SISTER CHROMATID EXCHANGE (SCE) IN BLOOD LYMPHOCYTES OF BOVINE LEUKAEMIA VIRUS INFECTED CATTLE

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1 Bull Vet Inst Pulawy 51, , 2007 SISTER CHROMATID EXCHANGE (SCE) IN BLOOD LYMPHOCYTES OF BOVINE LEUKAEMIA VIRUS INFECTED CATTLE MARIA SZCZOTKA, JAN STEC, JANUSZ KOCKI 1, AND JACEK KUŹMAK Department of Biochemistry, National Veterinary Research Institute, Pulawy, Poland 1 Department of Animal Genetics, Medical University of Lublin, Lublin, Poland szczotka@piwet.pulawy.pl Received for publication July 10, 2007 Abstract The exchanges between sister chromatids were detected with the fluorescence plus Giemsa technique, in which the thymine analogue, bromodeoxyuridine, was used for DNA labelling. This method was used to examine the influence of bovine leukaemia virus on chromosomal disorders in bovine blood lymphocytes. In metaphasal chromosomes of BLV infected lymphocytes, numerous sister chromatid exchanges (SCEs) were found. In chromosomal spreads of lymphocytes of BLV infected cows there were SCEs per metaphase. In the control animals, only a small number of spontaneous SCEs (0 2.0) were found (P<0.01). The obtained results indicated that BLV may cause chromosomal disorders in lymphocytes of leukaemic cattle. Key words: cattle, bovine leukaemia virus, lymphocytes, sister chromatid exchange. Bovine leukaemia virus (BLV) belongs to Retroviridae, and is closely related by genomic organisation and disease progression to human Tcell leukaemia viruses (HTLV1 and HTLV2) and simian Tcell leukaemia virus. BLV infection remains subclinical in the majority of cattle, but about one third of infected animals develop persistent lymphocytosis (PL). About 1%5% of infected animals develop lymphosarcoma with or without prior PL (12). The primary cellular target of BLV is the Blymphocyte (22). The susceptibility of other cells than B lymphocytes to BLV infection is less clear. Monocytes were first implicated as potential carriers of BLV in sheep on the basis of cell morphology and in situ hybridisation. Some authors reported that BLV was present in 5% to 40% of adherence purified monocytes, but not in T cells or granulocytes from BLV infected cattle with or without PL. T cell susceptibility for BLV infection was evidenced, when immunoaffinity depletion of B cells and monocytes from peripheral blood or positive selection of T cells with immunomagnetic beads were performed (28). Retroviruses are a family of RNA animal viruses that use reverse transcription in the replication. They have been classified as simpler or more complex on the basis of genetic organisation and replication. This virus population shows a great genetic variability. This retroviral genetic variation is the composite of three variables: the mutation rate per replication cycle, the number of replication cycles, and the selective advantage or disadvantage possessed by the variant viruses (4, 11, 24, 25, 27). In contrast to normal cells, cancer cells proliferate indefinitely and this characteristic may be a necessary condition for tumourigenesis (30). Inactivation of the p53 and Rb pathways allows the proliferation, but at the expense of escalating chromosomal instability, itself the result of further increases in unprotected chromosome ends. A crisis stage is reached when massive instability prohibits the generation of sufficient viable cells to sustain the proliferation. A small percentage of cells avoid crisis by reestablishing chromosome end protection (1). Mutagenesis can be assayed by sister chromatid exchange (SCE). The SCE is a common and readily quantifiable form of recombination in mammalian cells. The frequency of the SCE is particularly high within the subtelomeric regions of chromosomes. Some authors found, that recombination between telomeres of sister chromatids also occurs at unusually rates (1, 14, 15, 23). SCEs were present in the lymphocytes of patients with leukaemia (18, 20, 21, 29) and chromosomal instabilities were detected in cattle infected with bovine leukaemia virus (24, 27). SCEs are reciprocal exchanges of DNA segments between sister chromatid at identical loci during Sphase of the cell cycle. These changes are more sensitive indicators of mutagenic activity than chromosome breaks and they have become a major tool in mutagenesis research

2 326 (13).With the development of the thymidyne analogue bromodeoxyuridine (BUdR) and its subsequent use in DNA labelling experiments, the resolution of SCEs greatly improved in comparison to previous methods, in which the incorporation of radioactive nucleotides into replicating DNA were used. The fluorescence plus Giemsa technique for the scoring of SCEs was enhanced and for the first time, permanent staining of SCEs were demonstrated (13, 19, 20). The fluorescence plus Giemsa method involves two distinct steps: 1. In the first step, cells during the two complete cycles are stained with BUdR. Then, the cells are treated with colcemid to block them in metaphase. After BUdR exposure, the DNA of one chromatid of each chrosome contains bromouracil in one strand, while the DNA of its sister chromatid contains bromouracil in the both strands. 2. Then, the chromosomes are prepared from these cells and stained with the fluorescent dye Hoechst After staining, the BUdR is degradated with ultraviolet light and the chromosomes are stained according to the Giemsa method (19, 20). The aim of this work was the evaluation the influence of bovine leukaemia virus infection on SCEs formation in blood lymphocytes of cattle infected with BLV. Material and Methods Animals. The investigations were performed on the cattle from two herds naturally infected with BLV. Blood samples were taken from the jugular vein to the tubes containing EDTAK2 as anticoagulant Total blood white cell count, lymphocyte counts and Schilling formula were determined. The presence of specific anti BLV antibodies was monitored by AGID and ELISA methods using commercial kits. The presence of BLV provirus in blood lymphocytes was detected with PCR test. Cells. Lymphocytes were isolated from interphase after centrifugation the whole blood samples in density gradient (Histopaque). The cells were washed three times and concentration 1 x 10 6 per ml was prepared. SCE assay. The cells were incubated in RPMI medium supplemented with foetal calf serum and antibiotics for 48 h at 37 C. After this time, BUdR in final concentration of 10 µm was added to the growth medium. The cells were incubated in the dark at 37 C for a further 48 h (about 2 cell cycles). Before harvesting (16 h), depending on the cycling time, colcemid in final concentration 0.01 µg/ml was added to arrest the cells in metaphase. The cells were washed with PBS and pellet was suspended in 10 ml of growth medium and then the suspension was centrifuged at rpm for 5 min. Supernatant was discarded, but about 1 ml of the medium was left above the pellet, then cells were carefully mixed and suspended in the liquid. To each vessel, 10 ml of hypotonic solution of M KCl, prewarmed to 37 C was very slowly added and the cell suspension was incubated for 15 min at room temperature. After incubation, the cells were centrifuged for 15 min at rpm. Supernatant was removed, but about 1 ml was left above the pellet. Then, drop by drop 10 ml of icecold freshly prepared fixative mixture of methanol and acetic acid (3:1) with gently mixing the tubes after each drop was added and then the tubes were placed on ice for 10 min. The cells were stored in fixative overnight at 4 C. Preparation of slides. After fixation, the cells were gently mixed and 3 drops were dropped onto the clean surface of frosted slide. Slides were left for air drying at room temperature in the dark. Then, slides were stained with Hoechst fluorescent dye at a concentration of 20 µg/ml for 10 min and then 500 µl of 2 x SSC buffer (0.3 M sodium citrate/sodium chloride) was dropped onto each slide. Afterwards, the slides were covered with coverslips. To prevent evaporation, the edges of slides were sealed with nail varnish. The slides were placed for 60 min in shortwave ultraviolet (UV) box, with 4 cm distance between the source of UV and slide surface. After the coverslips were removed, the slides were washed 3 times in ultra pure water, 5 min per wash. The slides were covered with aluminium foil and left at room temperature in the dark for drying and then were stained with 3.5% Giemsa stain in Soerensen s buffer (ph 6.8) for 35 min. After that, the slides were rinsed in the tap water and dried 1 h at room temperature. Then, each slide was dipped into xylene, mounted in DPX, and covered with coverslips. The slides were placed overnight in a fume hood for drying. Under 40 x objective of a light microscope, the slides were examined for metaphase spreads under oil immersion and then scoring was performed in Olympus microscope equipped with the special computer analytical Karyotyping system. Results The presence of SCE. SCEs were observed in all BLV infected animals. The number of SCE was scored from 6.4 up to 13.0 per one metaphase. The chromosomes with multiple exchanges looked like harlequine (Figs 13). In the control group of animals, some spontaneously formed SCEs in metaphasal lymphocytes were observed, but these values were very low about 02.0 chromatid exchanges per metaphase (Tables 1 and 2 Haematological and serological results. In haematological examinations of BLV infected cattle, lymphocyte numbers were very different. In some animals the leukocyte number was very low and varied from 2.1 x 10 3 to 8.8 x 10 3 /µl of blood. In the other animals, lymphocytosis with ranges from 10.8 x 10 3 up to 20.3 x 10 3 /µl was observed. All examined animals were positive in ELISA, but in 5 animals from this group the results of PCR were negative (Tables 1 and 2).

3 327 Table 1 Sister chromatid exchanges in metaphasal lymphocytes of BLV infected and control cattle from herd I No. of animal Leukocytes Lymphocytes AGID ELISA PCR SCE per metaphase Animals No. 110 BLV infected group; animals No control. The mean SCEs value per metaphase for BLV infected cattle 9.3. The mean SCEs value per metaphase for control cattle 1.1. Table 2 Sister chromatid exchanges in metaphasal lymphocytes of BLV infected cattle from herd II No. of animal Leukocytes Lymphocytes AGID ELISA PCR SCE per metaphase The mean SCEs value per metaphase 11.1.

4 328 Fig. 1. Sister chromatid exchanges in metaphasal chromosomes of BLV infected cow. Fig. 2. Harlequin chromosome spreads in lymphocytes of BLV infected cow. Fig. 3. Chromosomal spreads in metaphase with sister chromatid exchanges. Discussion The determination of SCE is very useful in human medicine (5, 2, 6, 7, 15, 16, 26). SCE occurs in many haematological disorders and in different stages of leukaemia (13, 18, 21, 29). We found, that in animals infected with BLV, SCEs in blood lymphocytes were present. One SCE has been determined, when there was one area of dark staining and then light staining on one chromatid and, on the sister chromatid, was one area of light staining, and then place with dark staining. Each point of reduced staining is scored as one SCE. Staining with Giemsa stain underlined the differential incorporation of bromouracil into the sister chromatids. DNA that contains bromouracil quenches the fluorescence of HoechstDNA complexes. The chromatid containing bromouracil substituted in both strands show weak fluorescence and stains weakly with Giemsa. If chromatid contains bromouracil in only one strand, then more intensely degrades the BUdR, and subsequently stains darkly with Giemsa. When any sister chromatid exchange is present in chromosomes, this staining pattern is named as harlequin chromosomes Numerous SCEs in metaphasal chromosomes in peripheral blood lymphocytes of BLV infected cattle

5 329 were detected in both herds of the cattle. The SCE mean value was in the herd I and 10.5 in the herd II. There were no correlations in SCEs number in animals with lymphocytosis or in animals with aleukaemic form of the disease. In the control group, SCEs varied from 0 to 2.0. It is in agreement with observations made by some laboratories, that in cattle, sheep, and goat sister chromatid exchanges could be formed spontaneously, without the influence the external or internal factors (8, 9, 10). We had not in our experiment cows with lymphoma. Karyotypic instability of peripheral blood lymphocytes in cows infected with BLV was investigated by Dubik et al. (11). In animals with lymphocytosis, SCEs were found to be 7.2, but in cows with lymphosarcoma the values were 9.7. In the control group only 3.8 of SCEs were present. The mechanism of BLV infection and viral influence on the cell transformation and mutations is poorly understood. In some studies, the nonrandom chromosomal abnormalities in bovine lymphoma were detected (27). Metaphases in cells from lymphoid tumours from 20 dairy cows were banded and analysed after shortterm, unstimulated culture. Nineteen out of twenty cases exhibited clonal abnormalities, 17 were hyperdiploid, and 16 cases had extremely complex chromosomal changes. The most common abnormalities were the acquisition of additional small chromosomes 2329; trisomy of chromosomes 5 and 7, and Robertsonian translocations and isochromosome rearrangements involving chromosomes 10, 12, 23, and 26. Structural rearrangements of chromosomes 10, 12, 23, and 26 may reflect primary abnormalities occurring relatively early in the transformation, when trisomy 5 may be an extremely common secondary abnormality. Investigations performed by Tuna et al. (29) on metaphase preparation from peripheral blood lymphocytes of patients: 13 with acute nonlymphocytotic leukaemia, one with acute lymphocytic leukaemia, and one with Hodgkin s disease, before and after therapy showed, that mean SCE frequency in the cells was before therapy and 14.2 after therapy as compared with 7.87 in controls. The authors showed the effects of acute leukaemia and cytostatic drug therapy on chromosomes by SCE analysis. SCE values of patients with acute leukaemia were significantly higher than those of controls. This was more conspicuous in the cells which had undergone anticancer treatment. Popescu et al. (25) demonstrated that SCE formation in mammalian cells was modulated by deoxyribonucleotide pool imbalance. The formation of SCEs is closely related to DNA replication which is dependent on the proper balance of deoxyribonucleic triphosphates. The modulation of SCE by DNA precursors is consistent with the induction of BrdUrd mutagenesis in mammalian cells and raises the possibility that common DNA changes are responsible for the induction of SCE and mutation in mammalian cells. SCE has been shown to be an indicator of DNA damage (DNA instability) by various mutagenic and/or carcinogenic materials (3, 8, 23). A high frequency of SCEs has been reported in patients with various neoplastic diseases such as cancer of the cervix uteri (7), cutaneous malignant melanoma (26), breast cancer (16), ovarian cancer (6), and stomach cancer (5). In their lymphocytes, significant differences were shown in the SCE frequencies, where these exchanges were more numerous than in normal controls. On the basis of our experiments and works performed in other laboratories, SCE test can be useful for the determination of mutagenic activity and chromosomal abnormalities caused by viral or chemical factors. 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