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Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and P-N,N-Bis (2-Chloroethyl) Aminophenylacetic Acid in Cell Cultures in Vitro

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Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and P-N,N-Bis (2-Chloroethyl) Aminophenylacetic Acid in Cell Cultures in Vitro Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and p-N,N-bis (2-Chloroethyl) Aminophenylacetic Acid in Cell Cultures In Vitro aM. Efthimiou, aC. Andrianopoulos, aG. Stephanou, aN. A. Demopoulos, bS. S. Nikolaropoulos aDivision of Genetics, 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 drugs 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 chromosomes in micronuclei and an X-chromosome 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-tubulin. 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 metaphase 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 proteins and nucleic acids, it is reasonable tumors and its direct correlation with aneuploidy 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 chemotherapy 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 microtubules, 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 mitosis 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 microtubule 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 protein 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- Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No.1, January 2009 pp 267-268 60 Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and .... 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,
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