Teratogenesis, Carcinogenesis, and Mutagenesis 21:341–347 (2001)

© 2001 Wiley-Liss, Inc.

Micronucleus Incidence and TheirChromosomal Origin Related to Therapyin Acute Lymphoblastic Leukemia (ALL)Patients: Detection by Micronucleus andFISH Techniques

Hasan Acar, 1* Ümran Çali skan, 2 Sennur Demirel, 1 andDavid A. Largaesp– ada3

1Department of Medical Genetics, Selçuk University, Medical Faculty, Konya,Turkey

2Department of Pediatrics, Selçuk University, Medical Faculty, Konya, Turkey3Department of Genetics and Cell Biology and Development, University ofMinnesota Cancer Center, Minneapolis, Minnesota

Micronucleus assay and dual color-fluorescence in situ hybridization (DC-FISH),using centromere-specific and whole chromosome-specific painting probes, are con-sidered a useful screening test to determine the incidence of micronucleus, their ori-gin and contents. The patients with acute lymphoblastic leukemia (ALL), who hadundergone chemotherapy, were analysed before and after treatment with vincristine,methotrexate, daunomycin, prednisone, and asparaginase. The incidence of micronu-clei after the antileukemic agent treatment was significantly higher than before thetreatment. Application of DC-FISH using a combination of whole chromosome-spe-cific painting probes and the same chromosome-specific α-satellite centromeric probeshowed that there were no significant differences in the micronucleus incidence forany specific chromosome (chromosomes 7, 8, 11, 17, X, and Y). There were nosignificant differences between the incidence of centromere-positive micronuclei andthe incidence of centromere-negative micronucleus. We concluded that antileukemicagents induced the somatic genetic damage but this damage is not related to anyspecific chromosome studied. Teratogenesis Carcinog. Mutagen. 21:341–347,2001. © 2001 Wiley-Liss, Inc.

Key words: ALL; antileukemic agents; micronucleus; FISH

Contract grant sponsor: Selçuk University Research Fund; Contract grant number: 97/96.

*Correspondence to: Dr. Hasan Acar, Tibbi Genetik Bilim Dali, Selçuk Universitesi, Tip Fakültesi,42080, Konya, Tükiye. E-mail: yaseminsena@hotmail.com

342 Acar et al.

INTRODUCTION

Childhood acute lymphoblastic leukemia (ALL) is an accumulation of lympho-blasts and their progenitors. Current treatment protocols for childhood ALL involveprolonged exposure to a variety of chemotherapeutic agents. Chromosomal abnor-malities are thought to be important in the development and progression of cancerpathology [1]. The potential chromosomal effects of the antileukemic agents treat-ment are well known [2,3]. The clastogenic and mutagenic effects of methotrexateand vincristine were analysed by Sgura et al. and Pelz et al., respectively [4,5].

A micronucleus is produced either from a whole chromosome or a chromosomefragment. The failure of sister chromatids to separate properly and to move to oppo-site poles due to the lack of chromosome attachment to the spindle apparatus duringcell division, leads to one nucleus with a missing chromosome and one nucleus withan extra chromosome. In the latter cell, after nuclear membranes form around thesechromosomes, one chromosome is left behind, and is not incorporated into the daugh-ter nuclei formed. The micronucleus appears in the cytoplasm of daughter cells as asmall additional nucleus. These micronuclei also contain a product of chromosomebreakage termed acentric fragments. Acentric fragments also cannot be incorporatedinto the daughter nuclei formed. A similar mechanism operates for a lagging wholechromosome. Several cytological methods have been devised in the past to detectmitotic aneuploidy and acentric fragments in mammalian cells. The analysis of mi-cronucleus incidence is increasingly applied as a biomonitoring method for humanexposure to such agents because the micronucleus assay is relatively fast and inex-pensive [6]. Micronucleus formation reflects chromosomal abnormalities that origi-nated in the last in vivo division of nucleated cells, which are basically distinguishableby using FISH with chromosome-specific probes. Nevertheless, the conventional mi-cronucleus assay is not able to distinguish between micronuclei arising from thesetwo types of mutagenetic mechanism [7]. This technique also does not require highlyspecialised staff [8].

In the present study, we introduce a convenient method of detecting geneticlesions formed in vivo based on analysis of micronucleus. Cytogenetic analysis wasalso performed. To evaluate the contribution of the antileukemic agents treatment tothe incidence of micronucleus, we have compared the results obtained from bonemarrow samples before and after therapy. The origin of micronuclei and their con-tents as verified by using DC-FISH technique with a combination of whole chromo-some–specific painting and the chromosome-specific alpha satellite probes for thesame chromosomes.

MATERIALS AND METHODS

The study was performed on six childhood ALL patients. The ages of the pa-tients were between 1.3 and 4.8 years. The patients were treated with vincristine,methotrexate, daunomycin, prednisone, and asparaginase. Details of the patients aregiven in Table I.

Bone marrow was aspirated at diagnosis and after 1-month treatment with che-motherapeutic agents. Each aspirated bone marrow sample at diagnosis and after thetherapy was divided into two aliquots. One aliquot was directly processed for cyto-genetic analysis. The other was processed for a micronucleus and FISH study.

Micronucleus Incidence in ALL Patients 343

Metaphase Analysis

In order to figure out subjects with any constitutional chromosomal abnormali-ties such as accentric fragments, gaps, translocations, and chromosomal breakage,conventional cytogenetic analysis was done on bone marrow samples obtained be-fore treatment. Chromosome preparations were performed according to the standardbone marrow protocol. Metaphase spreads were analysed with the GTG-banding tech-nique. The results were also confirmed by using the FISH technique with wholechromosome–specific painting probes.

Micronucleus Assay

During this procedure, the bone marrow sample was also divided into two aliquots.One is processed for the conventional micronucleus assay where bone marrow cellswere treated with cold 0.05 M KCI and followed by centrifugation. The cell pelletwas fixed with a cold mixture of methanol: acetic acid (3:1) three times. The cellsuspension was dropped onto clean slides. The slides were aged at 37°C or at roomtemperature overnight, and stained with Giemsa dye. The slides were screened blindby two individuals. For each subject, 3,330 to 9,350 nucleated cells were analysed forthe presence of micronucleus at a final 100× magnification. The cells harbouring mi-cronucleus were recorded. For the micronucleus scoring, the micronucleus criteriadescribed by Countryman and Heddle, and Vian et al., [9,10] were used as: diameterless than 1/3 of the main nucleus, non-refractility, not touching, and the same colouras the nucleus or lighter [9,10]. The micronucleus incidence before and after treat-ment was compared by using Wilcoxon Signed Ranks (adjusted Z) test.

FISH Analysis

For FISH analysis, the other aliquot of the sample was processed, in order toverify the content of the micronucleus observed by the conventional micronucleusassay at diagnosis and after treatment. For each probe combination, separate slideswere prepared. Slides were denatured in 70% formamide/2 × SSC for 2–4 min at65°C, chilled with ice-cold 70% ethanol, and dehydrated with sequential washes in90 and 100% ethanol and air-dried.

The FISH study was performed using commercial whole chromosome–specific

TABLE I. Cytogenetic Results and Incidence of Micronuclei in Bone Marrow From Six ALLCases Treated With Antileukemic Agents†

Before therapy After therapy

Age Total No. of Total No. of

No. (year) Sex Cytogenetics cells MN % cells MN %

1 1.5 M 46, XY,i(17q) [10] 3,360 10 2.98 3,470 20 5.862 2.5 M 46, XY, t(8;?) [3] 3,505 5 1.43 4,412 12 2.723 3.0 F No metaphase 6,359 9 1.42 4,212 8 1.904 1.3 F 46, XX, [7]/PL [2] 3,330 4 1.20 9,435 35 3.715 4.8 F 46, XX, t(4;11) [4]/ 5,900 6 1.01 4,250 13 3.06

47, XX, t(4;11), +8 [3]6 2.5 F No metaphase 9,350 8 0.86 4,200 14 3.33

Mean ± standard devision:1.46 ± 0.79 3.41 ± 1.30*

†MN: micronucleus; PL: two metaphases are poliploidy.*Z = –2.201, P = 0.028.

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painting probes for chromosomes 7, 8, 11, 17, X, and Y labelled with FITC(CAMBIO), and chromosome–specific centromeric probes for the same chromosomeslabelled with Cy3 (CAMBIO). The probe mixture containing the whole chromo-some–specific painting and the chromosome-specific alpha satellite centromeric probefor the same chromosome in 50% formamide/2 × SSC/10% dextran sulphate, wasdenatured at 65°C for 10–15 min. The probe mixture was dropped onto the dena-tured slides under coverslips. Slides were sealed with rubber cement, and the hybrid-ization occurred in a water bath at 42°C, for at least 22 h. After hybridization, washeswere done in 2 × SSC twice and 50% formamide/2 × SSC twice, respectively, at42°C for 5 min each. The slides were then stained with a counterstain medium con-taining DAPI (4′, 6-diamidino-2-phenyl-indole). Slides were examined with anepifluorescence microscope (Nikon, Optiphot). For each probe combination, the slidewas screened to obtain about the same number of micronuclei, which were examinedby a conventional micronucleus assay. The data were analysed using the χ2 test.

RESULTS

Conventional cytogenetic analysis by trypsin G-banding on 6 cases with ALLbefore chemotherapy treatment were done. The results are presented in Table I. Thequality of the marrow preparations was not sufficient to exclude small rearrange-ments and unidentified marker chromosomes by cytogenetic analysis. Therefore, cy-togenetic results were confirmed by application of FISH. The FISH study revealedthat the second case had a small marker chromosome originating from chromosome17 in 2 of 3 metaphases, in addition to the findings by conventional cytogenetictechniques.

The micronucleus incidence was scored in the bone marrow of childhood ALLpatients before and after therapy. The micronucleus incidence was determined as thenumber of micronuclei per one thousand cells (Table I). The micronucleus incidenceincreased after the antileukemic agents treatment compared to before treatment (Z =–2.201, P = 0.028) (Table I). In contrast, one out of six cases (case 3) did not show asignificant difference between before and after antileukemic agent treatment whencompared to the other cases.

When DC-FISH with the combination of whole chromosome–specific paintingprobe for the chromosome 7, 8, 11, 17, X, and Y with α-satellite centromere-specificprobe for the same chromosomes was done, the results showed that the high incidenceof micronuclei observed after therapy was not related to any specific chromosome used(χ2 = 2.718, df = 5, P = 0.743) (Table II). The combination of a whole chromosome–specific painting probe and an alpha satellite centromeric probe for the same chromo-some revealed the origin of micronucleus. The results showed that there were nosignificant differences between the incidence of centromere-positive and -negative mi-cronuclei (χ2 = 1.451, df = 5, P = 0.919) (Table II).

DISCUSSION

It is well known that chromosome loss and chromosome damage, such as chro-mosome breaks and deletions, play a role in the formation of cells with micronuclei[11,12]. These aberrations are associated with the risk of cancer [13], where loss ofheterozygosity characterises tumour suppressor gene inactivation [14].

Micronucleus Incidence in ALL Patients 345

In this study, the micronucleus assay was performed in a conventional way with-out using the cytochalasin B method of Fenech and Morley [15]. It is not useful forour purpose, because the preserved cytoplasm interferes strongly with the FISH tech-nique, due to the difficulty of probe penetration efficiency. This method producesweak signals. In addition, recent studies have suggested that there are dissimilaritiesin the contents of the micronucleus, between binucleated cells and cell cultured withoutCyt-B, that may reflect either differences between first-division cells and culturedcells in general or consequences of the cytokinesis block [16]. In our study, a signifi-cant incidence of micronuclei was observed in bone marrow of childhood ALL pa-tients after the antileukemic agents treatment (P 0.028) (Table I). Similar results werereported in the literature [17–19]. This result suggests that the association of theantileukemic agents treatment with micronucleus frequency could reflect deficien-cies in DNA repair in leukaemia patients. In addition, the high micronucleus fre-quency occurs at the time the patients have been receiving methotrexate. This druginduces DNA-strand breaks due to a defective repair mechanism [5]. On the otherhand, Sgura et al. reported that the application of vincristine to lymphoid cells in-duced micronucleus formation harbouring predominantly whole chromosome [4].However, the age of the individual and the cell culture conditions, such as the mito-gen for stimulation of the cells, have been associated with micronucleus frequencies[12,20,21]. In this study, the range of ages was quite small, and also we used a directbone marrow preparation for the micronucleus assay. In contrast, case number 3 inour study showed no significantly different results before and after antileukemic agenttreatment when compared to the other cases. This variability among individuals maybe associated with different factors, one being a particular individual responsivenessto antileukemic agents or variation in the time of bone marrow sampling, althoughapparently not with differences in experimental protocol.

In addition, determination of whether the micronuclei scored represented wholechromosomes or accentric fragments could assist in interpreting the association ofthe antileukemic agents’ treatment on the leukemia. If the excess micronuclei pre-dominantly represented whole chromosomes, it would be more likely that they weregenerated by an aneuploidogenic effect of the antileukemic agents. On the other hand,if the micronuclei represented accentric fragments, their origin might include ge-nomic instability in bone marrow cells in leukaemia. To evaluate these possibilities,

TABLE II. Dual-Colour FISH Results of Six ALL Cases With a Combination of WholeChromosome–Specific Painting and the Same Chromosome Specific a-Satellite Probes*

MN-S

Total case (n) Chromosome no. Total cell Total MN MN-NS MN-S MN-C (+) MN-C (–)

6 7 27,850 95 86 9 4 56 8 25,150 85 78 7 3 46 11 30,238 102 94 8 5 36 17 22,985 78 69 9 5 46 X 28,850 97 85 12 7 52 Y 12,780 53 50 3 1 2

*MN: micronucleus; MN-NS: MN without signal; MN-S: MN with signals either with centromere orwithout centromere; MN-C (+): MN with centromere; MN-C (–): MN without centromere. χ2 = 2.718,df = 5, P = 0.743 (for the incidence of micronucleus with signals, MN-S); χ2 = 1.451, df = 5, P = 0.919(for the incidence of micronucleus with centromere, MN-C(+), and without centromere, MN-C)(–).

346 Acar et al.

we have designed this present study, since the conventional micronucleus assay isnot able to distinguish micronuclei arising from whole chromosomes (aneupolidogeniceffect of antileukemic agents) or accentric fragments (clastogenic effects of antileu-kemic agents).

The micronucleus assay and FISH technique were applied to verify the effectsof the antileukemic agents’ treatment. This report is the first to use these two meth-ods to verify the origin of micronuclei in childhood ALL patients, although it hasbeen used on radiation-induced [22,23] and vincristine-induced micronuclei [4], sincethe application of FISH analysis and the micronucleus assay can verify micronucleusorigin and their contents after treatment with chemical agents. Our FISH result showedthat there were no significant differences for any specific chromosome used. Therewere also no significant differences between the incidences of centromere-positiveand centromere-negative micronuclei. These observations suggest that the increasedmicronucleus frequency after therapy may have been attributable to the associationwith antileukemic therapy rather than to the leukemia. The patients might be predis-posed to a secondary cancer due to the antileukemic agents since they induce so-matic genetic damage. Högstedt et al. reported that ALL patients with highmicronucleus frequency in bone marrow have a short life expectancy [24]. In con-clusion, our data indicate that a more detailed study of the long-term effects of anti-leukemic agents in ALL patients using a combination of the FISH technique andmicronucleus assay is needed.

ACKNOWLEDGMENTS

We thank Ahmet Demirdelen and Osman Kurt for technical assistance, and Dr.Tülin Çora for consultation of cytogenetic analysis. We also thank Dr. Said Bodurfor statistical analysis of the data.

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