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). All six MGI- clones showed a loss of a piece of one taining Gurr's buffer at pH 6.8) (14). Well spread metaphase chromosome 2 and none of the four MGI+ clones showed this chromosomes were photographed and 20 karyotypes for each abnormality in chromosome 2. The size of the missing piece cell type were arranged according to the standard nomenclature varied in the six MGI- clones from a smaller to a larger piece for the normal mouse karyotype (17). (Table 2 and Fig. 1). In agreement with previous results (14), Assays for Fc and C3 Rosettes. Rosette formation for Fc MGI+D+ cells differed from MGI+D- cells, in that MGI+D+ with sheep erythrocytes coated with antibody (EA) or for C3 contained an additional piece on one chromosome 12 and with sheep erythrocytes coated with antibody and complement MGI+D- cells contained only one chromosome 9, one chro- (EAC) was tested by a modification of the method of Bianco mosome X, and unidentified chromosomes called C and D et al. (18) as described (10, 11). To determine the percentage (Table 2 and Fig. 1). of cells with a rosette, we mixed 108 EA or EAC with 106 cells The data in Table 2 and Fig. 1 also show that there were in to be tested, centrifuged the mixture at 500 X g and, without some clones other abnormalities including trisomy of chro- dispersing the pellet, incubated the reaction for 30 min at 370. mosomes 12 and 15. The least abnormal karyotypes were seen The pellet was then gently dispersed and the percentage of cells in MGI+ clones 11 and 12 with a modal number of 40 and one with 10 or more attached erythrocytes determined by counting abnormal chromosome 12, and MGI- clone 15 with a modal about 1000 cells. The reproducibility of the results was generally number of 41, one abnormal chromosome 2, and one abnormal ±10% when the percentage of rosette-forming cells was higher chromosome 8. Table 2. Chromosomes of MGI+ and MGI- clones

No. of chromosomes Clone Cell type no. 1 2 3 4 5 6 7 8 9 12 14 MGI+D+ 11 2 2 2 2 2 2 2 2 2 1 + B 2 MGI+D+ 12 2 2 2 2 2 2 2 2 2 1 + B 2 MGI+ D- 2 2 2 2 2 2 2 2 2 1 2 2 MGI+ D- 4 2 2 2 2 2 2 2 2 1 2 2 MGI-D)- 1 2 1 + F2 2 2 2 2 2 2 1 2 2 MGI-D- 6 2 1 + F2 1 2 1 + H Jl 2 2 2 2 2 MGI-D- 7 2 1 + F3 1 2 2 1 1 2 2 1 1 MGI-D- 8 1+E 1+F1 2 1+G 2 1+J2 2 2 2 2 2 MGI-D- 10 2 1 + F3 2 2 2 3 2 1 + K, 2 3 2 MGI-D- 15 2 1 + F2 2 2 2 2 2 2 + K2 2 2 2 * All other chromosome groups had the normal diploid banding pattern. Downloaded by guest on September 30, 2021 Cell Biology: Azumi and Sachs Proc. Natl. Acad. Sci. USA 74 (1977) 255

1 2 3 4 5 6 7 8 9 I- Table 3. Induction of EA and EAC rosettes on MGI+ mutants from MGI- clone 7

12 .1 15 18 1 0 % Cells with rosette 11 12 13 14 15 16 17 Is 19 X Y Control With MGI Clone .1 hoI.A I A i I I;i , 6 I A I in Cell type no. EA EAC EA EAC MGI- 7 1.8 5.0 2.0 5.4 MGI+ 7-M9 2.1 17.5 22.8 44.2 MGI+ 7-Mll 1.6 15.7 6.7 30.9 E 2 Fi F F3 4 G 5 H 6 rT MGI+ 7-M16 0.2 5.8 5.4 14.3 MGI+ 7-M4 1.3 7.3 13.3 35.3 MGI+ 7-M5 3.2 8.0 6.7 24.0

Cells were incubated for 4 days with 25% conditioned medium containing MGI.

6 J2 6 8 K1 8 8 K2 17 L 3 M 6 chromosome P that contained a chromosome 12 (Table 4). The gain of inducibility for rosettes by MGI was, therefore, associ- ated in all five mutants with a loss of either the one normal chromosome or one of these translocated chromosome 12s.

3 N6 3 P 12 7 Q 15 12 R15 Chromosomes of leukemias in vivo derived from the cultured clones a- !-3-} §:1' 44 To determine whether the chromosome banding pattern found in the cultured clones was maintained in leukemic cells growing in vio, we inoculated cells of the two MGI+D+ clones, two

16 S 19 12 8 C D T U V MGI+D- clones, and three MGI- clones (6,8, and 15) into mice and studied the banding pattern of the leukemias without any further culture. The loss of a piece in one chromosome 2 was found in all 10 tumors derived from MGI- cells. The only ka- FIG. 1. The Giemsa chromosome banding pattern of a normal ryotype changes from the cultured cells were a trisomy of male mouse (upper two rows) and of abnormal chromosomes found chromosome 12 and an abnormal chromosome S (Fig. 1) in one in the myeloid leukemic cells. C, D, T, U, and V are unidentified tumor from clone 6, and a trisomy of chromosome 15 in one chromosomes. X750. tumor from clone 8. The four tumors derived from MGI+ clones also showed the same karyotypes as the cultured cells, except Chromosomes of MGI+ mutants from MGI- clone 7 for one tumor from MGI+D- clone 2 which had a trisomy of Five MGI+ mutants that could be induced to form EA and EAC chromosome 15. rosettes after incubation with conditioned medium containing Chromosomes of leukemic cells before culture MGI (Table 3) were isolated from the MGI- clone 7. The MGI- clone 7 (Tables 1 and 4) had one chromosome 2 with a missing To determine whether MGI- cells also showed a loss of a piece piece, one normal chromosome 12, and four translocated of chromosome 2 before culture and whether this chromosome chromosomes including two translocated chromosomes 12 (P abnormality was present in lymphoid leukemias from the same and R. Fig. 1). All five MGI+ mutants still had the abnormal strain of mouse, we studied the chromosomes of the myeloid chromosome 2, but three mutants had lost one normal chro- leukemia that gave rise to clone 15 and of three independently mosome 12 and the other two mutants had lost the abnormal arising lymphoid leukemias before culture. The results show

Table 2. (Continued) % Cells Modal in chromosome groups* with chromo- karyo- some 15 17 19 X M N P Q R C D T U V type no. 2 2 2 2 0 0 0 0 0 0 0 0 0 0 95 40 2 2 2 2 0 0 0 0 0 0 0 0 0 0 85 40 2 2 2 1 0 0 0 0 0 1 1 0 0 1 95 41 2 2 2 1 0 0 0 0 0 1 0 0 0 0 75 39 2 1+L 2 2 0 0 0 0 0 0 0 0 0 0 95 39 2 2 2 1 1 0 0 0 0 0 0 0 0 0 80 38 2 2 2 1 0 1 1 1 1 0 0 1 0 0 85 39 2 2 2 1 0 0 0 0 0 0 0 0 0 0 100 39 3 2 1 2 0 0 0 0 0 0 0 0 1 0 90 43 2 2 2 2 0 0 0 0 0 0 0 0 0 0 100 41 Downloaded by guest on September 30, 2021 256 Cell Biology: Azumi and Sachs Proc. Natt. Acad. Sci. USA 74 (1977)

Table 4. Chromosomes of MGI+ mutants from MCl- clone 7

No. of chromosomes in chromosome groups* Cells Modal with chromo- Cell Clone N P Q R karyo- some type no. 2 3 6 7 12 14 X t(3;6) t(3;12) t(7;15) t(12;15) T type no. MGI- 7 1 + F3 1 1 1 1 1 1 1 1 1 1 1 85 39 MGI+ 7-M9 1 + F3 1 1 1 0 1 1 1 1 1 1 1 100 38 MGI+ 7-M11 1 + F3 1 1 1 0 1 1 1 1 1 1 1 90 38 MGI+ 7-M16 1 + F3 1 1 1 0 1 1 1 1 1 1 1 95 38 MGI+ 7-M4 1 + F3 1 1 1 1 1 1 1 0 1 1 1 95 38 MGI+ 7-M5 1 + F3 1 1 1 1 1 1 1 0 1 1 1 90 38 * All other chromosome groups had the normal diploid banding pattern. t = translocation.

(Table 5) that the myeloid leukemias that gave rise to clone 15 MGI+D+ cells showed other specific chromosome differences had the same loss of a piece of chromosome 2 as the cultured from MGI+D- cells, and that MGI+D+ cells can be induced to clone and that none of the three lymphoid leukemias had an undergo normal cell differentiation even though the cells do abnormal chromosome 2. One of the lymphoid leukemias not have a completely normal diploid chromosome banding showed a trisomy of chromosome 15, one a trisomy of chro- pattern. This shows that these malignant cells can be induced mosome 14 and a loss of one X chromosome, and the third a by MGI to have a normal differentiation phenotype without trisomy of chromosomes 5 and 15 or trisomy of chromosome a completely normal genotype. 5 and a loss of one X chromosome (Table 5). The loss of a piece of chromosome 2 in MGI- cells was present in myeloid leukemic cells before culture and in leuke- DISCUSSION mias produced in vvo from the cultured cells. This indicates that there was also a loss of this piece of chromosome 2 in cells We have analyzed the chromosome banding patterns of clones that are malignant in vio. The least abnormal karyotypes were of myeloid leukemic cells which differ in their ability to be found in MGI+D+ clones and their tumors which had one ab- induced to undergo normal cell differentiation. The results have normal chromosome 12 and in an MGI- clone with an abnor- shown that all six clones of MGI- cells derived from six inde- mal chromosome 2 and an abnormal chromosome 8. The ab- pendently produced myeloid leukemias had a piece missing normal chromosome 2, but not the abnormal chromosome 8, from one chromosome 2 and that this abnormality was not was found in all the MGI- clones and their tumors in vvo. It found in MGI+ myeloid leukemic clones or lymphoid leuke- can, therefore, be suggested that in addition to carrying genes mias. Five MGI+ mutants, derived from an MGI- clone with that control differentiation, chromosomes 2 and 12 also carry an abnormal chromosome 2, one normal chromosome 12, and genes that control the malignancy of myeloid leukemic cells. two translocated chromosomes 12, still had this piece missing The present experiments thus support the evidence obtained in one chromosome 2, but had also lost either the one normal with other types of tumors on the relationship between chro- or one of these translocated chromosome 12. These five mutants, mosome changes and the control of malignancy (6, 19-36). therefore, differed from the other MGI+ clones which did not In addition to these changes in chromosomes 2 and 12, there have a piece missing in one chromosome 2, in having either no was also in five of the myeloid and two of the lymphoid leu- normal and two translocated chromosomes 12 or only one kemias a loss of one chromosome X, in two of the myeloid leu- normal and one translocated chromosome 12. These results kemias a trisomy in chromosome 12, and in two of the myeloid indicate that genes controlling inducibility of EA and EAC and two of the lymphoid leukemias a trisomy of chiomosome rosettes by MGI, a stage in the differentiation of myeloid cells, 15. It is of interest that a trisomy of chromosomes 15 or 12 has are located on chromosomes 2 and 12. The data from the mu- also been observed in lymphoid leukemias in another strain of tants suggest that there are suppressor gene(s) on chromosome mouse (31). These results suggest that in addition to chromo- 12 that can suppress the inducing gene(s) on chromosome 2 and somes 2 and 12, chromosome 15 and the X chromosome may that inducibility by MGI is controlled by the balance between also carry genes that control malignancy. The present data can, these genes. therefore, be used to support the suggestion that there is more The results also indicate, as in previous experiments (14), that than one gene that controls malignancy (6, 22, 24, 26, 34, 35)

Table 5. Chromosomes of leukemias before culture % Cells Modal No. of chromosomes in chromosome groups* with chromo- Type of Tumor karyo- some leukemia no. 2 5 8 14 15 X type no. Myeloid 15 1 + F2 2 1 + K2 2 2 2 100 40 Lymphoid Li 2 2 2 2 3 2 95 41 Lymphoid L2 2 2 2 3 2 1 90 40 2 2 3 1 60 41 Lymphoid L3 42 3 Allothrcromsom22 2 2 1 40 40 *All other chromosome groups had the normal diploid banding pattern. Downloaded by guest on September 30, 2021 Cell Biology: Azumi and Sachs Proc. Nati. Acad. Sci. USA 74 (1977) 257

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