may present a normal unmethylated copy of this gene. 5-Azacytidine Combined With Etoposide 183 topoisomerase II inhibitors. Another aspect to be considered in order to interpret the present results is related to the evidence that the control of gene transcription is associated with the methylation status.. those located at X and ‘‘A’’ chromosomes. Analysis of cell cycle progression showed that each cell line presented characteristic cell cycle kinetics. and 13.0%. which were the target for 5-azaC. and that some ‘‘silent genes’’ (like the genes on the inactive X-chromosome) are hypermethylated. other authors have demonstrated that 5-azaC potentiates the induction of sister chromatid exchange by mitomycin C in human lymphocytes [38] and CHO cells [39]. [19] demonstrated that the combined treatment of 5-azaC with cytosine arabinoside (Ara-C) in K1 and XRS-5 cells induced a significant reduction in the frequencies of aberrations that were randomly distributed in the genome. in comparison with the control value (27. S and G2/M phases. In another study. considering the high level of induced-aberrations at preferential sites (X and ‘‘A’’ chromosomes). In addition. XRS-5 cells treated with each drug alone showed a G2-arrest. but with the possibility of being reactivated by demethylation induced by 5-aza-C treatment [6. other reports provided evidence supporting that XRS-5 cells harbor an intact but non- expressed KU80 allele. which is in accordance with the results of Poot et al. as well as the parameter analysed. as already mentioned. Variations on the cell cycle progression were observed in response to drug treatments.9. as mentioned before. However.8% of cells at G0/G1.05 mg/ml) post-treatment at G2-phase may be alternatively explained by the activation of DNA repair gene(s) in consequence of DNA demethylation. or making difficult the access to repair enzymes. respectively. while XRS-5 cells showed 49. i. the protective effect of 5-aza-C (4 mM) combined with VP-16 (0. [43]. it can be suggested that the synergistic effect of this compound with VP-16 may be a consequence of the characteristic chromatin structure of XRS-5 cells.

Alterations in the methylation pattern caused by 5-aza-C can lead to changes in chromatin structure. Costa Júnior and Sueli A. ACKNOWLEDGMENTS We are grateful to Luis A. 4. the drug combination caused a significant increase in the aberrations induced in those chromosomes.5 to 39. or 5 h). an increase in the mitotic indexes was observed. another possibility to explain the reduction in the CA frequencies is that 5-aza-C induces reactivation of certain genes involved in the repair process. or cell selection. the combined treatment (5-aza-C 4 mM þ VP-16) did not change the cell cycle kinetics. the combined treatments induced a significant reduction only in the aberrations induced in the X and ‘‘A’’ chromosomes (which are the main target for 5-azaC). and a concomitant reduction in the aberrations randomly induced in the genome. These results indicated that the interaction effect of the combined treatments was not consequence of the high drug cytotoxicity. protecting the DNA for the induction of damage by VP-16.6% reduction in the total CA frequencies). being compatible with the reduction in the total aberrations for the whole genome observed for the mutant cells. leading to the reduction in the aberrations induced by the combined treatments in K1 and XRS-5 cells. The data in the literature give the support to suggest that in cells submitted to the combined treatment with high drug concentrations (4 mM aza-C þ 0. in the mutant cells the hyper-condensation status of the chromosomes. In both cell lines. However. may make access to the elements of DNA repair machinery difficult. Alternatively. In the wild-type K1 cells. being higher at later times. 5-azaC incorporation into DNA could change the cleavable sites for the topo II inhibitor. and can inhibit the cell division at this phase.05 mg/ml VP-16).184 Guimarães et al. while in XRS-5 cells. on the basis of data in the literature. especially the X and ‘‘A’’ chromosomes. but the cell lines presented different cell response. as observed only in XRS-5 cells. These results were reinforced by the additional experiments. the induced-chromosome damage was not sufficient to cause a G2-arrest. However. . considering the typical chromosome morphology of the mutant cells. and the extent by which this drug incorporates into DNA in CHO-K1 and XRS-5 cells can be different. While in K1 cells 5aza-C treatment may facilitate the binding of DNA repair enzymes. leading to increased frequencies of aberrations in those chromosomes. since their chromosomes are normally super-condensed. CONCLUSIONS The present results demonstrate that treatment of CHO-K1 and XRS-5 cells with 5-aza-C can influence the induction of chromosome damage by the topo II inhibitor VP-16. thus reducing the induction of chromosome damage. in spite of the fact that 5-azaC can induce chromatin decondensation. to an extended G2 phase. XRS-5 cells submitted to the combined treatment (5-aza-C þ VP-16) showed a significant interaction effect (32. Neves for technical assistance. indicating that as a whole. showing a tendency to recover the cell cycle progression. independently of the harvesting time. this effect could occur to a less extent in XRS-5 cells. in which the cell cultures were harvested at different times (3.

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