Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1152-1156, February 1995 Medical Sciences Hypermutability of mouse chromosome 2 during the development of x-ray-induced murine myeloid leukemia KANOKPORN RITHIDECH, VICTOR P. BOND, EUGENE P. CRONKITE, MARGARET H. THOMPSON, AND JAMES E. BULLIS Brookhaven National Laboratory, Associated Universities, Inc., P.O. Box 5, Upton, NY 11973-5 Contributed by Eugene P. Cronkite, November 21, 1994 ABSTRACT In an effort to identify the precise role of a deletion at regions D-E of mouse chromosome 2 [del2(d-e)] during the development of radiation-induced myeloid leukemia, we conducted a serial sacrifice study in which metaphase chromosomes were examined by the G-banding technique. Such metaphase cells were collected from x-irradiated mice during the period of transformation of some of the normal hematopoietic cells to the fully developed leukemic phenotype. A group of 25 CBA/Ca male mice (1-12 weeks old) were exposed to a single dose of 2 Gy of 25-kilovolt-peak x-rays; 42 age-matched male mice served as controls. Groups of randomly selected mice were sacrificed at 2 hr, 1 week, and then at intervals of 3 months up to 24 months after x-irradiation. Slides for cytogenetic, hematological, and histological examination were prepared for each animal at each sacrifice time. An expansion of cells with lesions on one copy of chromosome 2 was evident in 2-25% of treated mice at each sacrifice time. The majority of such lesions were translocations at 2F or 2H, strongly suggesting hypermutability of these sites on mouse chromosome 2. No lesions were found in control mice. The finding leads to the possibility that genomic lesions close to 2D and 2E are aberrants associated with radiation leukemogenesis, whereas a single clone of cells with a del2(d-e) may lead directly to overt leukemia. The data also indicate that leukemic transformation arises from the cumulative effects of multiple genetic events on chromosome 2, reinforcing the thesis that multiple steps of mutation occur in the pathogenesis of cancer. The involvement of a specific chromosomal abnormality, a deletion at regions D-E of mouse chromosome 2 [del2(d-e)], in radiation-induced murine myeloid leukemia (ML) has been documented (1-5). Such findings strongly indicate that alterations of the genes within or close to 2(D-E) play a significant role in the development of radiation-induced murine ML. A potential role of the homeobox gene Hox4.1, mapped to 2D, for myeloid transformation has been suggested (6). Whether the induction of del2(d-e) is an early or late event in radiation leukemogenesis is unknown. Consequently, these findings prompted us to design a serial sacrifice study to investigate the precise contribution of genomic alterations on mouse chromosome 2 during the transformation of normal hematopoietic cells to the leukemic phenotype. A G-banding technique was used to determine such changes. We hypothesized that (i) a specific genetic change on chromosome 2 in target cells (bone marrow), associated with the initiation of ML, leads to the formation of aberrant cells and persists or expands during the promotion and progression to clinically diagnosable ML and (ii) genetic changes associated with the early stages of murine leukemogenesis may differ from those seen in diagnosed ML. The results confirmed these hypotheses. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact. MATERIALS AND METHODS In this study, 25 CBA/Ca male mice (1-12 weeks old) from our breeding colony were exposed to a single dose of 2. Gy of x rays from a 25-kilovolt-peak Maxitron x-ray machine (General Electric). One millimeter of Al and.5 mm of Cu were used as filtration; the dose rate was.33 Gy/min. This dose of x-rays is known to induce a 2-25% lifetime incidence of ML (7). Forty-two age-matched CBA/Ca male mice served as sham controls. Methods used for the animal handling, detection of ML, metaphase preparation of the cells, the G-banding analysis of mouse chromosomes, and histological and hematological evaluations have been presented in detail elsewhere (1). Groups of randomly selected mice (15 treated and 4 control mice) were killed at 2 hr, 1 week, and 3, 6, 9, 12, 15, 18, and 21 months after x-irradiation. The study was terminated at 24 months following the exposure. At each sacrifice time, slides of the bone marrow cells for cytogenetic analysis, blood smears for complete hematological evaluations, and tissue samples from the sternum, spleen, liver, kidney, lymph nodes, and tail for histological examination were prepared from each animal. The normal lifetime (about 14 days) incidence of ML in CBA/Ca male mice is about 1% (7). Thus, based upon the sample size and the time frame of this study, the probability of appearance of ML in the control mice would be most unlikely (<1-4). At each sacrifice time, 5 G-banded metaphase cells per mouse were analyzed. Although the primary focus was on chromosome 2, abnormalities in other chromosomes in each G-banded metaphase cell were also determined. In addition to the G-banding analysis, the mitotic index for each mouse was evaluated. Significant differences between mitotic indexes of the control and treated groups were determined by a onetailed Student's t test using the ABSTAT statistical analysis package (Anderson-Bell, Denver). The criterion for a statistical significance is a P value of.5. Bone marrow cells collected from tibias of each animal were used for cytospin slide preparation. These slides were examined along with the corresponding blood slides. Tissue samples (spleen, liver, sternum, tail, and kidney) from each animal were collected and processed for histological analysis. The specific chromosomal changes were related to the specific altered histological and hematological observations in the same animal. Comparisons of the alterations observed in animals sacrificed at earlier time points with those detected in ML cells should help define the critical alterations-i.e., biomarkers-that occur before the development of a diagnosed leukemia. Based upon our hypothesis regarding the expansion of aberrant cells and the lifetime incidence of ML caused by a single dose of 2. Gy of x rays (2-25%), we should expect that, on the average, cells with an abnormal chromosome 2 would be detectable in 2 or 3 out of 1 treated mice per sacrifice time. Although samples from 15 treated mice were collected for studies at each sacrifice time, the cytogenetic analysis was done on a minimum Abbreviation: ML, myeloid leukemia. 1152
Medical Sciences: Rithidech et al. Proc. Natl. Acad. ScL USA 92 (1995) 1153 of 1 exposed mice. Only 9 exposed mice were included in the analysis at 24 months after x-irradiation because the chromosome banding patterns in the rest of exposed mice were not of sufficient quality to permit exact identification of abnormalities. An expansion of cells with abnormal chromosomes was defined as two or more cells with either structural or numerical abnormalities on the same chromosome. To determine a clone of cells, we used the criterion suggested by Rowley and Potter (8). RESULTS Of the 25 x-irradiated mice, 15 were euthanized at each of the first nine sacrifice times and 75 mice died before the last sacrifice, leaving 4 mice for the final sacrifice. Among the 75 dead mice, 21 mice died of ML and the rest died of other causes-e.g., nephrosclerosis, pneumonia, and other types of cancers. Of the 42 control mice, 4 were euthanized at each of the first nine sacrifice times and the only diagnosis was hepatoma. Two mice died with hepatoma before the final sacrifice. Control mice in other studies died with hepatoma, nephrosclerosis, pneumonia, or neoplasms. Among the sacrificed mice, cytogenetic analyses were done in 89 treated and 4 control mice. Results showed that 21 out of 89 treated mice (24%) had acquired an abnormality (all types of aberrations) on one copy of chromosome 2. Three out of the 21 mice with ab2 were diagnosed as ML. No abnormal chromosome was found in control mice. There was no statistically significant difference between mitotic indexes of treated and control mice at each sacrifice time (P values ranging from.6 to.5). Over the course of the study, a total of 24 histologically confirmed cases of ML was detected among exposed mice, whereas no ML was found in the control group. Details of cytogenetic and peripheral blood values of mice with ML and without ML are given below. Table 1. Cytogenetic and peripheral blood data for each ML case C'ytogenetics Mice with ML. Although 24 cases of ML were detected in the treated group, the suddenness of unheralded deaths limited severely the collection of cells suitable for cytogenetic analysis. Hence, only in nine of the ML cases were cytogenetic studies possible. Peripheral blood and cytogenetic data on these nine ML cases are presented in Table 1. Six out of nine ML cases were detected at the terminal stage of the disease. The other three cases (cases 3, 8, and 9) were found in mice randomly selected for serial sacrifice. Among these three ML mice, one (case 3) was at the terminal stage of the disease whereas the other two cases evidenced only minimal infiltration of ML cells (i.e., they were in an early stage of the disease). The specific deletion, del2(d-e), was detected in cells from all nine mice with x-ray-induced ML. All ML mice with the terminal stage of the disease (cases 1-7) showed the del2(d-e) deletion in 1% of analyzed cells. On the other hand, the two mice in an early stage of ML had translocations or inversions involving one copy of chromosome 2, as well as deletion of chromosome 2, in some cells. There was no evidence of preferential involvement of other chromosomes among these translocations. In these two cases of ML, abnormalities on chromosome 2 (all types of aberrations) were found in 32% and 36% of cells scored, respectively. In addition to structural abnormalities, aneuploidy was observed in some mice with x-ray-induced ML. Although different chromosomes were involved in aneuploidy in different ML cases, the majority of the aneuploidy consisted of a gain of chromosome 19 (+ 19) or a loss of chromosome Y (-Y). There was no persistent pattern of involvement of other chromosomes, although there was evidence of clonality for a + 19 (case 8) or a -Y (case 9). However, these changes might be technical artifacts or random mitotic errors as seen in some cells from patients with acute ML (8). Polyploidy was detected in only one case (case 7) in some cells. Peripheral blood values (per mm3) % cells with Platelets, Metamyelocytes Age, one abnormal Leukocytes, Erythrocytes, no. X Neutrophils, and myelocytes, Mouse days Chromosome aberration(s) chromosome 2 no. x 1-3 no. X 1-6 1-3 no. x 1-3 no. x 1-3 1 347 del2(d-e), +6, +9, -16 1 41.9 9.32 99 21.37 8.38 2 448 del2(d-e) 1 43.2 5.48 331 17.71 7.34 3 472 del2(d-e) 1 11. 7.18 439 2.59 1.5 4 514 del2(d-e), -18, -Y 1 42.9 6.69 488 17.16 12.87 5 556 del2(d-e) 1 16.9 6.54 339 96.33 48.17 6 575 del2(d-e), +12, +16, +19, -Y 1 26. 6.38 52 11.31 4.42 7 78 del2(d-e), polyploidy 1 31.9 7.17 417 16.75 5.58 8* 743 del2d, four cells; del2(f-h), two 32 6.1 7.85 721 3.2.12 cells; del2(b-h), one cell; dup2(g-h), one cell; t(2;4)(d;c), three cells; t(2;14)(d;c), one cell; t(2;9)(h;e), two cells; t(2;x)(h;e), two cells; +3, two cells; +5, three cells; +6, two cells; +1, two cells; +13, two cells; +14, two cells; +16, three cells; +17, two cells; +18, two cells; +19, four cells 9* 744 del2d, eight cells; del2e, four 36 11.3 1.2 1995 5.2.56 cells; del2(d-e), three cells; dup2(f-g), one cell; t(2;16)(e;b-c), two cells; t(1;7)(b;e), one cell; +8, one cell; -6, one cell; -11, one cell; -14, one cell; -Y, three cells *ML with minimal tissue infiltration of leukemic cells.
1154 Medical Sciences: Rithidech et al. Proc. Natl. Acad. Sci. USA 92 (1995) o. o f t. O 'IC m o ~o o o o o r o o o o o ": C., o O o o F.) E " en It Cl N4 CD r1 en N C)l N C Id (N CI ',l in t- O- cl~ 'IC~ Cl -I oo tn cno b-o N _l Cl on Cl4 ec m N N ^ Cl No ~CflC en Cl4 Cl,ct '- Cl "- Cli ',n en fi N I Cl N-. Ct.. IRcn ro on ~-4l c o6 r-i c - Cl Cl ef^ N'... ~. o o6 6 ori 6 c C N N rs oi,i o: o! &- Cl4 CIA lr O o r. X. z = 6 o1 o '-4 O rd6 oo N Cti oc3 ~ Cl t- t- tf) e - o N o6 o6 9 *~~~ Cl C l V} E c CZ) Q) r O Cl Cl Al Cl elf CQ1 Cl O Cl -) t) o 1 LV A 4" C. ) U: OClCl 9 9 'I ' f Cl en -o " co.).x >~~ ' C_ ' CZ U: -4 N4 ", Cl4 ", a) 'I 9 2 d - C- 1 1. Ct <, Z CZ C) ~ F-._~~~~~~~~~~~~. _ l '-4 Cl '-4l C l -l N l NO men m en ccn enc m e \ b all o D 66o 6o o6 6 "1 -tit - I'l ~ > 8 o ol on on C o o o o 6 65 6 6 7 11 en~ o 6- co+ D '- Cl4 r-- Cl4 r- Cl l '4 -Q-o = CD -c " co 4C4O.. C' -o Ca) W r- Cl4 X~ * 4- c4+ O
Medical Sciences: Rithidech et al. Mice Without ML Having Different Types of Lesions on Chromosome 2. Among 89 of the mice serially killed for cytogenetic analyses, 19 had cells with one abnormal copy of chromosome 2. The lesions observed in these non-ml mice were not a del2(d-e) or the translocation found in ML mice. Table 2 presents all types of lesions detected on chromosome 2, as well as the peripheral blood data, of mice killed throughout the study. (The data were collected chronologically; thus, Table 2 includes results from 3 ML cases.) A few abnormalities on other chromosomes, not reported here, were also detected. At 2 hr after x-irradiation, a high frequency of aberration per cell made it difficult to identify exact regions of chromosome involvement in abnormalities. Nevertheless, it was clear that 2 out of 1 exposed mice (2%) acquired cells with damage on chromosome 2 (16% and 22% of cells scored, respectively) as well as damage on other chromosomes. The damage included stable and unstable abnormalities (both chromatid and chromosome types). The extremely low mitotic index in bone marrow cells from mice sacrificed 1 week after x-irradiation prevented cytogenetic evaluation. The fraction of bone marrow cells with chromosome 2 lesions (stable chromosome aberrations) progressively increased from 8% of analyzed cells at 3 months to 38% at 24 months, suggesting an expansion of cells with one abnormal chromosome 2. Hematological and histological evaluations indicated no abnormalities in mice having a small number of cells with ab2. It is evident that mice had at least 3% of cells with one abnormal chromosome 2 (all types of aberrations) prior to showing any abnormal hematological or histological phenotypes. Early in the leukemic transformation process, breakpoints on chromosome 2 were clustered in regions F and H (Fig. 1), resulting in cells with chromosomal exchanges, mostly translocations. The number of abnormalities associated with 2D and 2E increased with time. No abnormality in chromosome 2 was found in control mice. Mice Without ML or Abnormalities on Chromosome 2. Many types of chromosomal aberrations were observed in these mice. Such changes included translocations, deletions, inversions, and aneuploidy. Expansion of cells with abnormalities in other chromosomes, including chromosomes 1, 4, 5, 7, 8, 17, 18, 19, X, and Y, were observed in treated mice as well as in a few controls (data not shown). However, no persistent time pattern for these abnormalities was detected. In addition to these changes, chromatid breaks were occasionally found in some cells of a few exposed mice. Polyploidy was frequently detected among control and treated mice over the course of study. DISCUSSION Our data demonstrate clearly that del2(d-e) in one copy of chromosome 2 is the cytogenetic marker of x-ray-induced ML in the CBA/Ca mouse and that ML is a clonal disease. This del2(d-e) was observed in all nine ML mice. Other types of Proc. Natl. Acad. ScL USA 92 (1995) 1155 chromosome 2 abnormalities, mostly translocations, were also observed in some cells from the two ML mice in which minimal tissue infiltration of leukemic cells had occurred. These findings lead to the conclusion that cells with abnormalities in chromosome 2 other than del2(d-e) may be important in the transformation process of myeloid cells, even though they do not appear in the actual leukemic phenotype. Such lesions may be, however, genetic events that could be of importance for the identification of individuals at high risk for developing ML later in life. The persistent occurrence of cells with one abnormal chromosome 2 in 25% (the lifetime incidence of ML) of exposed mice supports this notion. Moreover, an increase over time of the number of cells with such lesions strongly suggests that mice having an abnormal chromosome 2 following x-irradiation are most likely to develop leukemia. The data also show that cumulative effects of different mutations are necessary for myeloid neoplastic transformation. The mouse 2D and 2E regions show genetic homologies with the human 2q24-32 and llpll-13 regions, respectively (9). Interestingly, the human 2q31 region has been found to be involved in a translocation with llpl3 in a patient with myelodysplastic syndrome (1). A loss of a tumor-suppressor gene is associated with an llpl3 deletion in Wilms tumor patients (11). Thus, further molecular analysis of the mouse 2(D-E) region and flanking regions may help identify the putative tumorsuppressor gene(s) for myeloid transformation. Our results are not inconsistent with "clonal evolution" leading to frank leukemia, even though the clonal expansion of del2(d-e) was detected only in mice with the full-blown ML phenotype. This inference was based upon the finding of the emergence of such a clone in the two ML mice with minimal tissue infiltration of leukemic cells (an early sign of the disease), together with the finding of del2(d-e) in all cells from frankly leukemic mice. However, mechanisms leading to the disappearance of translocations on chromosome 2 and the subsequent evolution of del2(d-e) in radiation leukemogenesis remain unknown. One can speculate that translocations or deletions on chromosome 2 were coincidentally induced by x rays and that the simultaneous existence of cells with an abnormal chromosome 2 in hematopoietic stem cells has a similar potential with respect to the genesis of ML. However, hematopoietic progenitor cells with a translocated chromosome 2 may have acquired a proliferative advantage but with a limited mitotic capacity. Thus, initially, cells with this specific lesion outgrew cells with others lesions but died out later. Eventually, these cells were dominated by one with more aggressive but less proliferative capacity-i.e., cells with del2(d-e). It is also possible that gene rearrangements in chromosome 2 resulting from translocations are important in the transformation process of myeloid cells but are incapable of maintaining the leukemic phenotype. Results also demonstrated the hypermutability of 2F and 2H. Multiple fragile sites or radiation-sensitive sites, containing interstitial telomere-like repeated [(TTAGGG)n] se- A A A A A A A A B B -- B= B - B B B * B C C C C C - C C C ie E ie ie ie i... E ie... E i D H **... *... H ***... H **-**H H _**** H H 3 months 6 months 9 months 12 months * 15 months 18 months 21 months 24 months FIG. 1. Localization of breakpoints (-) on chromosome 2 during the development of radiation-induced ML. *, excluding one mouse with frank leukemia phenotype [all cells containing del2(d-e)].
1156 Medical Sciences: Rithidech et al. quences, on mouse chromosome 2 have been suggested (12). In human beings, such regions of a chromosome (e.g., 2qll- 14) are prone to recombination, breakage, and fragility (see ref. 13 for review). These findings suggest possible association of the fragile sites or a telomere-like sequence with the susceptibility to neoplastic transformation. Thus, regions 2F and 2H might well be associated with the early stages of myeloid transformation. An increase in chromosomal aberrations has been found in old animals and human beings (14, 15). Thus, it is not surprising that expansions of cells with abnormalities in different chromosomes were observed in samples sequentially collected from exposed mice after x-irradiation. These cells may be necessary for maintaining normal balances of selfrenewal as well as differentiation of the myeloid lineage. We thank Prof. P. C. Vincent, M.D. (Royal Prince Hospital, New South Wales, Australia) and Prof. J. D. Rowley (University of Chicago) for valuable discussions. This research was supported by the U.S. Department of Energy under Contract DE-AC2-76CH16. 1. Rithidech, K., Bond, V. P., Cronkite, E. P. & Thompson, M. H. (1993) Exp. Hematol. 21, 427-431. Proc. NatL Acad Sci. USA 92 (1995) 2. Hayata, I. (1983) in Radiation-Induced Chromosome Damage in Man, 1983, eds. Ishihara, T. & Sasaki, M. S. (Liss, New York), pp. 277-297. 3. Breckon, G., Silver, A. & Cox, R. (1988) Kew Chromosome Conference III (Her Majesty's Stationery Office, London), pp. 179-184. 4. Trakhtenbrot, L., Krauthgamer, R., Resnitzky, P. & Haran- Ghera, N. (1988) Leukemia 2, 545-55. 5. Major, I. & Mole, R. H. (1978) Nature (London) 272, 455-456. 6. Blatt, C. & Sachs, L. (1988) Biochem. Biophys. Res. Commun. 156, 1265-127. 7. Cronkite, E. P. & Bond, V. P. (1988) National Cancer Institute Rep. Contract Y1--1712. 8. Rowley, J. D. & Potter, D. (1976) Blood 47, 75-721. 9. Siracusa, L. D. & Abbott, C. M. (1992) Mamm. Genome 3, S2-S43. 1. Hinoda, Y., Itoh, H., Takahashi, T., Adachi, M., Tsujisaki, M., Imai, K. & Yachi, A. (1992) Int. J. Hematol. 56, 95-97. 11. Heim, S. & Mitelman, F. (1987) Cancer Cytogenetics (Liss, New York). 12. Breckon, G., Pappworth, D. & Cox, R. (1991) Genes Chromosomes Cancer 3, 367-375. 13. Hastie, N. D. & Allshire, R. C. (1989) Trends Genet. 5, 326-33. 14. Fenech, M. & Morley, A. (1985) Mutat. Res. 148, 99-15. 15. Hando, J. C., Nath, J. & Tucker, J. D. (1994) Chromosoma 13, 186-192.