ORIGIN OF LEUKEMIC CELLS IN MOUSE LEUKEMIA INDUCED BY N-BUTYLNITROSOUREA*1. ed by cytotoxicity tests. Cells from leukemia without thymus involvement

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[Gann, 66, 37-42; February, 1975] ORIGIN OF LEUKEMIC CELLS IN MOUSE LEUKEMIA INDUCED BY N-BUTYLNITROSOUREA*1 Hayase SHISA, Yasue MATSUDAIRA, Hiroshi HIAI, and Yasuaki NISHIZUKA Laboratory of

1 [Gann, 66, 37-42; February, 1975] ORIGIN OF LEUKEMIC CELLS IN MOUSE LEUKEMIA INDUCED BY N-BUTYLNITROSOUREA*1 Hayase SHISA, Yasue MATSUDAIRA, Hiroshi HIAI, and Yasuaki NISHIZUKA Laboratory of Experimental Pathology, Aichi Cancer Center Research Institute*2 Administration of N-butylnitrosourea (BNU) induces leukemia in thymectomized C57BL/6J and C3Hf/Bi mice with almost the same high frequency as in H-T6T6) F1 mice receiving neonatal thymus tissues from C3Hf donors developed leukemias with or without marked enlargement of the grafts. The origin of leukemic cells was analysed by T6 marker chromosome and thymus allo-antigen theta in this hybrid system. ed by cytotoxicity tests. Cells from leukemia without thymus involvement cells, most probably bone marrow cells which are repopulating into the graft. When the mice were treated with BNU after the lymphoid elements in the grafted thymus had been replaced by host cells, leukemia mainly composed of host-origin cells developed. Leukemia in which neoplastic cells in the thymus grafts were of donor origin and those in other hematopoietic tissues were of host origin was found not infrequently. The present results mean that the target cells in BNU leukemogenesis are distributed within and outside the thymus and that some leukemias are of multifocal tissue origin. Continuous oral administration of N-butylnitrosourea (BNU) in drinking water to young adult mice yielded a high incidence of thymic lymphocytic leukemia.12,23) In this type of leukemogenesis, it is believed that the target organ of the chemical is the thymus, because initial microscopic changes suggestive of early leukemia are most frequently observed in this organ.19) It is possible to postulate that the bone marrow is also considered as a target organ, because (i) the thymic lymphocytes are continuously replaced by stem cells which are derived from bone marrow cells,3, 7,22) (ii) there is an increase in the incidence of thymic leukemia when bone marrow cells taken from carcinogen-treated mice are injected into the mice,1) (iii) frequency of chromosome aberrations evoked by exposure of the cells of the hematopoietic organs to 7,12- dimethylbenz[a]anthracene appears to be correlated to the incidence of carcinogeninduced leukemias, and cells of the bone marrow are most susceptible to chemicals in induction of chromosomal aberrations,9) and (iv) thymectomy shows no marked inhibitory effect on the development of leukemia induced by carcinogenic chemicals,5,8,12,13,23) and in thymectomized mice leukemias develop rather frequently from other hematopoietic tissues. It seemed of interest to make precise analysis of cellular origin in chemically induced mouse leukemias and to investigate whether or not the type of leukemias is related to the origin of leukemic cells of lymphatic series. The present paper will describe the origin of *1 This work was supported in part by a Grant-in-Aid from the Ministry of Education.

2 leukemic cells in BNU-induced leukemias analysed by T6 chromosome marker and thymus cell surface alloantigen theta. MATERIALS AND METHODS Animals Inbred mice of C57BL/6J, C3Hf/Bi, and CBA/H-T6T6 strains, raised in our laboratory, were used. C3Hf females were mated with CBA/H-T6T6 males and F1 hybrids were also used. The animals were fed on commercial compressed diet CMF (Oriental Yeast Co., Tokyo) and given tap water freely after BNU administration was completed. BNU Administration BNU was dissolved in drinking water at a concentration of 0.04%, and given orally to young adult animals for a period of 60 days. A fresh solution was given every morning. Surgical Treatment Thymectomy and thymus grafting were performed as previoulsy described.17) H. SHISA, ET AL. received parental C3Hf thymus of newborn mice of the same sex. Each animal received one whole thymus grafted subcutaneously into the right fat pad. Observation The check for tumor development, autopsy, and histological observations have been described previously.17) All the animals were killed at 10 months of age irrespective of the appearance of leukemia. Pooled data from males and females will be described because no sex difference was noted in leukemia susceptibility. described previously.6) Chromosome Analysis Chromosomal preparations were made with cells of bone marrow, spleen, thymus graft, and mesenteric lymph node of leukemic mice using the air-drying method.10) Colchicine was not injected into mice before sacrifice. Metaphase chromosomes were examined only on preparations in which the chromosomes were well spread. It is likely that the majority of the metaphase plates come from neoplastic cells in these preparations, although they do not permit the exact identification of leukemic cells. A minimum of 20 and usually metaphases per tissue were examined to determine the origin of the leukemic cells. RESULTS Leukemias in Thymectomized C57BL and C3Hf Mice C57BL and C3Hf mice were thymectomized at 32 days of age. The operated and non-operated mice were administered BNU starting from the age of 35 days. As shown in Table I, non-thymectomized mice developed leukemia at a high incidence of 90-96%. Most of the leukemias were typical lymphocytic or lymphoblastic leukemia with marked enlargement of the thymus. These were called thymic type leukemia.17) Their histological pictures were similar to those observed in Gross virus-induced and X-ray-induced leukemias, and were classified as "well-differentiated lymphocytic leukemia,"12) the starry sky pattern being common. In this type of leukemia, the cells possessed cytotoxicity test. Also, leukemia developed with 76-85% incidence in thymectomized mice. This incidence was slightly lower and the average latency longer than those in the non-thymectomized group. This type of leukemia was classified histologically as "poorly differentiated lymphocytic leukemia."12) Here, the leukemic cells did not possess the target cells in BNU leukemogenesis may be distributed outside the thymus also. CBA/H-T6T6) F1 Mice with Parental Neo- CBA/H-T6T6) F1 mice were thymectomized at 32 days of age and grafted with a thymus Table I. Incidence of Leukemia Induced by BNU in Thymectomized and Non-thymectomized Mice 38 Gann

3 CELL ORIGIN OF CHEMICALLY INDUCED MOUSE LEUKEMIA Table II. Incidence and Cell Origin of Leukemia Induced by BNU in Thymectomized F1 Mice Grafted with Neonatal Thymus of Parental Strain and Bone Marrow of C3Hf mice at 35 days of age. In Group II, F1 mice were thymectomized at 3 days of age and 2 days later grafted with a thymus from newborn C3Hf mice (Table II). BNU administration was started at the age of 37 days. This would mean that the mice of Group II received BNU when lymphoid elements of the grafted thymus had been replaced by host cells. It is well established that thymus lymphocytes of a grafted thymus are completely repopulated by cells derived from the host bone marrow within 21 days after grafting.2, neoplastic cells were derived from host F1 cells. Tumor cells of thymus and spleen of with a fairly high frequency. In addition, the majority of leukemia thus induced showed intensive involvement of the grafted thymus by massive infiltration of lymphocytic neoplastic cells. There is no doubt that this pattern exactly corresponds to the morphology of thymic type leukemia developing in nonthymectomized mice. Origin of Leukemic Cells (1) Leukemic Cells in Grafted Thymus: Twenty-one cases of leukemia, which developed in adult thymectomized and thymusgrafted group (see Group I in Table II), were analysed by T6 chromosome marker and cell- be divided into three subgroups, depending on the origin of leukemic cells in three organs; thymus graft, spleen, and bone marrow. As shown in Table III, neoplastic cells of these organs were of the same origin in Thymic I and III types. In 7 cases of Thymic I, all the leukemic cells were derived from donor thymus cells, and in 6 cases of Thymic III, all the cases of Thymic II, leukemic cells of the thymus were derived from the grafts, while cells of other organs were from F1 host cells. The leukemic cells of enlarged thymus had spleen of this subgroup were not checked for (2) Leukemic Cells in Non-thymic Leukemia: In 5 cases of leukemia without involvement of thymus grafts, termed non-thymic leukemia, all leukemic cells of three organs, 66(1)

4 H. SHISA, ET AL. T6T6) F1 Mice Grafted with Neonatal C3Hf Thymus a) Figures in parentheses indicate number of donor-type cells. lymph node, spleen, and bone marrow, were derived from host F1 cells. The leukemic cells Data obtained by chromosomal analysis on representative cases in each subgroup are given in Table IV. The age distribution of the four subgroups is illustrated in Fig. 1. Latent periods of Thymic I type were between 12 and 16 weeks after the start of BNU treatment, while those of Thymic III leukemia were prolonged to 18 weeks except in one case. Karyological Analysis of Leukemic Cells The results of chromosomal analysis of 21 cases of leukemic mice in Group I (see Table II) may be summarized as follows: Leukemias thus induced did not contain cells with marker chromosome as a rule. Seventeen leukemic cases (81%) showed only one modal number of chromosome. In one case, however, no modal number could be found due to a wide variation in the number of chromosomes (41-44) carried by different cells. Three cases showed bimodal numbers. Mouse No. 188 was a typical case (Table IV, Thymic II) where leukemic cells with normal karyotype were present in the thymus and derived from donor cells of the thymus graft, and those in spleen and bone marrow were of host cell Fig. 1. Age distribution of four types of leukemia Figures in squares indicate modal number of chromosomes. origin and had 41 modal chromosome number. As shown in Fig. 1, out of 12 leukemias which developed between 12 and 18 weeks, had a normal karyotype. Leukemias 11 with longer latent period (23-36 weeks) had a much higher incidence of abnormal chromosome components. DISCUSSION In agreement with previous studies,5,8,13,23) the present results clearly confirm that the target cells of leukemogenic chemicals are distributed within and outside the thymus. Our experiments with F1 hybrids, thymectomized and grafted with the parental thymus, 40 Gann

CELL ORIGIN OF CHEMICALLY INDUCED MOUSE LEUKEMIA indicated the existence of leukemic cells derived from grafted thymus cells and those from F1 host cells in the thymus.

5 CELL ORIGIN OF CHEMICALLY INDUCED MOUSE LEUKEMIA indicated the existence of leukemic cells derived from grafted thymus cells and those from F1 host cells in the thymus. In the case of leukemias of host origin, two possibilities are conceivable: (i) Normal bone marrow cells from the host repopulate the grafted thymus and then are transformed into neoplastic the host, most probably bone marrow cells, migrate into the grafted thymus after neo- mic cells, since the thymus framework may provide a favorable site for proliferation of leukemic cells.18) At present, it is impossible to determine these two possibilities. A tendency that most leukemias of donor origin precede those of host origin is perhaps because host cells, either normal or transformed, must migrate into the thymus to form a thymic neoplasm. Donor cell leukemias were not observed in mice to which BNU was given after lymphoid elements in the grafted thymus were replaced by host cells. Thus, it appears that the thymus and bone marrow can be regarded as the targets in chemical leukemogenesis. The possibility remains to be studied that the spleen and lymph node also do become the target organ of BNU exposure, as there are leukemias with just spleen or lymph node cells exhibiting abnormality.20) Experiments on this line are now underway. None of leukemias of Group I (Table II) showed mixed cell population of donor and host origins in the enlarged thymus. Chromosomal analysis revealed that the majority of leukemic mice bore tumor cells carrying only one modal number of chromosome set. It seems to be a result of selection mechanism of the host for proliferation of neoplastic cells. However, there are leukemias which have bimodal chromosome numbers, and this is the case of Thymic II, most typically in mouse No. 188 (Table IV). Such cases can be observed not infrequently in our other experimental groups.20) This strongly suggests multifocal tissue origin of leukemias. Our experiments on this line will be published elsewhere. Many early leukemias had normal karyotype and late-arising leukemias had a higher incidence of abnormal chromosomal elements (Fig. 1). This tendency agrees with the report of Ottonen and Ball14) and is also observed in viral leukemogenesis.15,21,24,25) It does not mean, however, that leukemic cells of grafted thymus cell origin always possess a normal karyotype and those of bone marrow cell origin, an abnormal chromosomal set. This problem requires further investigation. (Received August 28, 1974) REFERENCES 1) Ball, J. K., J. Natl. Cancer Inst., 41, 553 (1968). 2) Dukor, P., Miller, J. F. A. P., House, W., Allman, V., Transplantation, 3, 639 (1965). 3) Ford, C. E., Ciba Found. Symp. Thymus: Exp. and Clin. Studies, 131 (1966). 4) Green, I., J. Exp. Med., 119, 581 (1964). 5) Haran-Ghera, N., Kotler, M., Meshorer, A.J., J. Natl. Cancer Inst., 39, 653 (1967). 6) Hiai, H., Shisa, H., Matsudaira, Y., Nishizuka, Y., Gann, 64, 197 (1973). 7) Ilberg, P. L. J., Barnes, C. A., Int. J. Cancer, 5, 124 (1970). 8) Kirschbaum, A., Liebelt, A. G., Cancer Res., 15, 689 (1955). 9) Kurita, Y., Shisa, H., Matsuyama, M., Nishizuka, Y., Tsuruta, R., Yosida, T. H., Gann, 60, 91 (1969). 10) Kurita, Y., Sugiyama, T., Nishizuka, Y., Cancer Res., 28, 1738 (1968). 11) Metcalf, D., Wakonig-Vaartaja, R., Proc. Soc. Exp. Biol. Med., 115, 731 (1964). 12) Nishizuka, Y., Shisa, H., Gann Monograph, 12, 297 (1972). 13) Nishizuka, Y., Shisa, H., "Topics in Chemical Carcinogenesis", p. 493 (1972). University of Tokyo Press, Tokyo. 14) Ottonen, P. O., Ball, J. K., J. Natl. Cancer Inst., 50, 497 (1973). 15) Rich, M. A., Tsuchida, R., Siegler, R., Science, 146, 252 (1964). 16) Schlesinger, M., Hurvitz, D., J. Exp. Med., 127, 1127 (1968). 17) Shisa, H., Mie Med. J., 19, 89 (1969). 18) Shisa, H., Nishizuka, Y., Gann, 62, 407 (1971). 19) Shisa, H., Nishizuka, Y., Hiai, H., Matsudaira, Y., Gann Monograph, 17 (1975), in press. 20) Shisa, H., unpublished data. 66(1)

6 H. SHISA, ET AL. 21) Tsuchida, R., Rich, M. A., J. Natl. Cancer Inst., 33, 33 (1964). 22) Wallis, V., Davies, A. J. S., Koller, P. C., Nature, 210, 500 (1966). 23) Yokoro, K., Takizawa, S., Kawamura, Y., Nakano, M., Kawase, A., Gann, 64, 193 (1973). 24) Yosida, T. H., Law, L. W., Cytologia, 33, 256 (1968). 25) Joneja, M. G., Stich, H. F., Exp. Cell Res., 40, 148 (1965). 42 Gann

[GANN, 52, ; September, 1961]

[GANN, 52, ; September, 1961] [GANN, 52, 257-264; September, 1961] CHROMOSOMAL ALTERATION AND THE DEVELOPMENT OF TUMORS, VII. KARYOLOGICAL ANALYSIS OF SPONTA- NEOUS AND INDUCED LEUKEMIAS IN MICE1)2) YOSHINORI KURITA and TOSIHIDE H.