Proc. NatL. Acad. Sol. USA Vol. 74, No. 1, pp. 253-257, January 1977 Cell Biology Chromosome mapping of the genes that control differentiation and malignancy in myeloid leukemic cells (macrophage and granulocyte inducer/Fc and C3 receptors/differentiation mutants/mouse chromosomes 2 and 12) JUN-ICHI AZUM1 AND LEO SACHS Department of Genetics, Weizmann Institute of Science, Rehovot, Israel Communicated by George Klein, October 26,1976 ABSTRACT The chromosome banding pattern has been chromosome number, so that these cells appear to be particu- analyzed in clones of mouse myeloid leukemic cells that differ larly favorable for mapping the genes that control differen- in their ability to be induced to differentiate by the protein in- tiation and ducer MGI (macrophage and granulocyte inducer) None of the malignancy. clones had a completely normal diploid banding pattern. The It has been shown with chromosome banding that even clones studied were either MGI+ (that can be induced to form MGI+D+ cells that can be induced to differentiate normally Fc and C3 rosettes), a stage in the differentiation of myeloid did not have a completely normal diploid chromosome banding cells, or MGI- (that cannot be induced to form these rosettes) pattern and that there were specific chromosome differences All six cultured clones of MGI- cells from myeloid leukemias between clones of MGI+D+ and MGI+D- cells (14). The independently produced in six separate animals showed a loss present studies were undertaken to the of a piece of one chromosome 2 and this abnormal chromosome analyze chromosome was maintained in leukemias derived from the cultured cells. banding pattern of MGI- cells and to compare this with the This loss was not found in MGI+ clones orlymphoid leukemias. patterns of MGI+D+, MGI+D-, lymphoid leukemias, and Five MGI+ mutants, derived from an MGI- clone with a loss normal cells. We used six clones of MGI- cells derived from six of a piece of one chromosome 2, one, normal chromosome 12, separate animals with independently arising myeloid leukemias and two translocated chromosomes 12, maintained the abnor- and 5 MGI+ mutants from one of the MGI- clones. The chro- mal chromosome 2 but lost either the one normal or one of these mosome banding pattern of tissue culture was translocated chromosome 12. These results indicate that chro- clones compared mosomes 2 and 12 carry genes that control the differentiation with myeloid leukemias and lymphoid leukemias in vivo. The of myeloid leukemic cells and that inducibility by MCI is con- results indicate that the chromosomes carry genes that control trolled by the balance between these genes. We sugges that the differentiation of myeloid leukemic cells and also suggest these chromosomes also carry genes that control the mahgnancy which chromosomes carry genes that control the malignancy of these cells. of these cells. The development of an experimental system for the in vitro induction of differentiation in various types of white-blood cells MATERIALS AND METHODS (1-7), has made it possible to study the control mechanisms that regulate the differentiation of these cells and the blocks that can Cells and Cell Culture. Ten clones of myeloid leukemic cells occur in leukemia and nonmalignant diseases (6, 8-13). It has were used in the present study. Two clones (11 and 12) were been shown that some myeloid leukemic cells can be induced MGI+D+; two clones (2 and 4) were MGI+D-; and these four to differentiate normally by incubation with the differentiation clones were isolated (9) from a myeloid leukemic cell line in inducing protein MGI (macrophage and granulocyte inducer) culture obtained from a spontaneous myeloid leukemia in a (6, 8, 9) and that there are other myeloid leukemic cells with female SL mouse (15). The other six clones (1, 6, 7, 8, 10, and different blocks in differentiation (10, 12). Clones of one type 15) were separately cloned MGI- myeloid leukemic cell lines of cell (MGI+) can be induced by MGI to form Fc and C3 ro- established in Vitro from the first or second transplant gener- settes, which is a stage in the differentiation of myeloid cells, ation of six independently arising myeloid leukemias after ir- whereas clones of another type of cell (MGI-) cannot be in- radiation of female SJL/J mice with x-rays (16). The lymphoid duced by MGI for the surface changes that result in the for- leukemias studied were also obtained after irradiation of female mation of rosettes. The MGI+ clones can be divided into two SJL/J mice with x-rays and were in the first or second transplant types, one of which (MGI+D+), was also induced by MGI to generation. All the clones of myeloid leukemic cells grew in form mature macrophages and granulocytes, and the other suspension as myeloblasts to promyelocytes, and cells from all (MGI+D-) was induced to form Fc and C3 rosettes but not 10 clones produced myeloid leukemia after intravenous inoc- mature cells. The MGI- clones were all D- and could not be ulation into isologous adult mice. Cells were cultured in Eagle's induced to form rosettes or mature cells (10, 12). medium containing a 4-fold concentration of amino acids with The clonal origin and inheritance of the differences in in- vitamins (H-21, Grand Island Biological Co., New York) and ducibility by MGI suggested that there may be genetic differ- 10% inactivated fetal calf serum (560 for 30 min) for MGI- and ences between the different cell types and that it may be pos- similarly inactivated horse serum for MGI+ clones. The clones sible to map the chromosome location of the genes involved were in culture for about 6 months to more than 1 year. Five (14). The clones used, even those that had been in culture for MGI+ mutant cell lines from MGI- clone 7 were isolated either more than a year, still had a diploid or near-diploid modal after spontaneous mutation (7-M4 and 7-M5) or after muta- genesis with N-methyl-N-nitro-N-nitrosoguanidine (7-M9, Abbreviations: MGI, the protein inducer for rosettes sheep erythrocytes 7-M11, and 7-M16). The cells of clone 7 formed compact coated with antibody or with antibody and complement on MGI+ cells, colonies after incubation with conditioned medium containing and for the differentiation of D+ cells to mature macrophages and MGI. The five mutant colonies were isolated as diffuse colonies granulocytes; EA, sheep erythrocytes coated with antibody; EAC, sheep in which the differentiating cells migrate in the agar (. Lotem erythrocytes coated with antibody and complement. and L. Sachs, unpublished). 253 Downloaded by guest on September 30, 2021 254 Cell Biology: Azumi and Sachs Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Induction of EA and EAC rosettes on MGI+ than 10%. With lower frequencies of rosette-forming cells the but not on MGI- clones reproducibility was generally 120-30%. To assay for induction of rosettes by MGI, we incubated cells for 4 days with a 25% % Cells with rosette concentration of conditioned medium from lungs of mice in- as (11). This condi- Control With MGI jected with bacterial endotoxin described Clone tioned medium contains MGI. Cell type no. EA EAC EA EAC RESULTS MGI+D+ 11,12 0.3 3.0 19.0 50.0 MGI+ D- 2,4 0.1 1.5 16.0 43.0 Induction by MGI of EA and EAC rosettes on MGI+ MGI-D- 1 0 0 0 0 but not on MGI- clones MGI-D- 6 0.3 0.1 0.2 0 MGI-D- 7 1.8 5.0 2.0 5.4 The 10 clones of myeloid leukemic cells studied can be divided MGI-D- 8 0.1 0.1 0.1 0 into groups (four MGI+ and six MGI- clones) on the basis of MGI-D- 10 2.2 0.1 1.9 0 their ability to be induced to form EA and EAC rosettes by the MGI-D- 15 0 0.1 0.1 0 inducing protein MGI in conditioned medium (Table 1). As in previous experiments (10), two of the MGI+ clones (MGI+D+) Cells were incubated for 4 days with 25% conditioned medium can also be induced by MG1 to form mature granulocytes and containing MGI. macrophages and the other two MGI+ clones (MGI+D-) can be induced to form EA and EAC rosettes but not mature cells. Chromosome Analysis. Chromosome preparations were None of the six MGI- clones derived from six independently made from cultured cells or from leukemic spleen cells. The arising myeloid leukemias could be induced to form either EA cells were incubated in 5 ml of medium with one drop 0.01% rosettes, EAC rosettes, or mature cells. colcemide for 30 min at 370, then with 0.075 M KCJ hypotonic solution for 30 min at room temperature, and fixed in several Chromosomes of MGI+ and MGI- clones changes of glacial acetic acid-methanol (1:3, vol/vol). Drops The modal chromosome number of the 10 clones of.myeloid of the cell suspension were placed on a glass slide, air-dried, and leukemic cells ranged from 38 to 43. The Giemsa banding the chromosomes stained for Giemsa banding as described (14). pattern of the chromosomes has shown that none of the cells had The air-dried preparations were incubated in 0.12 M Na2HPO4 a completely normal diploid karyotype and that all the MGI- at 600 for 3 hr, rinsed with deionized water, and stained for 20 clones had a karyotype different from all the MGI+ clones min with Giemsa solution (4% of Gurr's Giemsa "R66" con- (Table 2).