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Home , Aneugen, Demecolcine, Spindle poison

Aneugenic Potential of the Analogues , and p-N,N-bis (2-Chloroethyl) Aminophenylacetic Acid in Cultures In Vitro

aM. Efthimiou, aC. Andrianopoulos, aG. Stephanou, aN. A. Demopoulos, bS. S. Nikolaropoulos aDivision of , Cell and Developmental Biology, Department of Biology, University of Patras, Greece bLaboratory of Medicinal Chemistry, Department of Pharmacy, University of Patras, Greece

ABSTRACT

Melphalan (MEL), chlorambucil (CAB) and p-N,N-bis(2- chloroethyl)aminophenylacetic acid (PHE) are nitrogen mustard analogues, which are clinically used as chemotherapeutic agents. They also exert carcinogenic activity. The aim of this study was to investigate the aneugenic potential of the above and the possible mechanism responsible for this activity. The Cytokinesis Block Micronucleus (CBMN) assay in combination with Fluorescence In Situ Hybridization (FISH) was used in human lymphocyte cultures to evaluate micronucleus (MN) frequency. Pancentromeric probe (α-satellite) was applied to identify in micronuclei and an X- specific centromeric probe was used to asses micronucleation and non-disjunction of this chromosome in binucleated cells. The effect of the above compounds on the organization of mitotic apparatus, as a possible target of chemicals with

aneugenic potential, was investigated in C2C12 mouse cell line by double immunofluorescence of α- and γ-. We found that the studied drugs increased MN frequency in a linear dose- dependent manner primarily by chromosome breakage and in a lesser extent by an aneugenic mechanism. Non-disjunction and micronucleation of X chromosome were also induced. Abnormal Key words: nitrogen mustard analogues, chromosome cells were linearly increased with concentration and malsegregation, abnormal centrosome number characterized by abnormal centrosome number. Interphase cells

Corresponding author: G. Stephanou, Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Patras 265 00, Greece. 59 Tel: +30 (2610) 969249, Fax: +30 (2610) 997185. E-mail: [email protected] Georgia Stephanou with micronuclei and abnormal centrosome number were also multinucleation. Recently, the discovery of centrosome observed. Since nitrogen mustards are highly reactive agents, hypertrophy or amplification (more than the usual one or with low selectivity and form covalent bonds with different two centrosomes per cell) in cells of a variety of human nucleophilic sites in and nucleic acids, it is reasonable tumors and its direct correlation with provided to consider that one possible pathway for nitrogen mustard one of the major pathways to aneuploidy in cancer cells.19,20 analogues to exert their aneugenic activity is through reaction Moreover, many studies have shown a strong association with nucleophilic moieties of proteins or genes that are involved between the occurrence of centrosome amplification or loss in the duplication and/or separation of centrosomes, resulting in and inactivating mutation of certain tumor suppressor abnormal centrosome number. Based on our results the proteins, most notably p53.21,22 carcinogenicity of nitrogen mustard analogues studied may be attributed not only to their activity to trigger gene mutation and Nitrogen mustards are bifunctional alkylating agents and chromosome breakage, but also to their aneugenic potential. one of the main classes of clinically used anticancer drugs. Further studies are warranted to clarify the above two Although they are also highly carcinogenic they are hypotheses. currently used for the treatment of various human cancers.23,24 Melphalan (MEL) and chlorambucil (CAB) 1. INTRODUCTION are chemotherapeutic agents that belong to the class of nitrogen mustards. They are carcinogenic to humans (Group Aneuploidy is associated with birth defects and 1).25,26 They are also mutagenic and clastogenic in various characterizes various syndromes. It is also known that the test systems including rat embryo cells and bone-marrow development of several types of cancer is associated with an cells, human lymphocyte cultures, Chinese hamster cells, aneuploid condition in somatic cells.1 Aneuploidy may be Drosophila, yeast and bacteria.25,26 Recent findings of our induced by aneugens, or may arise spontaneously. The gene group have shown that chemical analogues of nitrogen mutation hypothesis asserts that cancer is driven by mustards, including the active metabolite of chlorambucil, mutations to proto-oncogenes and tumor suppressor genes, p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE) with the aneuploidy a side effect of tumorigenesis.2 A great induced increased rate of chromosome delay in human number of recurrent chromosome aberrations have been lymphocyte cultures in vitro .27,28 determined in various tumor types.3 Recently, Duesberg et al.4 proposed that aneuploidy offers a mechanism of Since MEL and CAB are nitrogen mustards and are phenotype alteration which above a certain threshold based currently used in the clinical cancer as two on the percentage of affected chromosomes, is sufficient to anticancer drugs of choice, it would be of interest to study cause all cancer-specific phenotypes and is independent of their ability to exert aneugenic potential. For this purpose gene mutation. Aneuploidy could readily explain these we studied the ability of MEL, CAB and PHE to disturb phenotypes by altering the dosage and regulation of chromosome segregation. This was accomplished in human thousands of genes. lymphocyte cultures in vitro by the use of cytokinesis blocked micronucleus assay in combination with FISH analysis using Mitotic spindle, a dynamic bipolar array of , is centromeric probes to discriminate between clastogenic and one of the possible targets of aneugens.5,6 Correct aneugenic phenomena and specific chromosome X probe chromosome segregation during depends on the to investigate the capacity of the drugs to provoke non- formation of mitotic spindle, which is assembled during disjunction.29,30 We also investigated the effect of the above mitosis and meiosis.7 The centrosomes, which constitute compounds on the organization of mitotic apparatus, as a the major organizing centers, play an important possible target of chemicals with aneugenic properties. The role during mitosis and they contribute to control spindle analysis was performed in C2C12 mouse cell line using bipolarity, spindle positioning and cytokinesis.8 Complexes simultaneous immunofluorescence staining for α- and γ- of the γ-tubulin in the pericentriolar area are essential tubulin which constitute basic components of mitotic spindle. for microtubule nucleation and organization.9,10 Alterations in microtubule organization as a consequence of changes 2. MATERIALS AND METHODS in centrosomal function and number cause deleterious effects on cells. 2.1. Chemical compounds

A variety of agents such as colcemid,11 3,4- p-N, N-bis(2-chloroethyl)amino phenylacetic acid (PHE, dichloroaniline,12 thiabendazole,13 dimethylarsinic acid,14 CAS no. 103-82-2) was prepared according to the heat shock,15 cytomegalovirus,16 X-rays17 and fungicides18 procedure described in the literature.31 p-N,N-bis(2- have been demonstrated to induce multipolar spindles and chloroethyl)amino-L-phenylalanine (MEL, CAS no. 148-

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82-3 ) was prepared according to Bergel’s procedure32 and 2.3. Mitotic spindle morphology study p-N,N-bis(2-chloroethyl)amino phenylbutyric acid (CAB, CAS no. 305-03-3) were prepared according to Everett’s 2.3.1. Cell line culture and treatment procedure.33 The synthesized compounds were identified 1 13 and characterized by IR, H-NMR, C-NMR and LC-MS C2C12 cell line (DSMZ) derived from mouse myoblasts spectrometry. All the tested compounds were analytically was used in this study.34,35 These cells have the ability to ® pure (HPLC, Waters, MILLIPORE ). form an extensive microtubule network. Thus the C2C12 cell line is a good system to study alterations in the 2.2. Cytokinesis Block Micronucleus (CBMN) organization of mitotic apparatus by treating the cells with assay and chromosome segregation study the studied compounds. The cells were grown in a monolayer in essential medium and were routinely 2.2.1 Donors maintained in DMEM (Biochrom, Berlin, Germany) supplemented with 20% fetal bovine serum (FBS; Gibco- Three healthy, non smoking persons, one male and two Invitrogen), 2% chick embryo extract (CEE; Gibco- females, aged between 22 and 24 years old, who had received Invitrogen) and antibiotics, gentamycin (Gibco-Invitrogen, no treatment, had not been exposed to x-rays and had no CAS no. 1403-66-3) and fungizone (Gibco-Invitrogen, CAS o evidence or history of infection in the last 6 months prior to no. 1397-89-3), at 37 C in a 5% CO2 in humidified air. The the investigation, were used as blood donors to establish cells were seeded at a density of 1.8x105 cells/ml and grown whole blood lymphocyte cultures. on 22x22 mm glass coverslips in 35 mm Petri dishes and preincubated for 24 h after which the slides were placed in 2.2.2. Lymphocyte cultures, treatment and fresh medium. The cells were treated with MEL, CAB and micronucleus frequency PHE to give final concentrations of 1, 1.5, 2, 3 and 5 µM for 48h. After the treatment, the cells were washed twice with Analysis of MN was performed with the CBMN assay phosphate-buffered saline (PBS) and then fixed in absolute according to the recently published procedure.28 Separate methanol at -20 oC. (Sigma, CAS no. 477-30- cultures were established from each donor corresponding 05) at 0.03 µM was used as positive control. Experiments to the different experimental points, control and treated. were repeated at least twice unless otherwise indicated. The test chemicals were added into the cultures 41 hours after culture initiation to give final concentrations of 1, 2, 5 2.3.2. Fluorescence double staining for α- and and 10 µM . Slides were stained with 10% Giemsa (Ferak, γ- tubulin Berlin, Germany, CAS no. 51811-82-6), or were stored in the dark at 4 °C for FISH analysis. Cells were washed once in cold PBS and then incubated overnight at 4 °C with a diluted 1:200 mouse anti-γ-tubulin 2.2.3. FISH analysis monoclonal antibody (T6557, Sigma). Following PBS rinsing, the slides were incubated for 50 min in a humidified Pancentromeric probe. FISH was performed using an α- chamber at 37 °C with a diluted 1:20 FITC-conjugated satellite probe for all human directly labeled secondary anti-mouse IgG fragment antibody from sheep with FITC (Q-Biogene-MP Biomedicals, France, (F2266, Sigma). After extensive washing in cold PBS, the PAHC0001-G) according to the company’s proposed cells were kept for 50 min in the presence of Goat Serum protocol . (S9023, Sigma), a blocking reagent. The cells were washed twice in cold PBS and incubated overnight at 4 oC with a Specific chromosome X probe. Hybridization was diluted 1:100 mouse anti-α-tubulin monoclonal antibody performed at 37 °C overnight with a chromosome X α- (T5168, Sigma). Finally, the samples were incubated for 50 satellite digoxigenin-conjugated probe, DXZ1 (Oncor min at 37 °C with an anti-mouse antibody conjugated with Gaithersburg, MD, CP5060-DG.5) according to previously TRITC from goat diluted 1:20 (Sigma, T7782). DNA was published protocol.29 counterstained with DAPI.

In both FISH procedures counterstaining was performed 2.4. Slide scoring and microscopy using DAPI (Sigma, CAS no. 47165-04-8) after which the slides were air-dried and mounted in Vectashield Mounting 2.4.1. Micronucleus analysis and chromosome Medium (Vector, Burlingame, CA, CAS no. 152522-08-2). segregation The slides were kept in the dark at 4 °C and analyzed in Zeiss Axioskop fluorescence microscope. To determine MN frequencies at least one thousand

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No.1, January 2009 61 Georgia Stephanou binucleated cells were evaluated per each experimental immunofluorescence detection for α- and γ-tubulin was point. Standard criteria were used for the identification of performed using a Zeiss Axioscop epifluorescence MN.36 For assessment of possible effects on cell proliferation microscope equipped with an image analysis system (ISIS the Cytokinesis Block Proliferation Index (CBPI) was METAsystem). The bandpass filters used were of 546, 490 determined,37 which is given by the equation and 360 nm for green, blue and ultraviolet light respectively.

CBPI=[M1+2M2+3(M3+M4)]/N, where M1, M2, M3 and Slides were also analyzed with a Leica TCS-SP Confocal M4 corresponds to the number of cells with one, two, three Microscope, equipped with Argon Ion Laser and Helium- and four nuclei, respectively and N is the total number of Neon Laser covering wavelengths ranging from 450-650 cells. At least two thousand cells were scored per donor and nm. The system also contained Leica Confocal Software per experimental point. Coded slides were analyzed by one LCS. microscopist. Slides hybridized with the pancentromeric probe were analyzed for the presence (C+MN) or the 2.5. Statistical analysis absence (C-MN) of a signal for each experimental point. C+MN had the same staining intensity Statistical analysis was achieved by the G-test for as the main nucleus. MN were characterized only in those independence on 2 x 2 tables. This test is based on the cells whose nuclei contained clear pancentromeric signals. general assumption of the χ2 analysis, but offers theoretical Chromosome X malsegregation was considered in slides and computational advantages.39 In order to test if a dose- hybridized with X-chromosome specific probe, according to response relationship exists between test compound the following distribution patterns: binucleated cells with concentration and the studied end-point, linear regression normal signal distribution 2:2; single non-disjunction 3:1; analysis was performed and correlation coefficient (r) and double non-disjunction 4:0. Two signals very close or probability (P) were calculated. P values <0.05 indicated a touching each other or connected with a thin hybridized linear dose-response relationship.40 bridge were recorded as one and only two signals clearly separated from each other were recorded as two.38 Only 3. RESULTS binucleated cells where the total number of X signals was four, were recorded. The presence of X signal in MN was 3.1. Micronucleus frequency and FISH also scored. analysis in human lymphocyte cultures

2.4.2. Spindle morphology disturbances 3.1.1. Micronucleus frequency and cytotoxicity One thousand cells were analyzed to estimate Mitotic Index as percentage (%) of cells in mitosis. At least 1000 Data on micronucleus induction in whole blood cultures interphase cells were scored and analyzed for the presence determined by Giemsa analysis are presented in Table 1. of multiple signals of γ- tubulin, showing abnormal number Control values are coming from three different experiments of centrosomes. The ability of the studied compounds to for each donor, one for every compound. This also holds arrest cells in metaphase was evaluated by the distribution true in the analysis of chromosome breakage and delay. A of mitotic figures in at least 200 mitotic cells. A total of 200 statistically significant increase in BNMN and MN induction metaphase cells were analyzed for the presence of α- and γ- was observed for all studied compounds in both donors. At tubulin spindle alterations. were independently the lowest concentration, 1 µM, MEL induced a more than classified according to their spindle morphology and three two-fold increase in the frequencies of BNMN and MN, groups of abnormal configurations were discriminated. Non- while at the same concentration CAB and PHE increased congressed bipolar metaphases: Abnormal metaphases with the corresponding endpoints three or more times. Regression a bipolar exhibiting failure of chromosomes analysis showed a linear-dose response for BNMN induction to align on the metaphase plate and move on the mitotic by MEL for both donors. There was also linear increase in spindle. Monopolar metaphases: Metaphases showing only MN induction by CAB only for the first donor, while the one or two mitotic poles extremely close to each other. second donor showed a less clear dose-response for both Multipolar metaphases: Metaphases with more than two endpoints analyzed. A linear dose-response was also spindle poles. Mitotic cells characterized by abnormal observed for BNMN induction by PHE in both donors and centrosome number or the presence of nuclear material for MN in the second donor, while in the first donor a less that is not integrated in the main nucleus, were considered clear dose-response was identified in MN induction. With as abnormal cells. regard to CBPI an obvious decrease was shown in both donors for every studied compound. Microscopic analysis of FISH signals and

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Table 1. Spontaneous and induced frequencies of BNMN and MN in lymphocyte cultures of two donors treated in vitro with different concentrations of MEL, CAB and PHE

Chemical Donor Concentration Binucleated CBPI ‰ BNMN ‰ MN compound µM cells

0 3696 2.03 5.68 5.68 1 1290 1.76 12.40* 12.40*

1 2 1301 1.61 24.60* treg=4.31 26.90* treg=3.71 5 1143 1.57 51.62* R2=0.861 57.74* R2=0.821 10 1048 1.52 57.25* P=0.023 60.11* P=0.034

0 3528 2.14 6.23 6.23 1 1320 1.84 13.64* 13.64*

2 2 1371 1.78 18.96* treg=13.80 18.96* treg=13.95 5 1374 1.57 26.20* R2=0.984 26.93* R2=0.985 10 1116 1.51 50.18* P=0.001 52.87* P=0.001

0 3696 2.03 5.68 5.68 1 1192 1.70 15.94* 17.62*

1 2 1300 1.71 18.46* treg=2.66 18.46* treg=3.66 5 1041 1.56 48.23* R2=0.703 48.23* R2=0.817 10 1087 1.51 42.32* P=0.076 49.68* P=0.035

CAB 0 3528 2.14 6.23 6.23 1 1296 1.72 29.32* 32.41*

2 2 1424 1.68 34.41* treg=3.06 34.41* treg=3.13 5 1127 1.58 39.93* R2=0.758 39.93* R2=0.765 10 1075 1.53 53.02* P=0.055 56.74* P=0.052

0 3696 2.03 5.68 5.68 1 1269 1.73 22.85* 23.64*

1 2 1291 1.73 29.43* treg=3.23 29.43* treg=3.01 5 1214 1.58 37.89* R2=0.776 40.36* R2=0.751 10 1014 1.50 45.36* P=0.048 45.36* P=0.057

PHE0 3528 2.14 6.23 MEL 6.23 1 1310 1.78 16.79* 16.79*

2 2 1145 1.66 20.09* treg=3.32 20.09* treg=3.85 5 1207 1.60 33.97* R2=0.786 34.80* R2=0.832 10 1077 1.52 35.28* P=0.045 38.07* P=0.031

CBPI: cytokinesis block proliferation index; BNMN: binucleated micronucleated cells; MN: micronuclei.The frequencies are estimated per thousand binucleated cells.* 0.001< p <0.03 in comparison with untreated cultures (G-test).

3.1.2. Chromosome breakage versus MN with no hybridization signal was the major type of the chromosome delay induced MN indicating a strong clastogenic activity of the compounds under study. In addition the frequencies of the Only the highest concentration was studied for the three MN exhibiting hybridization signal were statistically compounds in the same donors, at which the highest MN significantly increased for all studied compounds in both induction was identified in order to discriminate donors indicating an aneugenic potential. chromosome breakage versus chromosome delay (Table 2).

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Table 2. Spontaneous and induced frequencies of micronuclei with (C+MN) and without (C - MN) a hybridization signal in lymphocyte cultures of two donors treated in vitro with 10 µM of MEL, CAB and PHE, as evaluated after FISH analysis using an α-satellite probe for all human centromeres

Chemical Donor Concentration Binucleated MN % C+MN % C-MN ‰ C+MN ‰ C-MN compound µM cells

0 6413 42 26.19 73.80 1.71 4.83 1 10 1023 51 17.65 82.35 8.79* 41.06*

MEL 0 6238 56 23.21 78.79 2.08 6.89 2 10 1416 81 20.99 79.01 12.00* 45.20*

0 6413 42 26.19 73.80 1.71 4.83 1 10 1013 60 21.67 78.33 12.83* 46.40*

CAB 0 6238 56 23.21 78.79 2.08 6.89 2 10 1036 68 19.12 80.88 12.55* 53.09*

0 6413 42 26.19 73.80 1.71 4.83 1 10 1854 98 16.33 83.67 8.63* 44.23*

PHE 0 6238 56 23.21 78.79 2.08 6.89 2 10 1008 55 12.73 87.27 6.94* 47.62*

MN: micronuclei; % C+MN, % C - MN: percentage of micronuclei with and without a centromeric signal in the total detected micronuclei, respectively.Frequencies are expressed per thousand binucleated cells.* 0.0001< p <0.01 in comparison with untreated cultures (G-test).

3.1.3. Chromosome X malsegregation followed a linear dose dependent pattern. A weak mitotic arrest was observed for MEL at 2, 3 and 5 µM and for CAB Chromosome X aneuploidy rate was evaluated in a third at 3 and 5 µM in comparison to the strong mitotic arrest of young female donor. An elevated chromosome X non- Demecolcine at 0.03 µM (Figure 3). No statistically disjunction was observed in parallel with increased rate of significant increase of metaphase percentage was observed micronucleation of the same chromosome (Table 3). Total for PHE. As shown in Figure 4, the formation of abnormal malsegregation provoked by the three pharmaceutical mitotic cells was provoked in a statistically significant manner compounds was about 4-fold for MEL and CAB and 3-fold starting from the lower tested concentration of MEL (1 µM) for PHE compared with control. Chromosome X and from 1.5 µM concentration of CAB and PHE. The malsegregation is depicted in Figure 1. enhancement of the percentage of abnormal mitotic cells was linearly correlated with the concentration for all studied 3.2. Immunostaining of α- and γ-tubulin in drugs.

C2C12 mouse cell line 3.2.2. γ-tubulin signals in metaphase cells 3.2.1. Mitotic Index, mitotic arrest and abnormal mitotic cells Treatment of cells with MEL and CAB resulted in a statistically significant increase of the percentage of abnormal

Treatment of C2C12 mouse cells with the three compounds metaphases including bipolar, non-congressed, monopolar resulted in decreased Mitotic Index (Figure 2). MEL and polypolar metaphases (Table 4). In contrast, treatment seemed to be the most toxic agent at 2, 3 and 5 µM of cells with PHE increased only the percentage of monopolar concentrations, while CAB and PHE decreased the Mitotic and multipolar metaphases. At 5 µM MEL and PHE induced Index in a parallel way at least for the two higher a high percentage of abnormal metaphases, which was at concentrations, 3 and 5 µM. The decrease of Mitotic Index the same level as obtained by a much lower concentration

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Table 3. Spontaneous and induced frequencies of chromosome X micronucleation and non-disjunction in lymphocyte cultures of a female donor (donor 3) treated in vitro with 10 µM of MEL, CAB and PHE, as evaluated after FISH with chromosome specific centromeric probe.

Chemical Binucleated ‰ ‰ MN BNMNX+ % ‰ NDX Total compound cells BNMN BNMNX+ BNMNX+ 3:1 % Malsegregation %

Untreated 3783 9.25 9.52 9 0.24 2.43 32 0.86 1.10

MEL 1188 78.28* 86.70* 12 1.01 10.93* 34 3.10* 4.11*

CAB 1091 99.91* 112.74* 11 1.01 10.58* 31 2.98* 3.99*

PHE 1075 57.67* 60.46* 9 0.84 8.81* 22 2.15* 2.99*

BNMN: binucleated micronucleated cells; MN: micronuclei; BNMNX+: binucleated cells displaying chromosome X micronucleation; NDX: binucleated cells displaying chromosome X non-disjunction; The Total Malsegregation rate for chromosome X is given by the sum of % BNMNX+ plus % NDX Frequencies are expressed per thousand binucleated cells. * 0.0001< p <0.01 in comparison with untreated cultures (G-test).

Figure 1 FISH with pancentromeric probe (A1) and specific centromeric probes for chromosome X (A2, A3) in human lymphocyte cultures. A1: Binucleated cell with micronucleus exhibiting hybridization signal (MEL 10 µM), A2: Non-disjunction for chromosome X (MEL 10 µM) and A3: Chromosome X micronucleation (CAB 10 µM). of Demecolcine, 0.03 µM. This can be explained by the tubulin spots are shown in Figure 5 (B1-B7). fact that MEL and PHE are primarily and express their aneugenic activity at a considerably higher 3.2.3. γ-tubulin signals and micronuclei in concentration than a classic aneugen, such as Demecolcine. interphase cells. Disturbance of microtubule It is worthwhile to mention that, at the lower concentrations arrays of the investigated compounds, the percentage of monopolar metaphases was higher than the percentage of multipolar Abnormal interphase cells were considered in relation to ones, while at higher concentrations the opposite held true. the number of γ-tubulin signals and lagging nuclear material- In addition the enhancement of the percentage of micronuclei. Cells with micronuclei were significantly multipolar, as well as of total abnormal metaphases (sum of increased in cultures treated with the compounds. The same the three abnormal types) was linearly related to the was true for cells with multiple signals of γ-tubulin and also concentration. Mitotic cells with abnormal numbers of γ- for cells characterized by both anomalies (Table 5).

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2 Figure 2. Effects of MEL, CAB and PHE on the Mitotic Index of C2C12 mouse cells. (R =0.802, P=0.016, treg=-4.03 for 2 2 MEL, R =0.952, P=0.001, treg=-8.92 for CAB and R =0.805, P=0.015, treg=-4.06 for PHE). Demecolcine (DEM) was used as a positive control.

Figure 3. Distribution of mitotic figures -prophase, metaphase, ana-telophase- in C2C12 mouse cells treated with different concentrations of MEL, CAB and PHE. Demecolcine (DEM) was used as a positive control.

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Figure 4. Abnormal mitotic cells in C2C12 mouse cells after treatment with different concentrations of MEL, CAB and 2 2 2 PHE. (R =0.910, P=0.003, treg=6.34 for MEL, R =0.920, P=0.002, treg=6.80 for CAB and R =0.805, P=0.015, treg=4.07 for PHE). Demecolcine (DEM) was used as a positive control.

µ Figure 5. Abnormal mitotic figures and abnormal interphase C2C12 mouse cells. B1: Multipolar metaphase (MEL 3 M), B2: Monopolar metaphase (MEL 1.5 µM), B3: Tripolar metaphase (PHE 1 µM), B4: Tetrapolar metaphase ( MEL 3 µM), B5: Tetrapolar prophase (CAB 3 µM), B6: Tripolar (PHE 1.5 µM), B7: Telophase with MN and abnormal number of signals for γ-tubulin (CAB 2 µM), B8: Interphase cells with abnormal number of centrosomes (overlay in confocal microscope), C1-C4: Microtubule network in control cells and cells treated with 3 µM of C2: PHE, C3: CAB and C4: MEL

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No.1, January 2009 67 Georgia Stephanou = 6.17 = 4.18 =0.905 =0.967 =0.814 = 10.81 2 2 2 reg reg P=0.003 P=0.000 P=0.014 reg t R R t R t a a 88.09*, 100.00*, 16.56±0.72 13.12±1.04 18.49*±0.77 27.20*±3.65 56.67*±7.79 19.33*±0.39 28.11*±1.28 38.40*±3.27 52.14*±1.62 21.35*±3.02 21.91*±2.49 26.59*±1.42 83.65*±11.10 =7.02 = 4.59 = 4.23 =0.925 =0.840 =0.817 2 2 2 reg reg reg P=0.002 P=0.010 P=0.013 t R t R t R s a percentage (%) in the total detected a a 93.44*, 50.00*, 2.91±0.38 2.60±0.13 3.63±0.34 1.86±0.94 3.40±0.36 15.05±9.21 7.16*±3.41 9.27*±1.16 8.62*±1.43 32.59*±9.56 29.62*±6.94 12.25*±1.05 69.56*±14.66 metaphase mouse cells treated with different concentrations 12 C 2 a a 0 4.76 - tubulin γ γ γ γ γ

0.98±0.98 2.30±0.90 6.66*±0.09 3.19*±1.59 6.81*±0.63 3.53*±0.56 4.52*±0.77 5.47*±1.41 5.83*±0.43 4.69*±2.54 4.58*±1.05 4.53*±0.47 3.72*±0.12 - and α α α α α

a a 6.56 33.33 6.67±0.98 9.58±0.95 8.92±0.48 10.43±0.03 11.19±1.50 13.42±3.84 12.05±1.56 13.11±2.58 8.95*±4.56 15.47*±3.28 23.31*±1.69 17.84*±2.79 17.27*±2.39 Bipolar non- Monopolar Multipolar abnormal metaphases Total a a 0 48.90 81.52±0.77 72.80±3.65 43.33±7.79 83.45±0.71 80.67±0.39 71.90±1.27 61.60±3.27 47.86±1.62 86.87±1.04 78.65±3.02 78.05±2.49 73.41±1.41 10.72±10.72 6.53±5.37 3.14±1.74 79.60±17.80 89.28±10.72 16.36±11.09 70 61 42 450 354 299 422 440 283 159 190 449 437 348 268 M metaphases metaphases congressed 0 903 86.52±2.53 8.63±0.64 1.42±1.15 3.43±0.98 13.48±2.53 1 2 5 1 2 3 5 1 2 3 5 3 µ µ µ µ µ 0.03 516 1.5 1.5 1.5 < 0.05 in comparison with untreated cultures (G-test). <

b

< p MEL CAB PHE DEM DEM was used as positive control. Results from one culture only. metaphases. Table 4. Analysis of aberration induction on mitotic spindle and centrosome organization in C Table Mean of at least two independent cultures (± standard error). The groups normal and abnormal metaphase cells are expressed a a b * 0.0001 Chemicalcompound Concentration Total Normal Untreated of MEL, CAB and PHE, as evaluated after double immunofluorescence for

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No.1, January 2009 68 Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and .... = 4.31 = 9.30 = 8.40 =0.823 =0.956 =0.946 2 2 2 reg reg reg P=0.013 P=0.001 P=0.001 t R R R t t a a cells(%) 97.86*, 98.13*, 7.48*±1.26 9.09*±1.49 5.94*±0.26 9.61*±1.07 6.23*±0.68 9.80*±0.34 32.08*±0.33 42.98*±1.87 87.32*±0.34 40.96*±0.05 60.59*±9.61 97.25*±1.87 38.71*±18.15 a a , a -tubulin γ γ γ γ γ 1.18* 13.69*, 54.42*, as a percentage (%) in the total detected interphase 0.39*±0.12 1.23*±0.04 0.35*±0.05 0.51*±0.15 4.97*±0.04 9.07*±0.12 0.37*±0.09 0.89*±0.30 4.24*±0.34 6.49*±2.23 64.30±24.11 94.45±2.62 26.58*±0.53 46.14*±16.96 = 5.67 = 7.01 = 5.12 =0.889 =0.925 =0.871 2 2 2 reg reg reg P=0.005 P=0.002 P=0.006 t R t R t R - tubulinfor signals γ γ γ γ γ a a , a for Abnormal cells with (%)abnormal Total 7.61* 84.24*, 42.32*, 4.65*±0.72 3.94*±0.99 2.87*±0.08 4.68*±0.90 3.95*±0.88 6.78*±0.31 25.92±19.40 13.46*±0.48 17.74*±3.37 48.15*±0.24 21.71*±0.59 38.22*±7.83 mouse cells treated with various concentrations of MEL, and PHE, as CAB 50.49*±15.55 12 C 2 a a 0.20 1.11*, 0.63±0.46 2.45*±0.44 4.33*±0.36 2.72*±0.13 4.42*±0.02 1.90*±0.58 2.13*±0.34 13.66*±0.77 16.17*±1.39 12.59*±0.64 15.01*±0.21 15.88*±4.02 34.31*±22.54 - tubulin γ γ γ γ γ a a - and 1.87 2.14 α α α α α 2.76±1.87 cells (%)cells MNsignals Multiple MN and multiple Interphase 92.52±1.26 90.90±1.49 94.06±0.26 90.40±1.07 67.93±0.33 57.02±1.87 12.68±0.34 93.77±0.68 90.21±0.34 59.05±0.05 39.41±9.60 5.55±2.62 4.24±2.39 61.29±18.14 99.21±0.16 0.27±0.10 0.53±0.19 0 0.79±0.16 2100 2189 1274 2164 1015 2021 2195 2095 2030 2050 3161 2021 2051 2030 1075 M cells 0 4158 1 2 5 1 2 3 5 1 2 3 5 3 µ µ µ µ µ 0.03 3261 1.5 1.5 1.5 < 0.001 in comparison with untreated cultures (G-test) <

micronuclei.

b

< p MEL CAB PHE MN: DEM DEM was used as positive control. Results from one culture only. * 0.0001 Table 5. Analysis of the induction aberrations interphase cells in C Table Mean of at least two independent cultures (± standard error). The groups normal and abnormal interphase cells are expressed a b cells. Untreated Chemical Concentration Interphase Normal compound evaluated after double immunofluorescence for

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No.1, January 2009 69 Georgia Stephanou

Interphase cells with abnormal numbers of γ-tubulin signals referred that the direct and indirect genotoxic potential of are shown in Figure 5 (B8, overlay in confocal microscope). some antineoplastic drugs, including CAB, evaluated by Regression analysis showed a linear dose response for the means of loss of heterozygosity of the reporter white gene in percentage of cells with multiple signals of γ-tubulin and for Drosophila, could be predominantly due to whole X the percentage of the total abnormal cells for all studied chromosome loss as well as through mitotic inter- and intra- compounds. We also observed a decrease of microtubule chromosomal recombination.44 In addition nitrogen network of α-tubulin in the treated cultures in comparison mustard, the common molecule of the studied compounds, with control (Figure 5, C1-C4), which may indicate that induced a 6-fold increase in aneuploid frequency in a the studied anticancer drugs provoked disturbance in human lymphoblastoid cell line by enhancing the formation microtubule arrays. of cells with two Y chromosomes.45

4. DISCUSSION The results of this study, which support chromosome malsegregation effect by MEL, CAB and PHE, confirm and Alkylating agents are employed in cancer chemotherapy extend our previous findings according to which nitrogen and their chemotherapeutic and cytotoxic effects are mustard analogues provoke chromosome delay in human directly related to the alkylation of DNA. Targets other lymphocyte cultures in vitro.27,28 The molecular mechanism than DNA have been also proposed by several authors. through which the alkylating agents induce chromosome Bonnati et al.41 suggested that modified purines are malsegregation is not well understood. Campagna et al.,46 aneugenic in mammalian cells in culture. Nusse et al.42 suggested a role for mismatch repair in induction of showed that the ethylating agent diethylsulphate induces centromere-containing micronuclei by methylnitrosourea, chromosome loss in V79 cells. In this paper we studied the with DNA being the main target, while diethylsulphate ability of MEL, CAB and PHE to induce aneugenic provoked the same effect in a mismatch repair independent phenomena in human lymphocyte cultures and to affect manner, possibly via inhibition of the S6K1 protein kinase mitotic spindle organization in a mouse cell line. cascade, raising the intriguing question of the benefits of alkylating agents with multiple targets in chemotherapy. We found that MEL, CAB and PHE are capable of inducing increased micronucleus frequency in a dose We also evaluated the aneugenic potential of the studied dependent way. In addition it was revealed by FISH analysis anticancer drugs through their effect on tubulin morphology. that, at the higher concentration studied, the induced We found that MEL, CAB and PHE caused a weak micronuclei are the result of two different phenomena, metaphase arrest and induced abnormal interphase and mainly of chromosome breakage and in a lesser extent of metaphase cells. In addition we indicated that the three chromosome delay. Frequencies of micronuclei containing compounds affect the centrosome organization, since acentric chromosome fragments were about 4-fold (MEL treated cells in interphase as well as in metaphase exhibited and CAB) and 6-fold (PHE) higher than the frequencies abnormal number of centrosomes. Centrosome abnormalities of micronuclei containing centromeric hybridization signal are accompanied by dramatic changes in the number and in both studied donors. These observations show the potent distribution of nucleated microtubules.47 Monopolar clastogenic potential, but additionally point out aneugenic metaphases or spindles with only one centrosome dominated properties of the studied molecules. We also found, after when the cells were treated with lower chemical FISH analysis with chromosome X specific probe, that concentration. Monopolar spindles may arise either due to chromosome X micronucleation as well as chromosome X failure of centrosome duplication or due to defects in non-disjunction are induced by all studied compounds. centrosome separation.48 Higher chemical concentration Chromosome non-disjunction seems to take place in a higher gave increased frequency of multipolar metaphases. The rate than chromosome X micronucleation. Total presence of more than two centrosomes in the same mitosis malsegregation (sum of chromosme X micronucleation and promotes the formation of multipolar spindles and non-disjunction) frequency was increased 3- 4-fold in consequently results in unequal distribution of chromosomes relation to control. to the daughter cells. This loss or gain of chromosomes can cause genetic instability such as elimination of tumor Our results are in accordance with previously published suppressor genes or other imbalance that stimulate decreased findings. Phousongphouang et al.43 found that in MCL-5 apoptotic response to cellular damage, or accumulation of and h1A1v2 human B-lymphoblastoid cell lines, MEL chromosomes with growth promoting genes.47,49 On the caused a significant increase of micronuclei due to other hand it has been suggested that inhibition of chromosome loss, although micronuclei were induced can favor asymmetric division and generation of aneuploid primarily due to chromosome breakage. Recently it was cells.50 In addition centrosome defects have been reported

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No.1, January 2009 70 Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and .... in various human tumors suggesting that they may 174-181. contribute to genetic instability in cancer.19,22 MEL and CAB, 11 C. Sellitto, R. Kuriyama. Distribution of pericentriolar material in multipolar spindles induced by colcemid treatment in Chinese although used as anticancer drugs, are in hamster ovary cells, J. Cell Sci. 89 (1988) 57-65. animals and various case reports suggest sufficient 12 K. Salassidis, M. Bauchinger. Mitotic spindle damage induced carcinogenicity in humans.25 by 3,4-dichloroaniline in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes, Mutagenesis 5 (1990) 367-370. Based on our results their carcinogenicity may be attributed 13 C. Pisano, A. Battistoni, A. Antoccia, F. Degrassi, C. Tanzarella. not only to their activity to provoke gene mutation and Changes in microtubule organization after exposure to a chromosome breakage, but also to their aneugenic properties. benzimidazole derivative in Chinese hamster cells, Mutagenesis. Since nitrogen mustards are highly reactive agents and form 15 (2000) 507-515. 14 T. Ochi. Induction of centrosome injury, multipolar spindles covalent bonds with nucleophilic sites in proteins and nucleic and multipolar division in cultured V79 cells exposed to sites,51 it is reasonable to say that one possible pathway for dimethylarsinic acid: role for microtubules in centrosome nitrogen mustard analogues to exert their aneugenic activity dynamics, Mutat Res. 454 (2000) 21-33. is through reaction with nucleophilic moieties of proteins or 15 C.A. Vidair, S.J. Doxsey, W.C. Dewey. Heat shock alters centrosome organization leading to mitotic dysfunction and genes that are involved in the duplication or separation of cell death, J. Cell Physiol. 154 (1993) 443-455. centrosome, resulting in abnormal number of centrosomes. 16 V.B. Bystrevskaya, T.V. Lobova, V.N. Smirnov, N.E. Makarova, Further studies are warranted to clarify the above A.A. Kushch. Centrosome injury in cells infected with human hypotheses. cytomegalovirus, J. Struct. Biol. 120 (1997) 52-60. 17 C. Sato, R. Kuriyama, K. Nishizawa. Microtubule-organizing centers abnormal in number, structure, and nucleating activity ACKNOWLEDGEMENTS in x-irradiated mammalian cells, J. Cell Biol. 96 (1983) 776-782. 18 A. Can, D.F. Albertini. M-phase specific centrosome- This research was partially supported by the European Social microtubule alterations induced by the fungicide MBC in human granulosa cells, Mutat Res. 373 (1997) 139-151. Fund (ESF), Operational Program For Educational and 19 B.R. Brinkley, T.M. Goepfert.A. Supernumerary centrosomes Vocational Training II (EPEAEK II), particularly the Program and cancer: Boveri’s hypothesis resurrected, Cell Motil. 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