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Preleukemic proliferative changes in murine bone marrow after single and multiple 7,12-dimethylbenz(a)anthracene (DMBA)-applications
Leukemia Research Vol. 17, No. 1, pp. 43 49, 1993. Printed in Great Britain.
0145 2t26/93 $6.00 + .00 © 1993 Pergamon Press Ltd
PRELEUKEMIC PROLIFERATIVE CHANGES IN MURINE BONE MARROW AFTER SINGLE AND MULTIPLE 7,12-DIMETHYLBENZ(a)ANTHRACENE (DMBA)-APPLICATIONS H.-P. PETERSON, A. FELDGES, K.-H. VON WANGENHEIM and L. E. FEINENDEGEN Institute of Medicine, Research Center Jiilich GmbH, Jiilich, F.R.G. (Received 2 July 1991. Revision accepted 9 September 1992)
Abstract--The effect of a leukemia-inducing treatment on early changes in kinetic parameters of murine bone marrow cells were investigated. Mice were treated i.p. one, four and eight times at biweekly intervals with 1 mg DMBA. Up to nine weeks after the last injection, CFU-S number, proliferation ability of bone marrow cells (PF), cell doubling time (td) and the compartment ratio (CR) were measured. Following multiple DMBA injections, CFU-S number and PF were decreased whereas CR and td increased, thus indicating persisting stem cell injury and proliferative compensation in the hemopoietic amplification compartment. A single DMBA injection had no effect. It is concluded that a first DMBA injection induces cytotoxic (and genotoxic) damage in the bone marrow leading simultaneously to a strong proliferation stimulus and a hindered proliferation ability of HSC, some of which will be predisposed for further mutagenic treatment. The following DMBA injections meet strongly proliferating HSCs, thus enhancing the probability for the loss of proliferation control/ terminal differentiation. Key words: Dimethylbenz(a)anthracene, DMBA, leukemogenesis, neoplasia, hematopoiesis, proliferation, stem cells, ionizing radiation.
INTRODUCTION
time course of a developing leukemia. Up to eight injections of 1 mg D M B A at biweekly intervals result in leukemias in rodents in almost 100% of the animals treated [3, 4]. In the present investigation mice were given this schedule of injections. In addition to the proliferation ability, other parameters such as the number of stem cells (CFUS 7d) and the relative contribution to early spleen repopulation by direct stem cell progeny and by more differentiated hemopoietic progeny (compartment ratio, CR) were determined. We found that multiple D M B A applications persistently reduced the number of HSC, weakened the proliferation ability of HSC and their progeny and increased the compartment of progenitor cells-----comparable to the effects of ionizing irradiation. We conclude that a multiple D M B A treatment induced unspecific genetic damage in HSC thus increasing the chance of mutating proliferation-controlling genes and thereby creating an early step in leukemogenesis: uncontrolled proliferation.
THE PROLIFERATION ability of murine hemopoietic stem cells (HSC) and their progeny can be me_asured by transplanting bone marrow cells of donor mice to lethally irradiated syngeneic recipient mice and measuring the increasing number of proliferating donor cells in the recipient spleens by incremental uptake of the 125-I-labelled thymidine analogue iododeoxyuridine (IUdR) [8]. Using this method, reduced proliferation ability--or 'stem cell q u a l i t y ' has been observed following sublethal gamma- and neutron-irradiation up to 1 yr post exposure [1, 2]. This long lasting effect has been explained by persisting genetic damage in surviving HSC. Since leukemogenesis is characterized by a growth--or proliferation--advantage of the progeny of the neoplastic clone, we asked whether and when proliferation changes might be measurable in the Abbreviations: CFU-S, colony forming unit spleen; CR, compartment ratio; DMBA, 7,12-dimethylbenz(a)anthracene; GM-CSF, granulocyte/macrophage-colony stimulating factor; HSC, hemopoietic stem cells; IUdR, 5(125-I)iodo-2-deoxyuridine; PF, proliferation factor; td, cell doubling time; SEM, standard error of the mean. Correspondence to: Dr Hans-Peter Peterson, Institute of Medicine, Research Center Jtilich, P.O. Box 1913, D5170 Jiilich, F.R.G.
M A T E R I A L S AND M E T H O D S Mice Female C57BL/6 mice, 10-12 weeks of age, were used both as donors (controls and DMBA treatment) and recip-
43
H.-P. PETERSONet al.
44
ients (measurement of effects). The mice were given food and acidified water ad libitum. Following eight Gy irradiation from a Cs-137 gamma source, the recipients used for the proliferation test received, in addition, 0.5 g neomycin, 0.5 g tetracycline and 25 g sucrose/litre drinking water to prevent infection, and two days prior to IUdR labeling 0.1% NaI to dilute the inorganic iodine pool [5].
DMBA application A special 15% fat emulsion with 7,12-dimethylbenz(a)anthracene (DMBA), 5 rag/g, was a gift of Prof. C. Huggins, Chicago and Dr K. Gries, the Upjohn GmbH, Heppenheim. Since i.v. injections caused irritation and obturation of the veins, thus preventing multiple injections, 0.2 ml emulsion (=1 mg DMBA) per mouse were injected intraperitoneally in groups of 6-14 mice per experiment. The controls were from the same batch of animals and sham injected with saline. Counts in the peripheral blood, preparation of cell suspensions and CFU-S determination At the time of sacrifice, as stated under results, blood was taken from tail veins. Leukocytes were counted by means of a Coulter Counter and blood smears were prepared for differential counts. The bone marrow was flushed from the femurs into ice cold Hank's solution and the cell numbers were determined by use of a Coulter Counter. For the CFU-S assay [6] and the proliferation test, bone marrow of at least three treated donors and saline injected controls was pooled respectively. For CFU-S determination 40,000 nucleated cells in (J.2 ml were injected into the tail vein of 10-15 recipients each, 2 h after irradiation. Spleen colonies larger than 0.25 mm in diameter were counted under a dissection microscope at day seven after transfusion. Day seven CFU-S were chosen because, according to experience with radiation studies, the effect of the treatment was more pronounced than with more primitive CFU-S [7]. The assay system for determination of proliferative ability of donor bone marrow and the compartment ratio According to published procedures [8], from controls and DMBA-treated mice bone marrow cell suspensions with 1.0, 1.5 and 2.25 x 10° nucleated cells were transfused into 12 lethally irradiated recipients each. Six mice from each group were i.p. injected with 185 kBq (5 ~tCi) 5(125-I) iodo-2-deoxyuridine (IUdR) at day three after cell transfusion, the other six mice were given the same injection at day five. Two hours later, their spleens were fixed and washed for 48 h in 4% formaldehyde and measured for incorporated activity. Day five counts were divided by day three counts to give a proliferation factor (PF) which indicates the relative increase in proliferating cells in the spleen within 48 h. From PF, a doubling time of cells (td) was calculated according to the formula [1, 81: In 2 ta = 48 h In P----~ The compartment ratio (CR) was calculated from the splenic IUdR-activity at day three or day five per transfused
CFU-S [21: cpm/spleen at day three (or day five) after marrow transfusion CR= number of CFU-S transfused
110
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10 15 20 Weeks offer first DMBA injection
I
I
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25
FIG. 1. Changes in body weight following increasing numbers of DMBA injections, % of controls. The arrows pointing to the abscissa refer to the DMBA injections. Vertical bars indicate + SEM when larger than the symbols drawn.
The CR thus indicates the size of the hemopoietic amplification compartment normalized to the size of the stem cell compartment transfused. The size of the latter was estimated by calculating the number of transfused CFU-S from parallel CFU-S assays without considering seeding efficiency. An increase in CR above 100% of control indicates an increased contribution from the amplification compartment to splenic repopulation.
Statistical methods Student's t-test was used. Since PF was independent of graft sizes within the cell numbers transfused [see 1], the results from the three transfusion groups per experiment were treated as independent repetitions following normalization to the corresponding average control value taken as 100%.
RESULTS
General response to D M B A treatment Body weight. Figure 1 shows the relative changes in b o d y weight in the course of time of the experiment. Following an initial decrease up to 5 weeks after the beginning of the D M B A injections, the b o d y weight increased again up to the 13th week, that is one week after the seventh D M B A injection. T h e reasons were irritations in the p e r i t o n e u m and c o n s e q u e n t develo p m e n t of ascites. With subsequent loss in weight there was an increasing death rate. A b o u t 50% of the D M B A treated mice died. Peripheral blood counts. Table 1 lists the changes in peripheral blood leukocytes. N o change was seen three weeks after one D M B A injection. O t h e r investigators have r e p o r t e d that peripheral blood values may partially recover with time after 1-4 D M B A injections [9, 10]. With eight D M B A injections the d a m a g e was quite obvious in that leukocyte n u m b e r s decreased to 60% of controls 9 weeks after the last
Cell proliferation in leukemogenesis
45
TABLE 1. LEUKOCYTES/MM 3 IN PERIPHERAL BLOOD X 10 -3
No. of DMBA injections
Weeks after last injection
1
3
8 8
3 9
Control
DMBA
% of control
8.5 +- 0.4 8.0 +- 0.2 4.7 --+0.1
8.3 -+ 0.3 6.2 -+ 0.7 2.8 -+ 0.3
97 78 60
Tables 1 to 6, means +- SEM *p < 0.05; **p < 0.01; ***p < 0.001
TABLE 2. NUCLEATED CELLS/FEMUR × 10 -6
No. of DMBA injections
Weeks after last injection
No. of experiments
Control
DMBA
% of control
1 1 1 4 8 8
3 9 15 3 3 9
4 1 1 3 3 1
23.3 -+ 2.8 19.9 24.9 22.5 +- 1.7 25.7 + 1.8 26.8
21.7 - 3.8 22.2 23.0 26.9 --- 2.7 25.1 + 2.1 32.2
92 111 92 120 99 120
injection, or 23 weeks after the beginning of the treatment. This data indicates a persisting injury in the hemopoietic system. Beginning two weeks after the fourth D M B A injection, a reduction in the ratio of lymphocytes to granuiocytes became apparent (data not shown). One week after the sixth D M B A application lymphocytes were reduced by 50% and about 20% immature myeloid precursors appeared. Three and eight weeks after eight D M B A injections lymphocytopenia persisted. Simultaneously the number of mature granulocytes decreased, whereas the immature myeloic cells became more prominent. Three weeks after the eighth D M B A injection one mouse developed a malignant non-Hodgkin lymphoma, classified as a lymphoblastic lymphoma. The animal showed splenomegaly (163mg), low body weight and increased lymphocyte count in the peripheral blood (28,000/mm3).
Effect of D M B A on the bone marrow Bone marrow cellularity. In spite of severe disturbances, as expressed by the changes in the peripheral blood, the cellularity of the bone marrow showed no significant change or hyperplasia at various times (Table 2). CFU-S concentration. The CFU-S concentration in the bone marrow was severely reduced after multiple DMBA injections (Table 3). At three weeks after the last of these injections the effects were similar, but increased with time to a reduction of CFU-S 7d
to 31% of controls. One D M B A injection, apparently, did not induce persistent damage. As is seen from Fig. 2, the femoral CFU-S content (open circles) shows comparable responses. Proliferative ability. In Fig. 2 the effect of D M B A treatment on the proliferative ability (PF, closed circles) and on CFU-S content per femur (open circles) is shown in percent of controls. In Table 4 the absolute values for PF are listed. While after one D M B A injection no significant effect occurred, multiple D M B A injections caused a significant reduction in PF and femoral CFU-S content that persisted after eight injections for at least three weeks. At all times of observation, the effect on PF was less pronounced compared to that on CFU-S per femur. This appears to express a recovery or compensatory mechanism regarding proliferation of CFU-S progeny, as it is seen from the response of CR. Cell doubling time. From PF the mean cell doubling time can be calculated as shown under Material and Methods. Cell doubling time (to) is prolonged at any time of observation after D M B A treatment; it approximates the severity of damage shown by the other parameters (Table 5). Apparently, the difference is larger with multiple doses of D M B A (1.01.3 h) as compared to a single dose (0.2-0.4 h). Radiation studies had previously shown [ 1, 11] that the prolongation of td was caused to a minor extent by a longer mitotic cycle and mainly by an increasing number of cells either leaving the spleen between
H.-P. PETERSONet al.
46
TABLE 3. CFU-S/106 NUCLEATEDCELLS No. of DMBA injections
Interval in weeks
No. of experiments
Control
DMBA
1
3
3
155 _+ 27
138 +_ 32
1
9
1
108
111
1 4 8 8
15 3 3 9
1 3 3 1
132 162 _+28 168 -+ 28 212
125 68 +- 4 99 --- 23 66
,
,
60
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1 x DMBA
• PF
o CFU-S / Femur I
0/r
I
I
I
I
I
I
3
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1tt
I
100 |
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6O ~0
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o
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5
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I
15
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20
25
DISCUSSION
Weeks offer first DHBA injection
FIG. 2. Changes in proliferative ability (closed circles) and CFU-S per femur (open circles), after a single (A), four (B) and eight DMBA injections (C), following various times of recovery after the last injection. The numerals next to the averages: number of experiments. No vertical bars: no determination of SEM. *: p < 0.05, **: p < 0.01.
day three and five probably due to differentiation a n d / o r experiencing cessation of cell proliferation. Compartment ratio. The changes in the comp a r t m e n t ratio (CR) are shown in Table 6. With a
TABLE 4.
88 103 95 44** 58* 31
reduction of CFU-S n u m b e r and PF, the CR, obtained at day three and five after m a r r o w transplantation, is always elevated following multiple D M B A injections. This indicates that the donor's insufficiency of the stem cell c o m p a r t m e n t is compensated by stimulation of cell proliferation in the hemopoietic amplification c o m p a r t m e n t as is also indicated by lack of persisting suppression of m a r r o w cellularity (Table 2). As a consequence, the PF is elevated due to cells from the amplification compartment seeding and dividing in the spleen after transplantation. In those instances where the C R of D M B A - t r e a t e d groups was significantly increased, the C R at day three (in % of controls) was always higher than that at day five.
100 / '
/,0 2o 1
% of control
Overt leukemia is characterized by increasing amounts of immature cells in bone m a r r o w and peripheral blood. This must be due to a loss of proliferative control and/or terminal differentiation ability in a target stem cell and its daughters leading to their growth advantage over normal cells. It was the aim of this investigation to look for early detectable effects on bone m a r r o w cell proliferation following single or multiple exposures to the leukemia-inducing agent D M B A . We used a sensitive test system which has been shown to detect early and
PROLIFERATION
FACTOR
(PF)
PF No. of DMBA injections
Weeks after last injection 3 9 15 3 3 9
No. of experiments
Control 25.7 + 2.8 22.2 +_ 6.0 28.3 -+ 1.5 18.0 _+ 1.3 19.4 _ 1.0 20.5 + 3.4
DMBA 22.5 21.6 24.2 14.1 15.0 15.5
+_ 1.8 -+ 2.9 + 0.7 ___2.2 +_ 0.9 _+ 1.7
% of control 88 97 86 78** 77* 76
Cell proliferation in leukemogenesis
47
TABLE 5. CELL DOUBLING TIME (td) (h) t d (h)
No. of DMBA injections
Weeks after last injection
No. of experiments
1 1 1
3 9
4 1 1
15 3 3 9
4 8 8
3 3 1
Control
DMBA
10.3 +- 0.3 1(I.7 -+ 0.9 10.0 -+ 0.2 11.5 -+ 0.3 11.3 -+ 0.3 11.0 -+ 0.6
10.7 -+ 0.2 10.9 -+ 0.5 10.4 -+ 0.1 12.8 +- 0.8 12.3 -+ 0.3 12.3 -+ 0.6
Increase in td (h) (1.4 0.2 (1.4
1.3 1.0 1.3
TABLE 6. COMPARTMENT RATIO (CR)
3d CR No. of DMBA injections
Interval in weeks
No. of experiments
1 1 1
3 9 15
3 1 1
4 8 8
3 3 9
3 3 1
Control 8.2 11.4 9.9 6.4 7.3 5.9
+ 2.8 + 7.1 -+ 0.8 +- 0.9 -+ 0.8 +- 0.2
late effects of D N A - d a m a g i n g treatments [13, 1, 2]. In order to avoid the immediate cytotoxic effects of D M B A [9, 10] the first tests were done three weeks after the (last) injection of D M B A . A single D M B A injection had no lasting effect on femoral CFU-S numbers and on proliferation ability (Fig. 2). Seidel [9] found a recovery of femoral CFU-S 9d numbers of 70 and 90% of control at one and three weeks after exposure. This corresponds to our data showing that the stem cell c o m p a r t m e n t can fully recover from a single D M B A dose within three weeks. A single dose to neonatal mice, however, reduced the CFU-S numbers for about eight weeks [26]. At three weeks after multiple doses of D M B A the stem cell c o m p a r t m e n t was still significantly reduced. Corresponding data have been reported by Seidel [9]. Following exposure to a different carcinogen, ionizing radiation, a reduction of self renewal of CFU-S at the advantage of the committed progenitor cells was measured [11, 12]. Since peripheral blood parameters and femoral cellularity showed no significant depression at three weeks after exposure, a compensating increased proliferation must have taken place. Similar to the reduced n u m b e r of stem cells, the quality of stem cells (and their progeny), measured as PF, was significantly reduced following multiple
DMBA 7.9 +_ 2.6 10.4 -+ 0.1 12.3 + 1.0 15.8 -+ 2.0 9.5 -+ 0.6 14.9 _+ 1.0
5d CR % 97 91 124 261"** 132"* 254*
Control
DMBA
187 -+ 35 232 +_ 26 280 + 27 112 + 15 143 -+ 12 122 _+_24
163 +- 26 224 _+ 29 298 + 27 213 + 20 143 +_ 9 228 +- 12
% 91 97 106 199"** 102 186"
D M B A injections but not following a single injection. Since PF can be transformed to the mean doubling time of the proliferating bone m a r r o w cells (see Materials and Methods), this means a prolongation of the mean cell doubling time. A similar effect on bone marrow cells was found after exposure to ionizing radiation [1, 2]. A reduced PF cannot be simply due to the reduced stem cell c o m p a r t m e n t since it has been shown earlier that PF is independent from the CFU-S n u m b e r transplanted within a relatively broad range [1]. One has to assume that a relatively enlarged compartment of more mature cells seed and proliferate in the transplanted spleens. This is reflected by the C R values, which have been taken as a measure for the relative c o m p a r t m e n t sizes of stem cell and progenitor cell c o m p a r t m e n t [2]. Following multiple D M B A doses, C R was always (with one exception, see Table 6) significantly elevated, whereas following a single dose no significant effect was observed. Thus, contrary to a single D M B A exposure, multiple exposures lead to a reduced pool size of HSC, a reduced HSC quality and an increased progenitor pool at three weeks after the last exposure and later on.
The significance of these D M B A induced changes in the kinetics of hemopoietic stem cell proliferation can be understood from radiation experiments:
48
H.-P. PETERSONet al.
approximately the same degree of persisting changes in these kinetic parameters was reached after recovery periods between 3 and 12 weeks following a single whole-body exposure of mice to ionizing irradiation [1, 2]. From these studies it was concluded that the irradiation induced unspecific genetic lesions in hemopoietic stem cells, which transmitted this damage to their progeny. Similar to ionizing radiation, D M B A is a potent inducer of chromosome aberrations [15-19]. For example, following a single injection of 50mg D M B A / k g in rats, 54% of cells in metaphase showed chromosome aberrations, at the average of 2.2 aberrations per cell [15]. Up to eight repetitions of this dose, as given in our experiments, must have left nearly no cell without genetic injury. These chromosome aberrations may explain the impediment of the cell proliferation observed. Multiple intravenous injections of D M B A lead to neoplasms of the hemopoietic system in 100% of all cases [20, 21], whereas a single injection produced only 6% [4]. This holds for adult animals. However, if applied to neonatal mice, a high incidence of leukemia can be observed after a single D M B A dose [26, 27]. Leukemic cells have been found as early as seven days after the last D M B A dose [22] and often the induced leukemias are manifest within 100 days after the first dose [4, 20, 23]. Within the period studied we observed one animal with a lymphoblastic lymphoma three weeks after the eighth D M B A injection. This type of leukemia is not surprising since thymic lymphomas/leukemias predominate in murine carcinogen-induced neoplasms [24]; in rats, D M B A induces leukemias of several different types, predominantly a stem-cell leukemia, associated with erythroblastosis [4]. Fohlmeister [10] induced erythroblastic leukemias in rats. It can therefore be assumed that the target cell for D M B A in rodents is an early HSC with a broad differentiation potential. In conclusion, D M B A induces leukemia by introducing D N A damage in hemopoietic cells predisposing some of them for the loss of differentiation control/terminal differentiation after additional injury. The reduced stem cell compartment and the hindered proliferation ability following multiple D M B A treatments will enforce a proliferation stimulus affecting all hemopoietic cells including the 'predisposed cells'. This promotion increases the probability of further mutagenic effects and thus the probability for the loss of proliferation control/ terminal differentiation. By this way a leukemic clone may arise whose cells will replace the normal population. The velocity with which this process will occur (and be detected), probably depends on the pro-
liferation control genes hit and the species/strainspecific activity of the immune defence system of the organism resulting in a rather wide spectrum of latency periods. This general scheme corresponds to the development of radiation-induced lymphomas in mice [25]. Acknowledgements--The investigations were supported by the Bundesamt ft~r Zivilschutz of the F.R.G. The authors are indebted to Prof. Dr C. B. Huggins, Chicago and Dr K. Gries, the Upjohn GmbH, Heppenheim, for the gift of DMBA emulsion, and Prof. Dr P. Pfitzer and Dr A. Schmitt-Graef, Institute for Cytopathology, University of Diisseldorf, for cytological diagnosis of the lymphoblastic lymphome.
REFERENCES 1. Wangenheim K.-H. v., Peterson H.-P. & Feinendegen L. E. (1986) Residual radiation effect in the murine hematopoietic stem cell compartment. Radiat. Environ. Biophys. 25, 93. 2. Peterson H.-P., Wangenheim K.-H. v. & Feinendegen L. E. (1989) Early and late effects in the bone marrow of mice following 2 Gy (6 MeV) neutron irradiation. Radiat. Environ. Biophys. 28, 291. 3. Huggins C. (1991) Personal communication. 4. Huggins C. B. & Sugiyama T. (1966) Induction of leukemia in rat by pulse doses of 7,12-dimethylbenz(a)anthracene. Proc. natn. Acad. Sci. U.S.A. 55, 74. 5. Hughes W. L., Commefford S. L., Gitlin P., Krueger R. C., Schultze B., Shah V. & Reilly P. (1965) Deoxyribonucleic acid metabolism in vivo: I. Cell proliferation and death as measured by incorporation and elimination of iododeoxyuridine. Fed. Proc. 23, 640. 6. Till J. E. & McCulloch E. A. (1961) A direct measurement of the radiation sensitivity of normal bone marrow cells. Radiat. Res. 14, 213. 7. Peterson H.-P., Wangenheim K.-H. v. & Feinendegen L. E. (1986) Persisting radiation effect on murine 7day and 12-day CFU-S. Expl Hemat. 14, 776. 8. Hiibner G. E.,Wangenheim K.-H. v. & Feinendegen L. E. (1981) An assay for the measurement of residual damage of murine hematopoietic stem cells. Expl Hemat. 9, 111.
9. Seidel H. J. (1977) The effect of dimethylbenz(a)anthracene on murine hematopoiesis. Z. Krebsforsch. 89, 221. 10. Fohlmeister I., Hohentanner O. & Porr A. (1986) Recovery patterns of rat hemopoietic stem cells between pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA) applied in a leukemogenic regimen. J. Cancer Res. clin. Oncol. 111,237. 11. Wangenheim, K.-H. v., Peterson H.-P. & Feinendegen L. E. (1990) Radiation sensitivity on the hemopoietic system. In The Hemopoietic Stem Cell. Seidel H. J. (Ed.), p. 143. Universitfitsverlag, Ulm. 12. Wangenheim, K.-H. v., Peterson H.-P., Cronkite E. P. & Feinendegen L. E. (1987) 5-Fluorouracil treatment after irradiation impairs recovery of bone marrow functions. Radiat. Environ. Biophys. 26, 163.
Cell proliferation in leukemogenesis 13. Hiibner G. E. & Cronkite E. P. (1982) Drug-induced residual damage of murine hematopoietic stem cells measured by a new assay. Leukemia Res. 6, 815. 14. Wangenheim K.-H. v., Cronkite E. P., Peterson H.-P., Hiibner G. E. & Feinendegen L. E. (1987) Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors. Leukemia Res. 11,345. 15. Ito Y., Ueda N., Maeda S., Murao S., Sugiyama T., Lee H. & Harvey R. G. (1988) Induction of chromosomal aberrations in rat bone marrow cells and mutations in Salmonella typhimurium by benz[a] anthracene derivatives. Mutation Res. 206, 55. 16. Falzon M., Vu V. T., Roller P. P. & Thorgeirsson S. S. (1987) Relationship between 7,12-dimethyl- and 7,8,12-trimethylbenz(a)anthracene DNA adduct formation in hematopoietic organs and leukemogenic effects. Cancer Letts 37, 41. 17. Sugiyama T., Kurita Y. & Nishizuka Y. (1969) Biologic studies on 7,12-dimethylbenz(a)anthracene-induced rat leukemia with special reference to the specific chromosomal abnormalities. Cancer Res. 29, 1117. 18. Sugiyama T. (1971) Specific vulnerability of the largest telocentric chromosome of rat bone marrow cells to 7,12-dimethylbenz(a)anthracene. J. Natl Cancer Inst. 47, 1267. 19. Wiener F., Spira J., Ohno S., Haran-Ghera N. & Klein G. (1978) Chromosome changes (trisomy 15) in murine T-cell leukemia induced by 7,12 dimethylbenz(a)anthracene. Int. J. Cancer 22, 447. 20. Huggins C. B., Grand L. & Ueda N. (1982) Specific
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induction of erythroleukemia and myelogenous leukemia in Sprague Dawley rats. Proc. natn. Acad. Sci. U.S.A. 79, 5411. Uematsu K. (1981) Early changes of hematopoietic tissues in mice treated with intravenous pulse-doses of 7,12-dimethylbenz(a)anthracene. Acta Path. Jpn 31, 799. Haran-Ghera N., Ben-Yaakov M., Chazan R. & Peled A. (1975) Pathways in thymus- and bone marrowderived lymphatic leukemia in mice. Bibl. Haemat. 40, 133. Huggins C., Grand L. & Oka H. (1970) Hundred day leukemia: preferential induction in rat by pulse-doses of 7,8,12-Trimethylbenz(a)anthracene. J. exp. Med. 131,321. Furmanski P. & Rich A. M. (1982) Neoplasms of the hematopoietic system. In The Mouse in Biomedical Research, Foster H. L., Small J. D. & Fox J. G. (Eds), Vol IV, pp. 352-371. Boniver J., Humblet C., Rongy A. M., Delvenne C., Delvenne P., Greimers R., Thiry A., Courtoy R & Defresne M. P. (199(I) Cellular events in radiationinduced lymphomagenesis. Int. J. Radiat. Biol. 57,693. Ball J. K., Hoshino S. & McCarter J. A. (1973) Depressive effect of 7,12-Dimethylbenz(a)anthracene and ionizing radiation on bone marrow colony-forming cells. J. Natl Cancer Inst. 51, 1491. Nishizuka Y. & Shisa H. (1968) Enhancement of 7,12dimethylbenz(a)anthracene leukemogenesis in mice by neonatal injection of cortisone acetate. Br. J. Cancer 22, 29(/.
0145 2t26/93 $6.00 + .00 © 1993 Pergamon Press Ltd
PRELEUKEMIC PROLIFERATIVE CHANGES IN MURINE BONE MARROW AFTER SINGLE AND MULTIPLE 7,12-DIMETHYLBENZ(a)ANTHRACENE (DMBA)-APPLICATIONS H.-P. PETERSON, A. FELDGES, K.-H. VON WANGENHEIM and L. E. FEINENDEGEN Institute of Medicine, Research Center Jiilich GmbH, Jiilich, F.R.G. (Received 2 July 1991. Revision accepted 9 September 1992)
Abstract--The effect of a leukemia-inducing treatment on early changes in kinetic parameters of murine bone marrow cells were investigated. Mice were treated i.p. one, four and eight times at biweekly intervals with 1 mg DMBA. Up to nine weeks after the last injection, CFU-S number, proliferation ability of bone marrow cells (PF), cell doubling time (td) and the compartment ratio (CR) were measured. Following multiple DMBA injections, CFU-S number and PF were decreased whereas CR and td increased, thus indicating persisting stem cell injury and proliferative compensation in the hemopoietic amplification compartment. A single DMBA injection had no effect. It is concluded that a first DMBA injection induces cytotoxic (and genotoxic) damage in the bone marrow leading simultaneously to a strong proliferation stimulus and a hindered proliferation ability of HSC, some of which will be predisposed for further mutagenic treatment. The following DMBA injections meet strongly proliferating HSCs, thus enhancing the probability for the loss of proliferation control/ terminal differentiation. Key words: Dimethylbenz(a)anthracene, DMBA, leukemogenesis, neoplasia, hematopoiesis, proliferation, stem cells, ionizing radiation.
INTRODUCTION
time course of a developing leukemia. Up to eight injections of 1 mg D M B A at biweekly intervals result in leukemias in rodents in almost 100% of the animals treated [3, 4]. In the present investigation mice were given this schedule of injections. In addition to the proliferation ability, other parameters such as the number of stem cells (CFUS 7d) and the relative contribution to early spleen repopulation by direct stem cell progeny and by more differentiated hemopoietic progeny (compartment ratio, CR) were determined. We found that multiple D M B A applications persistently reduced the number of HSC, weakened the proliferation ability of HSC and their progeny and increased the compartment of progenitor cells-----comparable to the effects of ionizing irradiation. We conclude that a multiple D M B A treatment induced unspecific genetic damage in HSC thus increasing the chance of mutating proliferation-controlling genes and thereby creating an early step in leukemogenesis: uncontrolled proliferation.
THE PROLIFERATION ability of murine hemopoietic stem cells (HSC) and their progeny can be me_asured by transplanting bone marrow cells of donor mice to lethally irradiated syngeneic recipient mice and measuring the increasing number of proliferating donor cells in the recipient spleens by incremental uptake of the 125-I-labelled thymidine analogue iododeoxyuridine (IUdR) [8]. Using this method, reduced proliferation ability--or 'stem cell q u a l i t y ' has been observed following sublethal gamma- and neutron-irradiation up to 1 yr post exposure [1, 2]. This long lasting effect has been explained by persisting genetic damage in surviving HSC. Since leukemogenesis is characterized by a growth--or proliferation--advantage of the progeny of the neoplastic clone, we asked whether and when proliferation changes might be measurable in the Abbreviations: CFU-S, colony forming unit spleen; CR, compartment ratio; DMBA, 7,12-dimethylbenz(a)anthracene; GM-CSF, granulocyte/macrophage-colony stimulating factor; HSC, hemopoietic stem cells; IUdR, 5(125-I)iodo-2-deoxyuridine; PF, proliferation factor; td, cell doubling time; SEM, standard error of the mean. Correspondence to: Dr Hans-Peter Peterson, Institute of Medicine, Research Center Jtilich, P.O. Box 1913, D5170 Jiilich, F.R.G.
M A T E R I A L S AND M E T H O D S Mice Female C57BL/6 mice, 10-12 weeks of age, were used both as donors (controls and DMBA treatment) and recip-
43
H.-P. PETERSONet al.
44
ients (measurement of effects). The mice were given food and acidified water ad libitum. Following eight Gy irradiation from a Cs-137 gamma source, the recipients used for the proliferation test received, in addition, 0.5 g neomycin, 0.5 g tetracycline and 25 g sucrose/litre drinking water to prevent infection, and two days prior to IUdR labeling 0.1% NaI to dilute the inorganic iodine pool [5].
DMBA application A special 15% fat emulsion with 7,12-dimethylbenz(a)anthracene (DMBA), 5 rag/g, was a gift of Prof. C. Huggins, Chicago and Dr K. Gries, the Upjohn GmbH, Heppenheim. Since i.v. injections caused irritation and obturation of the veins, thus preventing multiple injections, 0.2 ml emulsion (=1 mg DMBA) per mouse were injected intraperitoneally in groups of 6-14 mice per experiment. The controls were from the same batch of animals and sham injected with saline. Counts in the peripheral blood, preparation of cell suspensions and CFU-S determination At the time of sacrifice, as stated under results, blood was taken from tail veins. Leukocytes were counted by means of a Coulter Counter and blood smears were prepared for differential counts. The bone marrow was flushed from the femurs into ice cold Hank's solution and the cell numbers were determined by use of a Coulter Counter. For the CFU-S assay [6] and the proliferation test, bone marrow of at least three treated donors and saline injected controls was pooled respectively. For CFU-S determination 40,000 nucleated cells in (J.2 ml were injected into the tail vein of 10-15 recipients each, 2 h after irradiation. Spleen colonies larger than 0.25 mm in diameter were counted under a dissection microscope at day seven after transfusion. Day seven CFU-S were chosen because, according to experience with radiation studies, the effect of the treatment was more pronounced than with more primitive CFU-S [7]. The assay system for determination of proliferative ability of donor bone marrow and the compartment ratio According to published procedures [8], from controls and DMBA-treated mice bone marrow cell suspensions with 1.0, 1.5 and 2.25 x 10° nucleated cells were transfused into 12 lethally irradiated recipients each. Six mice from each group were i.p. injected with 185 kBq (5 ~tCi) 5(125-I) iodo-2-deoxyuridine (IUdR) at day three after cell transfusion, the other six mice were given the same injection at day five. Two hours later, their spleens were fixed and washed for 48 h in 4% formaldehyde and measured for incorporated activity. Day five counts were divided by day three counts to give a proliferation factor (PF) which indicates the relative increase in proliferating cells in the spleen within 48 h. From PF, a doubling time of cells (td) was calculated according to the formula [1, 81: In 2 ta = 48 h In P----~ The compartment ratio (CR) was calculated from the splenic IUdR-activity at day three or day five per transfused
CFU-S [21: cpm/spleen at day three (or day five) after marrow transfusion CR= number of CFU-S transfused
110
100 -~ 9o
g
\v,,t
Bo 7o OMBA -injec lions 3 4 5 6
2
I !!! i
i
i
5
i
!! l
7 i
!!
B
t
I
I
I
[
L
I
10 15 20 Weeks offer first DMBA injection
I
I
I
I
l
25
FIG. 1. Changes in body weight following increasing numbers of DMBA injections, % of controls. The arrows pointing to the abscissa refer to the DMBA injections. Vertical bars indicate + SEM when larger than the symbols drawn.
The CR thus indicates the size of the hemopoietic amplification compartment normalized to the size of the stem cell compartment transfused. The size of the latter was estimated by calculating the number of transfused CFU-S from parallel CFU-S assays without considering seeding efficiency. An increase in CR above 100% of control indicates an increased contribution from the amplification compartment to splenic repopulation.
Statistical methods Student's t-test was used. Since PF was independent of graft sizes within the cell numbers transfused [see 1], the results from the three transfusion groups per experiment were treated as independent repetitions following normalization to the corresponding average control value taken as 100%.
RESULTS
General response to D M B A treatment Body weight. Figure 1 shows the relative changes in b o d y weight in the course of time of the experiment. Following an initial decrease up to 5 weeks after the beginning of the D M B A injections, the b o d y weight increased again up to the 13th week, that is one week after the seventh D M B A injection. T h e reasons were irritations in the p e r i t o n e u m and c o n s e q u e n t develo p m e n t of ascites. With subsequent loss in weight there was an increasing death rate. A b o u t 50% of the D M B A treated mice died. Peripheral blood counts. Table 1 lists the changes in peripheral blood leukocytes. N o change was seen three weeks after one D M B A injection. O t h e r investigators have r e p o r t e d that peripheral blood values may partially recover with time after 1-4 D M B A injections [9, 10]. With eight D M B A injections the d a m a g e was quite obvious in that leukocyte n u m b e r s decreased to 60% of controls 9 weeks after the last
Cell proliferation in leukemogenesis
45
TABLE 1. LEUKOCYTES/MM 3 IN PERIPHERAL BLOOD X 10 -3
No. of DMBA injections
Weeks after last injection
1
3
8 8
3 9
Control
DMBA
% of control
8.5 +- 0.4 8.0 +- 0.2 4.7 --+0.1
8.3 -+ 0.3 6.2 -+ 0.7 2.8 -+ 0.3
97 78 60
Tables 1 to 6, means +- SEM *p < 0.05; **p < 0.01; ***p < 0.001
TABLE 2. NUCLEATED CELLS/FEMUR × 10 -6
No. of DMBA injections
Weeks after last injection
No. of experiments
Control
DMBA
% of control
1 1 1 4 8 8
3 9 15 3 3 9
4 1 1 3 3 1
23.3 -+ 2.8 19.9 24.9 22.5 +- 1.7 25.7 + 1.8 26.8
21.7 - 3.8 22.2 23.0 26.9 --- 2.7 25.1 + 2.1 32.2
92 111 92 120 99 120
injection, or 23 weeks after the beginning of the treatment. This data indicates a persisting injury in the hemopoietic system. Beginning two weeks after the fourth D M B A injection, a reduction in the ratio of lymphocytes to granuiocytes became apparent (data not shown). One week after the sixth D M B A application lymphocytes were reduced by 50% and about 20% immature myeloid precursors appeared. Three and eight weeks after eight D M B A injections lymphocytopenia persisted. Simultaneously the number of mature granulocytes decreased, whereas the immature myeloic cells became more prominent. Three weeks after the eighth D M B A injection one mouse developed a malignant non-Hodgkin lymphoma, classified as a lymphoblastic lymphoma. The animal showed splenomegaly (163mg), low body weight and increased lymphocyte count in the peripheral blood (28,000/mm3).
Effect of D M B A on the bone marrow Bone marrow cellularity. In spite of severe disturbances, as expressed by the changes in the peripheral blood, the cellularity of the bone marrow showed no significant change or hyperplasia at various times (Table 2). CFU-S concentration. The CFU-S concentration in the bone marrow was severely reduced after multiple DMBA injections (Table 3). At three weeks after the last of these injections the effects were similar, but increased with time to a reduction of CFU-S 7d
to 31% of controls. One D M B A injection, apparently, did not induce persistent damage. As is seen from Fig. 2, the femoral CFU-S content (open circles) shows comparable responses. Proliferative ability. In Fig. 2 the effect of D M B A treatment on the proliferative ability (PF, closed circles) and on CFU-S content per femur (open circles) is shown in percent of controls. In Table 4 the absolute values for PF are listed. While after one D M B A injection no significant effect occurred, multiple D M B A injections caused a significant reduction in PF and femoral CFU-S content that persisted after eight injections for at least three weeks. At all times of observation, the effect on PF was less pronounced compared to that on CFU-S per femur. This appears to express a recovery or compensatory mechanism regarding proliferation of CFU-S progeny, as it is seen from the response of CR. Cell doubling time. From PF the mean cell doubling time can be calculated as shown under Material and Methods. Cell doubling time (to) is prolonged at any time of observation after D M B A treatment; it approximates the severity of damage shown by the other parameters (Table 5). Apparently, the difference is larger with multiple doses of D M B A (1.01.3 h) as compared to a single dose (0.2-0.4 h). Radiation studies had previously shown [ 1, 11] that the prolongation of td was caused to a minor extent by a longer mitotic cycle and mainly by an increasing number of cells either leaving the spleen between
H.-P. PETERSONet al.
46
TABLE 3. CFU-S/106 NUCLEATEDCELLS No. of DMBA injections
Interval in weeks
No. of experiments
Control
DMBA
1
3
3
155 _+ 27
138 +_ 32
1
9
1
108
111
1 4 8 8
15 3 3 9
1 3 3 1
132 162 _+28 168 -+ 28 212
125 68 +- 4 99 --- 23 66
,
,
60
'°I
1 x DMBA
• PF
o CFU-S / Femur I
0/r
I
I
I
I
I
I
3
/,o
x D['l BA
1tt
I
100 |
3{'--.----
6O ~0
2o ! o
o
I t lI
5
1 1 It l 10
I
15
I
20
25
DISCUSSION
Weeks offer first DHBA injection
FIG. 2. Changes in proliferative ability (closed circles) and CFU-S per femur (open circles), after a single (A), four (B) and eight DMBA injections (C), following various times of recovery after the last injection. The numerals next to the averages: number of experiments. No vertical bars: no determination of SEM. *: p < 0.05, **: p < 0.01.
day three and five probably due to differentiation a n d / o r experiencing cessation of cell proliferation. Compartment ratio. The changes in the comp a r t m e n t ratio (CR) are shown in Table 6. With a
TABLE 4.
88 103 95 44** 58* 31
reduction of CFU-S n u m b e r and PF, the CR, obtained at day three and five after m a r r o w transplantation, is always elevated following multiple D M B A injections. This indicates that the donor's insufficiency of the stem cell c o m p a r t m e n t is compensated by stimulation of cell proliferation in the hemopoietic amplification c o m p a r t m e n t as is also indicated by lack of persisting suppression of m a r r o w cellularity (Table 2). As a consequence, the PF is elevated due to cells from the amplification compartment seeding and dividing in the spleen after transplantation. In those instances where the C R of D M B A - t r e a t e d groups was significantly increased, the C R at day three (in % of controls) was always higher than that at day five.
100 / '
/,0 2o 1
% of control
Overt leukemia is characterized by increasing amounts of immature cells in bone m a r r o w and peripheral blood. This must be due to a loss of proliferative control and/or terminal differentiation ability in a target stem cell and its daughters leading to their growth advantage over normal cells. It was the aim of this investigation to look for early detectable effects on bone m a r r o w cell proliferation following single or multiple exposures to the leukemia-inducing agent D M B A . We used a sensitive test system which has been shown to detect early and
PROLIFERATION
FACTOR
(PF)
PF No. of DMBA injections
Weeks after last injection 3 9 15 3 3 9
No. of experiments
Control 25.7 + 2.8 22.2 +_ 6.0 28.3 -+ 1.5 18.0 _+ 1.3 19.4 _ 1.0 20.5 + 3.4
DMBA 22.5 21.6 24.2 14.1 15.0 15.5
+_ 1.8 -+ 2.9 + 0.7 ___2.2 +_ 0.9 _+ 1.7
% of control 88 97 86 78** 77* 76
Cell proliferation in leukemogenesis
47
TABLE 5. CELL DOUBLING TIME (td) (h) t d (h)
No. of DMBA injections
Weeks after last injection
No. of experiments
1 1 1
3 9
4 1 1
15 3 3 9
4 8 8
3 3 1
Control
DMBA
10.3 +- 0.3 1(I.7 -+ 0.9 10.0 -+ 0.2 11.5 -+ 0.3 11.3 -+ 0.3 11.0 -+ 0.6
10.7 -+ 0.2 10.9 -+ 0.5 10.4 -+ 0.1 12.8 +- 0.8 12.3 -+ 0.3 12.3 -+ 0.6
Increase in td (h) (1.4 0.2 (1.4
1.3 1.0 1.3
TABLE 6. COMPARTMENT RATIO (CR)
3d CR No. of DMBA injections
Interval in weeks
No. of experiments
1 1 1
3 9 15
3 1 1
4 8 8
3 3 9
3 3 1
Control 8.2 11.4 9.9 6.4 7.3 5.9
+ 2.8 + 7.1 -+ 0.8 +- 0.9 -+ 0.8 +- 0.2
late effects of D N A - d a m a g i n g treatments [13, 1, 2]. In order to avoid the immediate cytotoxic effects of D M B A [9, 10] the first tests were done three weeks after the (last) injection of D M B A . A single D M B A injection had no lasting effect on femoral CFU-S numbers and on proliferation ability (Fig. 2). Seidel [9] found a recovery of femoral CFU-S 9d numbers of 70 and 90% of control at one and three weeks after exposure. This corresponds to our data showing that the stem cell c o m p a r t m e n t can fully recover from a single D M B A dose within three weeks. A single dose to neonatal mice, however, reduced the CFU-S numbers for about eight weeks [26]. At three weeks after multiple doses of D M B A the stem cell c o m p a r t m e n t was still significantly reduced. Corresponding data have been reported by Seidel [9]. Following exposure to a different carcinogen, ionizing radiation, a reduction of self renewal of CFU-S at the advantage of the committed progenitor cells was measured [11, 12]. Since peripheral blood parameters and femoral cellularity showed no significant depression at three weeks after exposure, a compensating increased proliferation must have taken place. Similar to the reduced n u m b e r of stem cells, the quality of stem cells (and their progeny), measured as PF, was significantly reduced following multiple
DMBA 7.9 +_ 2.6 10.4 -+ 0.1 12.3 + 1.0 15.8 -+ 2.0 9.5 -+ 0.6 14.9 _+ 1.0
5d CR % 97 91 124 261"** 132"* 254*
Control
DMBA
187 -+ 35 232 +_ 26 280 + 27 112 + 15 143 -+ 12 122 _+_24
163 +- 26 224 _+ 29 298 + 27 213 + 20 143 +_ 9 228 +- 12
% 91 97 106 199"** 102 186"
D M B A injections but not following a single injection. Since PF can be transformed to the mean doubling time of the proliferating bone m a r r o w cells (see Materials and Methods), this means a prolongation of the mean cell doubling time. A similar effect on bone marrow cells was found after exposure to ionizing radiation [1, 2]. A reduced PF cannot be simply due to the reduced stem cell c o m p a r t m e n t since it has been shown earlier that PF is independent from the CFU-S n u m b e r transplanted within a relatively broad range [1]. One has to assume that a relatively enlarged compartment of more mature cells seed and proliferate in the transplanted spleens. This is reflected by the C R values, which have been taken as a measure for the relative c o m p a r t m e n t sizes of stem cell and progenitor cell c o m p a r t m e n t [2]. Following multiple D M B A doses, C R was always (with one exception, see Table 6) significantly elevated, whereas following a single dose no significant effect was observed. Thus, contrary to a single D M B A exposure, multiple exposures lead to a reduced pool size of HSC, a reduced HSC quality and an increased progenitor pool at three weeks after the last exposure and later on.
The significance of these D M B A induced changes in the kinetics of hemopoietic stem cell proliferation can be understood from radiation experiments:
48
H.-P. PETERSONet al.
approximately the same degree of persisting changes in these kinetic parameters was reached after recovery periods between 3 and 12 weeks following a single whole-body exposure of mice to ionizing irradiation [1, 2]. From these studies it was concluded that the irradiation induced unspecific genetic lesions in hemopoietic stem cells, which transmitted this damage to their progeny. Similar to ionizing radiation, D M B A is a potent inducer of chromosome aberrations [15-19]. For example, following a single injection of 50mg D M B A / k g in rats, 54% of cells in metaphase showed chromosome aberrations, at the average of 2.2 aberrations per cell [15]. Up to eight repetitions of this dose, as given in our experiments, must have left nearly no cell without genetic injury. These chromosome aberrations may explain the impediment of the cell proliferation observed. Multiple intravenous injections of D M B A lead to neoplasms of the hemopoietic system in 100% of all cases [20, 21], whereas a single injection produced only 6% [4]. This holds for adult animals. However, if applied to neonatal mice, a high incidence of leukemia can be observed after a single D M B A dose [26, 27]. Leukemic cells have been found as early as seven days after the last D M B A dose [22] and often the induced leukemias are manifest within 100 days after the first dose [4, 20, 23]. Within the period studied we observed one animal with a lymphoblastic lymphoma three weeks after the eighth D M B A injection. This type of leukemia is not surprising since thymic lymphomas/leukemias predominate in murine carcinogen-induced neoplasms [24]; in rats, D M B A induces leukemias of several different types, predominantly a stem-cell leukemia, associated with erythroblastosis [4]. Fohlmeister [10] induced erythroblastic leukemias in rats. It can therefore be assumed that the target cell for D M B A in rodents is an early HSC with a broad differentiation potential. In conclusion, D M B A induces leukemia by introducing D N A damage in hemopoietic cells predisposing some of them for the loss of differentiation control/terminal differentiation after additional injury. The reduced stem cell compartment and the hindered proliferation ability following multiple D M B A treatments will enforce a proliferation stimulus affecting all hemopoietic cells including the 'predisposed cells'. This promotion increases the probability of further mutagenic effects and thus the probability for the loss of proliferation control/ terminal differentiation. By this way a leukemic clone may arise whose cells will replace the normal population. The velocity with which this process will occur (and be detected), probably depends on the pro-
liferation control genes hit and the species/strainspecific activity of the immune defence system of the organism resulting in a rather wide spectrum of latency periods. This general scheme corresponds to the development of radiation-induced lymphomas in mice [25]. Acknowledgements--The investigations were supported by the Bundesamt ft~r Zivilschutz of the F.R.G. The authors are indebted to Prof. Dr C. B. Huggins, Chicago and Dr K. Gries, the Upjohn GmbH, Heppenheim, for the gift of DMBA emulsion, and Prof. Dr P. Pfitzer and Dr A. Schmitt-Graef, Institute for Cytopathology, University of Diisseldorf, for cytological diagnosis of the lymphoblastic lymphome.
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Cell proliferation in leukemogenesis 13. Hiibner G. E. & Cronkite E. P. (1982) Drug-induced residual damage of murine hematopoietic stem cells measured by a new assay. Leukemia Res. 6, 815. 14. Wangenheim K.-H. v., Cronkite E. P., Peterson H.-P., Hiibner G. E. & Feinendegen L. E. (1987) Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors. Leukemia Res. 11,345. 15. Ito Y., Ueda N., Maeda S., Murao S., Sugiyama T., Lee H. & Harvey R. G. (1988) Induction of chromosomal aberrations in rat bone marrow cells and mutations in Salmonella typhimurium by benz[a] anthracene derivatives. Mutation Res. 206, 55. 16. Falzon M., Vu V. T., Roller P. P. & Thorgeirsson S. S. (1987) Relationship between 7,12-dimethyl- and 7,8,12-trimethylbenz(a)anthracene DNA adduct formation in hematopoietic organs and leukemogenic effects. Cancer Letts 37, 41. 17. Sugiyama T., Kurita Y. & Nishizuka Y. (1969) Biologic studies on 7,12-dimethylbenz(a)anthracene-induced rat leukemia with special reference to the specific chromosomal abnormalities. Cancer Res. 29, 1117. 18. Sugiyama T. (1971) Specific vulnerability of the largest telocentric chromosome of rat bone marrow cells to 7,12-dimethylbenz(a)anthracene. J. Natl Cancer Inst. 47, 1267. 19. Wiener F., Spira J., Ohno S., Haran-Ghera N. & Klein G. (1978) Chromosome changes (trisomy 15) in murine T-cell leukemia induced by 7,12 dimethylbenz(a)anthracene. Int. J. Cancer 22, 447. 20. Huggins C. B., Grand L. & Ueda N. (1982) Specific
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induction of erythroleukemia and myelogenous leukemia in Sprague Dawley rats. Proc. natn. Acad. Sci. U.S.A. 79, 5411. Uematsu K. (1981) Early changes of hematopoietic tissues in mice treated with intravenous pulse-doses of 7,12-dimethylbenz(a)anthracene. Acta Path. Jpn 31, 799. Haran-Ghera N., Ben-Yaakov M., Chazan R. & Peled A. (1975) Pathways in thymus- and bone marrowderived lymphatic leukemia in mice. Bibl. Haemat. 40, 133. Huggins C., Grand L. & Oka H. (1970) Hundred day leukemia: preferential induction in rat by pulse-doses of 7,8,12-Trimethylbenz(a)anthracene. J. exp. Med. 131,321. Furmanski P. & Rich A. M. (1982) Neoplasms of the hematopoietic system. In The Mouse in Biomedical Research, Foster H. L., Small J. D. & Fox J. G. (Eds), Vol IV, pp. 352-371. Boniver J., Humblet C., Rongy A. M., Delvenne C., Delvenne P., Greimers R., Thiry A., Courtoy R & Defresne M. P. (199(I) Cellular events in radiationinduced lymphomagenesis. Int. J. Radiat. Biol. 57,693. Ball J. K., Hoshino S. & McCarter J. A. (1973) Depressive effect of 7,12-Dimethylbenz(a)anthracene and ionizing radiation on bone marrow colony-forming cells. J. Natl Cancer Inst. 51, 1491. Nishizuka Y. & Shisa H. (1968) Enhancement of 7,12dimethylbenz(a)anthracene leukemogenesis in mice by neonatal injection of cortisone acetate. Br. J. Cancer 22, 29(/.