25. p53 Deficiencv Backcrossed into C3H/He Mice Produced High-incidence of Stemcell Leukemia after lrradiation
Kazuko Yoshida, Shirou Aizawa and Yoko Hirabayashi* (*Visiting Scientist, National Institute of 1-Iealth Sciences)
Keywords: p53 homo-deficient mice, stem-cell leukemia
The mice lacking a p53 suppressor gene were developed independently in three laboratories by agene-targeting strategy. Although these strategies differed, they were essentially all null mutants; the resulting homozygous deficient mice all are highly predisposed to malignancy, most often malignant lymphomas. Other types of tumors, including osteosarcomas, and hemangiosarcomas, also develop with some frequency. These mice have a mixed inbred genetic background of C57BL/6 and 129/SV or CBA. Purely bred C57BL/6 mice themselves are highly predisposed to lymphoma; therefore, the high frequency of lymphoma in p53-deficient mice reflects this genetic background. To test the effect of the genetic background on the spectrum of tumors, we ohtained pure C3H/He p53-deficient mice by crossing p53-deficient C57BL mice back to C3H/He. C3H/He mice show a relatively high incidence of radiationinduced myeloid leukemta.
Bone marrow cells were harvested from p53homodefieient C311/lleNirsMs male mice, and 1x1O6 cells were injected through a tail vein of the lethally irradiated mice (transplantation assay). The hematopoietic cells in these recipient mice were replaced with p53 homo-deficient cells within 4 weeks after transplantation; then, they were further exposed to whole-body irradiation at 1, 3, or 5 Gy Large numbers of mice are required for studies of carcinogenesis, and the transplantation assay is a good tool to meet this need, because 40-50 mice can be unil'ormly repopulated with bone marrow from one p53 homodeficient mouse. In addition, analysis of the function of p53 gene is limited to the hematopoietic tissue.
When mice with p53 deficiency were backcrossed into C311/ Ile mice, there was a dramatic differ ence, with 77% developing undifferentiated leukemias compared with 22.2% whith lymphomas. The leukemias were negative for either the T cell markers (Thyl.2 and CD3), or for the B cell mark ers (B220 and s-IgM) , and also, for myeloid lineage cell markers (Mac-1 and Gr-1). The leukemia cells only expressed c-Kit and Pgpl. c-Kit is the receptor for the stem cell factor and it was expressed on al most all hematopoietic progenitors cells. Pgpl was positive for all bone-marrow cells at varying intensi ties; however, lymphoid cells appeared dull, CFU-s were intermediate, myeloidlineage-committed stem cells were brighter, and mature myeloid cells were very bright. Therefore, this undifferentiated leukemia seemed to be a stem cell leukemia with myeloid differentiation. These leukemic cells occasionally showed a minimum differentiation, with megakaryocytic, myeloblastic, and erythroblastic elements observed histologically in the leukemic infiltrate. Further, TER119 was positive in 35% of the cases when the enlarged splenic cells were examined.
By contrast, thymic lymphomas, the second most common malignancy in the p53 deficient C3H/He mice, appeared relatively later than the stem cell leukemias. Thymic lymphoma has rarely been seen in wild type C3H/He mice. Even with the same p53 deficiency, the incidence of the thymic lymphoma in the C3H/He mice was much less than in C57BL animals, probably because of the high incidence of myeloid prone tumors of the C3H/He mice.
We also found that radiation dramatically decreased the latency of leukemia in p53 homozygous deficient mice, and depended on the dose of radiation; the largest decrease in the 50% incidence, from 118 days to T4 days, appeared in the 5 Gy irradiated group.