Chromosome changes in butylnitrosourea (BNU)-induced mouse leukemia

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<ul><li><p>Znt. J . Cancer: 30, 511-516 (1982) </p><p>CHROMOSOME CHANGES IN BUTYLNITROSOUREA MOUSE LEUKEMIA F. CARBONELL, H.J. SEIDEL, S. SAKS and L. KREJA Department of Clinical Physiology and Occupational Medicine, University of Ulm, UlmlDonau, Germany. </p><p>G-banding analysis was carried out in 45 mice after ex- p u r e to the leukemogen butylnitrorourea (BNU). Of 27 animals without clinical signs of leukemia, 2 had chromosomal rearrangements (7%) while, of 18 leukemic mice, 5 showed abnormal karyotypes (28%). The chro- mosomal abnormalities were not random and showed preferential changes of chromosomes Nor. 14 and IS. The organ distribution of the chromosomal abnormalities showed a preferential involvement of the thymus with a secondary presence of chromosome anomalies in peri- pheral lymphoid organs and the bone marrow. The find- ings are discussed in relation to the recent reports on the specificity of triromy I5 in murine leukemia and lym- phoma </p><p>From the published results of a series of chromo- some studies in murine leukemias it is apparent that trisomy 15 is a very common and probably specific finding in T-cell leukemias, whether these are spon- taneous or radiation-, virus- or chemically induced (Dofuku et al., 1975; Chan et al., 1979; Wiener et al., 1978 a, b , 1980; Herbst et al., 1981). Trisomy 15 has been described for B-cell lymphomas (Wiener et.al, 1981) and chromosome 15 rearrangements were also involved in plasmocytomas (Yoshida et al., 1978; Ohno et al., 1979). We are studying T-cell leuke- mogenesis by butylnitrosourea (BNU) in BDF, mice (Shisa and Matzudaira, 1975; Seidel, 1980) and the purpose of this paper is to analyse the chronology, incidence and type of chromosomal aberrations in these leukemias and their organ distribution before and after the manifestation of leukemia. This study on primary leukemias contributes also to the question of the specificity of trisomy 15 in mouse leukemias. The results show a non-random distribution of chromo- somal aberrations, but in addition to the observation of trisomy 15, trisomy 14 was also frequently involved. </p><p>MATERIAL AND METHODS </p><p>Mice Female C57BLl6J X DBN2 hybrid (BDF,) mice </p><p>(Gl. Bomholtgard, Ry, Denmark) of 20-23 g body weight were kept under the standard conditions of the Central Animal Facility, University of Ulm. They were fed commercial pellets and acidified tap water (pH=5) containing 0.02% of BNU (Serva, Heidel- berg, Germany). The BNU solution was freshly pre- pared three times a week. Experimental procedures </p><p>The mice were exposed to BNU for 12 weeks and then given normal drinking water. This regimen leads to 90-100 % deaths from leukemia in these mice within the following 8-40 weeks with a median survival time of about 20 weeks. The leukemias of our laboratory have been classified as T-cell lymphomas by means of anti-T cell serum and immune-peroxidase staining (Thierfelder , personal communication). Chromosom- </p><p>(BNU)-INDUCED </p><p>Oberer Eselsberg, D 7900 </p><p>a1 analysis was performed at pre-determined intervals more than 3 weeks after withdrawal of BNU and also in individual mice with clinical signs of lymphoma, such as thymic lymphoma and enlargement of the spleen and lymph nodes. Cytogenetic analysis </p><p>Mice were injected intraperitoneally with 0.2 ml of a colchicine solution (50 pg) 2 h prior to killing. The different organs were finely minced in Hanks' solu- tion. Cells were pelleted and resuspended in hy- potonic KCI solution (75 m ~ ) for 15 min at 37" C, fixed in acetic acid: methanol (1:3) and spread on glass slides. G-banding was performed by a modification of the method of Seabright (1971). Chromosomes were identified according to the criteria of the Committee on Standardized Genetic Nomenclature for Mice (1979). An average of 20 metaphases was studied per mouse and per organ. </p><p>RESULTS </p><p>A total of 45 mice were analysed; 27 had no clinical signs of leukemia (i.e. no enlargement of the thymus or any other lymphoid organ) while the other 18 showed typical thymic lymphomas with or without in- volvement of other organs. The number of animals with chromosome abnormalities, as shown in Table I, comprised 2 out of the 27 mice in the pre-clinical phase (7%) and 5 out of the 18 animals with lym- phomas (28 %). The specific chromosomal aberrations are listed in Table 11. </p><p>TABLE 1 - CYTOGENETIC ANALYSIS OF THYMUS, BONE MARROW, SPLEEN AND LYMPH NODES (IF ENLARGED) </p><p>OF MICE AFTER BNU ~~ </p><p>Animals studied Normal Abnormal No' kaniotvue (%) k a w o t m (%) </p><p>Mice without lymphoma 27 25 (93) 2 (7) Mice with lymphoma 18 13 (72) 5 (28) </p><p>Of the mice without lymphoma but with chromo- somal aberrations, one showed a trisomy 14, resulting from an isochromosome 14 in the thymus and the spleen (Fig. 1). The bone marrow metaphases were normal. In the second mouse, all thymic metaphases had from one to three non-identified metacentric marker chromosomes; the spleen and marrow were cytogenetically normal. All other 25 mice killed in the pre-clinical phase had normal karyotypes in the thy- mus, spleen and bone marrow. </p><p>Of the lymphoma-bearing mice, two (Nos. 5 and 7) were characterized by a trisomy 14 (Fig. 2). Mouse No. 4 was trisomic for chromosomes 14 and 15 in all </p><p>Received: June 30, 1982. </p></li><li><p>512 CARBONELL ET AL. </p><p>1 2 3 4 5 </p><p>6 7 8 9 10 </p><p>J </p><p>11 12 13 14 15 </p><p>16 17 18 19 X </p><p>FIGURE 1 - G-banding karyotype of a metaphase from the thymus of animal No. 1. Note the presence of an isochromosome 14 resulting in trisomy 14. </p><p>analysed metaphases. Mouse No. 6 showed two dif- ferent patterns of aberrations; one clone [T(4;?), T(4;?), +1, +15] was predominant in the thymus, spleen and bone marrow (Fig. 3), and the second clone [T(4;?), T(4;?), +15, -18, +mar] was found in the enlarged lymph nodes. </p><p>Organ involvement showed the following pattern. The thymus always had cytogenetically abnormal clones, even in the two mice without thymic lym- phomas, as did also the lymph nodes, which were studied only when grossly enlarged. The bone marrow </p><p>was free of abnormal metaphases in both mice without thymic lymphomas and in mouse No. 7 with clinical leukemia and was only partly involved in mice Nos. 3 and 4. The spleen had the abnormal clone in three out of the four mice in which it could be studied. </p><p>DISCUSSION </p><p>In this study 45 mice were analysed after exposure to BNU which causes T-cell lymphomas. Abnormal karyotypes were observed in 5 out of 18 mice with </p></li><li><p>CHROMOSOMES IN MOUSE LEUKEMIA 513 </p><p>1 2 3 4 5 </p><p>6 7 8 9 10 </p><p>11 12 13 </p><p>16 17 18 </p><p>14 15 </p><p>* * </p><p>19 X </p><p>FIGURE 2 - G-banding karyotype of a metaphase from a lymph node of animal No. 7. Note the presence of trisomy 14. </p><p>clinical signs of leukemia, evidenced by enlargement of the thymus and other lymphoid organs, and in 2 out of 27 mice without such organ enlargement. This indi- cates that the majority of these leukemias had a nor- mal karyotype. In our study the observed changes had a non-random distribution, thus confirming other re- ports (Chan et al., 1979; Dofuku ef al., 1975; Herbst et al., 1981; Spira et al., 1980; Uenaka et al., 1978; Wiener et al., 1978a, b). However, not only was trisomy 15 present but trisomy 14 also played an im- portant role in our BDFl mice after exposure to BNU. </p><p>Trisomy 14 had not been described in murine T-cell leukemias, but the recent results of Spira et al. (1981) suggested that chromosome No. 14 may have a specific function at least in the progression of malig- nancy. </p><p>BNU induces leukemias in rats and mice, the rat leukemias being myeloidferythroid in type. Chro- mosomal changes were not found in the 9 rat leuke- mias studied by Mori and Sazaki (1974). However, Uenaka et al. (1978) found that out of 34 rat leukemias 17 had normal karyotypes, 12 had a mixture of normal </p></li><li><p>514 CARBONELL ET AL. </p><p>1 </p><p>6 </p><p>11 </p><p>16 </p><p>2 3 4 5 </p><p>7 8 9 10 </p><p>13 14 15 12 </p><p>17 18 19 X </p><p>FIGURE 3 - G-banding karyotype of a metaphase from the thymus of animal No. 6. Note the rearrangement of both chromosomes 4 and the trisomies 1 and 15. </p><p>and aneuploid metaphases, and 5 were completely ab- normal. The most frequent rearrangement was triso- my 2. Wiener et al. (1980) studied three BNU-induced leukemias in AKR X CBAT6T6 mice; all three ani- mals had trisomy 15. In another report, a B-cell tumor with trisomy 14 was described after ethylnitrosourea, but this chromosome study was done after four pas- sages of the tumor (Wiener et al., 1981). Most em- phasis has been given to trisomy 15. This has been described in spontaneous T-cell lymphomas of the AKR mouse (Dofuku et al., 1975; Herbst et al., 1981), </p><p>and in a variety of T-cell leukemias induced by RadLV (Wiener et al., 1978a) or dimethylbenzanthracene (Wiener et al., 1978b; Chan et al., 1979; Spira et al., 1980). Other examples in which trisomy 15 has been observed are B-cell lymphomas (Fialkow et al., 1980; Wiener et al., 1981). Rearrangements between chromosome 15,6 and 12 have been described in plas- mocytomas (Shepard et al., 1978; Yoshida et al., 1978; Ohno et al., 1979). </p><p>The problem in the comparison of all these results and their significance for the leukemogenic process is </p></li><li><p>CHROMOSOMES IN MOUSE LEUKEMIA 515 </p><p>TABLE n - CHROMOSOMAL ABERRATIONS IN MICE AFTER BNU </p><p>Modal % Metaphases </p><p>number anomalies Material' chromosome with chromosome Mouse TgF Clinical status BNU NO. </p><p>5 12 </p><p>6 17 </p><p>40 100 Isochromosome 14 40 100 Isochromosome 14 </p><p>40-41 100 1 to 3 marker chr. </p><p>1 3 Normal thy SPl </p><p>2 6 Normal thy </p><p>3 3 Leukemic thy </p><p>4 12 Leukemic thy SPl </p><p>b.m. 40 0 Normal </p><p>40 0 Normal 40 48 T(5;?) 40 45 T(5;?) </p><p>42 100 +14,+15 42 100 +14,+15 </p><p>1.n. 42 100 +14,+15 b m . 40,42 75 Normal I +14,+15 </p><p>Leukemic SPl 41 100 +14 1.n. 41 100 +14 </p><p>b.m./spl </p><p>b.m. SPl - - No analyzmetaph. </p><p>thylspl - - No analyzmetaph. 42 100 T(4;?),T(4;?) ,+I ,+15 42 100 T(4;?) ,T(4;?) ,+ 1 ,+15 </p><p>b.m. 42 100 T(4;?),T(4;?),+1,+15 1.n. 41 100 T(4;?) ,T(4;?) ,+ 15 ,-18 ,+mar. </p><p>41 100 +14 41 100 + 14 1.n. </p><p>Leukemic thy SPI </p><p>7 17 Leukemic thy </p><p>b.m.lsp1 40 0 Normal </p><p>'thy, thymus; spl, spleen; b.m., bone marrow; l a . , lymph nodes. </p><p>that the material is not very homogeneous; it includes only a few primary leukemias and the majority of the studies were made after transplantation or on in vitro cell lines. These procedures can be accompanied by the occurrence of secondary chromosomal changes. Nevertheless our data support the specific role of chromosome 15 and also 14 in primary murine T-cell leukemias to which most attention should be paid. </p><p>Our data on the organ distribution of the chromo- somal abnormalities contribute to the question of the origin of this T-cell lymphoma and the subsequent or- gan involvement. The thymus was involved in all cases where it could be studied; it was also the site of the abnormal clones in the two cases without macroscopic signs of the disease in which the bone marrow was still </p><p>cytogenetically normal. In 4 out of 5 animals, all metaphases of the thymus were of the leukemic clone; the other organs had a mixed population of cytogenet- ically normal and abnormal metaphases in two cases. In one animal a clonal evolution was seen in a lymph node. This general pattern suggests the secondary in- volvement of peripheral lymphoid organs and the bone marrow. </p><p>ACKNOWLEDGEMENT </p><p>This study was supported by the Deutsche For- schungsgemeinschaft (SFB 112) and the Ministry of the Interior. The cooperation of Drs. E. Herbst and S. Dzarlieva is gratefully acknowledged. We thank Mrs. Barthel, Mrs. E. Kratt, Mrs. K. Neuhaus and Mrs. K. Steinhoff for their excellent technical assistance. </p><p>REFERENCES </p><p>CHAN, F.P.H., BALL, J.K., and SERGOVICH, F.R., Trisomy 15 in murine thymomas induced by chemical carcinogens, X-irradia- tion, and an endogenous murine leukemia virus. 1. nat. Cancer Znst., 62, 605-610 (1979). </p><p>OHNO, S., BABONITS, M., WIENER, F., SPIRA, J . , and KLEIN, G. , Nonrandom chromosome changes involving the Ig gene-carrying chromosomes 12 and 6 in pristane-induced mouse plasmocy- tomas. Cell, 18, 1001-1007 (1979). </p><p>COMMlTIEE ON STANDARDIZED GENETIC NOMENCLATURE FOR MICE, New rules for nomenclature of genes, chromosome anomalies and inbred strains. Mouse News Letter, 61, 4-16 (1979). DOFUKU, R. , BIEDLER, J.L., SPENGLER, B.A. , and OLD, L.J., Trisomy of chromosome 15 in spontaneous leukemia of AKR mice. Proc. nat. Acad. Sci. (Wash.), 72, 1514-1517 (1975). FIALKOW, P.J., REDDY, A.L., and BRYANT, J . I . , Clonal origin and trisomy of chromosome 15 in murine B-cell malignancies. Int. J. Cancer, 26, 603-608 (1980). HERBST, E.W., GROPP, A., and TIETGEN, C., Chromosome rear- rangements involved in the origin of trisomy 15 in spontaneous leukemia of AKR mice. Znt. J . Cancer, 28, 805-810 (1981). MORI, M., and SASAKI, M., Chromosome studies on rat leukemias and lymphomas, with special attention to fluorescent karyotype analysis. J . nut. Cancer Znst., 52, 153-160 (1974). </p><p>SEABRIGHT, M., A rapid banding technique for human chromo- somes. Lancet, 2,971-972 (1971). SEIDEL, H.J., Haemopoiesis and immune status during BNU- induced leukaemogenesis in mice. J . Cancer Res. clin. Oncol., </p><p>SHEPARD, U.S., PETIENGILL, O.S., WORSTER-HILL, D.H., and SORENSON, G.D. , A specific chromosome breakpoint associated with mouse plasmocytomas. J . not. Cancer Znst., 61, 255-258 (1978). SHISA, H., and MATSUDAIRA, Y. , Origin of leukemic cells in mouse leukemia induced by N-butylnitrosonrea. Gann, 66,37-42 (1975). SPIRA, .I., BABONITS, H., WIENER, F. , OHNO, S. , WIRSCHLJBSKI, Z., HARAN-GHEM, N., and KLEIN, G. , Nonrandom chromosom- al changes in thy-1-positive and thy-I-negative lymphomas in- </p><p>98, 31-39 (1980). </p></li><li><p>516 CARBONELL ET A L </p><p>duced by 7,12-dimethylbenzanthracene in SJL mice. Cancer Res., 40, 2609-2616 (1980). SPIRA, J . , WIENER, F., BABONITS, M., GAMBLE, J . , MILLER, J., and KLEIN, G., The role of chromosome 15 in murine leukemogenesis. I. Contrasting behaviour of the tumor vs. nor- mal parent-derived chromosomes No. 15 in somatic hybrids of varying tumorigenicity. Inr. J . Cancer, 28, 785-798 (1981). UENAKA, H., UEDA, N. , MAEDA, S. , and SUGIYAMA, T., Involve- ment of chromosome 2 in chromosome changes in primary bukemia induced in rats by N-nitroso-N-butylurea. J. nut. </p><p>WIENER, F., OHNO, S . , SPIRA, J . , HARAN-GHERA, N., and KLEIN, G., Chromosome changes (trisomies 15 and 17) associ- ated with tumor progression in leukemias induced by radiation leukemia virus. J . nut. Cancer Inst., 60, 227-237 (1978~). </p><p>CffRCer IRSf . , 60, 1399-1404 (1978). </p><p>WIENER, F. , SPIRA, J . , OHNO, S . , HARAN-GHERA, N., and KLEIN, G., Chromosome changes (trisomy 15) in murine T-cell leukemia induced by 7,12-dimethylbenz(a)antracene (DMBA). Int. J . Cancer, 22, 447-453 (19786). WIENER, F. , BAFIONITS, M. , SPIRA, J., BREGULA, U. , KLEIN, G., MERWIN, R.M., ASOFSKY, R. , LYNES, M., and HAUGHTON, G., Chromosome 15 trisomy in spontaneous and carcinogen-induced murine lymphomas of B-cell origin. Inr. 1. Cancer, 27, 51-58 (1981). WIENER, F., SPIFU, J . , BABONES, M., HARAN-GHERA, N., and KLEIN, G., Non-random duplication of chromosome 15 in murine T...</p></li></ul>

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