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Exacerbating factors of radiation-induced myeloid leukemogenesis
Leukemia Research Vol. 17, No. 5, pp. 437-440, 1993. Printed in Great Britain.
0145-2126/93 $6.00 + .00 © 1993 Pergamon Press Ltd
E X A C E R B A T I N G FACTORS OF R A D I A T I O N - I N D U C E D M Y E L O I D LEUKEMOGENESIS KAZUKO YOSHIDA, KUMIE NEMOTO, MAYUMI NISHIMURAand MASATOSHI SEKI Division of Physiology and Pathology, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku Chiba-shi Chiba, 263 Japan (Received 6 August 1992. Revision accepted 19 December 1992)
Abstract--The spontaneous incidence of myeloid leukemia in female mice was slightly higher than in male mice, whereas the radiation-induced incidence was significantlylower than in male mice. We also examined whether the incidence of myeloid leukemia was related to inflammatory response. Mice had a piece of cellulose acetate membrane inserted into the peritoneal cavity to cause inflammation. This did not affect the incidence of myeloid leukemia in unirradiated mice at all, but in 2.84 Gy irradiated mice the incidence (35.9% in male, 26.0% in female mice) increased significantlycompared with irradiated-only mice (23.9% and 12.0%, respectively). From these results, the physiological fluctuation of humoral factors by means of inflammatory response is considered to increase the development of radiation-induced myeloid leukemia. Key words: Myeloid leukemia, inflammatory response, tissue injury, sex difference, exacerbating factors, radiation.
not inflammatory reaction would act as a promoter of radiation-induced leukemogenesis. When a piece of cellulose acetate membrane (CAM) was inserted into the peritoneal cavity of mice, CAM became covered by macrophage-fibroblast cell layers because of inflammatory reaction [5]. Such cell layers could act as an artificial hematopoietic microenvironment. Colonies consisting of hematopoietic progenitor cells were formed on the macrophage-fibroblast cell layers when bone marrow cells were transplanted into the peritoneal cavity of CAM-inserted mice [5, 6]. We used this system to induce inflammatory reaction, and the mice were then exposed to about 3 Gy of irradiation, the most effective dose for leukemogenesis. It is well known that androgens can stimulate hematopoiesis. In addition, they can also increase the number of hematopoietic stem cells [6-8]. We therefore tried to determine whether the incidence of radiation-induced myeloid leukemia differed between female and male mice.
INTRODUCTION WE HAVEreported that the spontaneous incidence of myeloid leukemia in C3H/He male mice was less than 1%, but that it could be increased considerably by whole body irradiation [1]. The incidence of myeloid leukemia increased in proportion to the radiation dose, and the most effective dose was seen to be about 3 Gy, with an incidence of 23.9%. The incidence of myeloid leukemia was influenced by the animal facility conditions, i.e. germ free mice rarely developed leukemia, or the incidence of myeloid leukemia was observed to decrease with improvements of the animal facility conditions [2]. Our previous report clearly demonstrated that the administration of prednisolone acetate (which synthesizes glucocorticoids) after irradiation resulted in a significant increase in the incidence of myeloid leukemia [1]. The mechanism of this promoting effect is not clearly understood, but it may be related to the recovery of hematopoiesis after irradiation, because it has been reported that glucocorticoids suppress the production of growth factor (IL-3, CSF) in mRNA as well as in serum [3, 4]. These results suggest that the stimulation or suppression of hematopoiesis may play a role in the development of myeloid leukemia. Then, we examined whether or
MATERIALS AND METHODS Animals Male and female C3H/He mice, bred at our institute, 8-10 weeks old, were used. They were maintained under clean conventional conditions. Irradiation Mice were exposed to 2.84Gy of whole body X-
Abbreviations: CAM, cellulose acetate membrane; FSD, focus surface distance. 437
438
K. YOSHIDA et al. -7d
GROUPS
i
Od i
CONTROL SACRIF~ED
>
C.A.M.
HEMATOLOGICAL AND HISTOPATHOLOGICAL
EXAMINATION 2.84GY
C.A.M.+2.84GY DIED
HISTOPATUOLOGICAi.
EXAMINATION 2.84GY+C.A.M.
FIG. 1. Experimental groups and procedures. Control: nonirradiated; CAM: CAM insertion only; 2.84 Gy: 2.84 Gy irradiation only; CAM + 2.84 Gy: CAM insertion 7 days before irradiation; 2.84 G y + CAM: CAM insertion immediately after irradiation.
irradiation at a dose rate of 0.614 Gy/min with 200 KV, 20 mA, 0.5 mm AI + 0.5 mm Cu filters and an FSD (focus surface distance) of 56 cm.
Experimental schedule Experimental groups and schedules are illustrated in Fig. 1. A 2 × 2 cm sheet of CAM (Joko Co. Ltd) was inserted into the peritoneal cavity of mice. This laparotomy was done under pentobarbitone anesthesia, and the mice lost little blood because it was performed along the media line of the abdomen. The experimental groups consisted of nontreated mice (control), CAM only inserted mice (CAM), 2.84 Gy irradiation only mice (2.84 Gy), CAM insertion at 7 days before irradiation mice (CAM + 2.84Gy), and CAM insertion immediately after 2.84 Gy irradiation mice (2.84 Gy + CAM). All experimental groups consisted separately of male and female mice. All mice were monitored throughout their life time except for those showing symptoms of advanced leukemia such as anemia and palpable splenomegaly, which were sacrificed at the agonal period and then examined hematologically. All mice were examined by standard histological procedures. The criteria of myeloid leukemia were published elsewhere [9]. RESULTS
The effects on inflammatory response on myeloid leukemogenesis The results of all experiments are shown in Table 1. In male mice, the spontaneous incidence of myeloid leukemia was less than 1% and the insertion of CAM alone did not affect this rate. However, the incidence was significantly increased in the three kinds of irradiated groups. A m o n g these, the insertion of CAM 7 days before irradiation did not increase the incidence of myeloid leukemia compared with the radiation only group (22.4 and 23.9%, respectively). On the
other hand, the incidence was significantly increased by the insertion of C A M immediately after irradiation compared with the other two irradiated groups (36.5%). In female mice, the incidence of myeloid leukemia of both the non-treated and CAM insertion groups was about 4%. The incidence of myeloid leukemia was also increased by radiation only, but the difference was not statistically significant by the Z2 test (12.0%) in this sample size. However, the incidence of myeloid leukemia was increased significantly compared with the control groups when the mice had the CAM inserted into the peritoneum 7 days before or right after irradiation (16.0% p < 0.05, 26.0% p < 0 . 0 1 , respectively). In particular, the development of myeloid leukemia in the C A M inserted mice after irradiation showed a significant difference compared with the irradiation only group ( p < 0.05).
Sex difference on myeloid leukemogenesis The spontaneous incidence of myeloid leukemia of female mice was slightly higher than male mice, but the difference was not statistically significant. In spite of the fact that development of myeloid leukemia in unirradiated female mice was slightly higher than in male mice, the incidence did not increase to the same level as that of male mice when the mice were irradiated at 2.87 Gy, or with radiation plus CAM insertion. The difference between male and female irradiated only groups was statistically significant by the ~ test ( p < 0.1).
Life shortening The survival curves for all experimental groups of
Modifying factors of radiation-induced myeloid leukemogenesis
439
TABLE 1. INCIDENCE OF MYELO1D LEUKEMIA AFTER 2.84 Gy IRRADIATIONOR 2.84 Gy IRRADIATION PLUS C A M INSERTION
Exp. groups
No. of leukemic mice
No. of mice
Incidence of leukemia (% S.E.)
Mean survival days (S.E.)
Male Control CAM 2.84 Gy C A M + 2.84 Gy 2.84 Gy + C A M
110 49 109 49 104
1 0 26 *ae 11 a 38 a~
0.9 _+ 0.9 0 23.9 _+ 4.1 a~ 22.4 - 6.0 a 36.5 -+ 4.7 a~
657.8 690.5 565.4 526.5 528.7
-+ 12.8 -+ 18.9 +- 14.5 **f +- 27.5 f +- 12.9 f
Female Control CAM 2.84 Gy C A M + 2.84 Gy 2.84 Gy + C A M
49 49 50 50 50
2 2 6e 8° 13ad
4.1 4.1 12.0 16.0 26.0
672.9 638.9 504.4 451.4 484.0
+- 23.3 -+ 22.8 _+ 18.0 f +_ 18.0 f - 22.1 f
_+ 2.8 _+ 2.8 +_ 4.6 c +_ 5.2 b +- 6.2 ad
*abcde: ~(2 test for the incidence of myeloid leukemia. a p < 0.01, b p < 0.05: statistically significant differences between non-irradiated groups and irradiated groups, c p < 0.05, a p < 0.1: statistically significant differences between 2.84 Gy and C A M plus 2.84 Gy groups. **~: generalized Wilcoxon test for survival curve, p < 0.001: statistically significant differences between non-irradiated and irradiated groups.
100
~:2~q.~ ~_~'t~
A c~
200
400
600
800
DAYS AFTER IRRADIATION
100
B ?
ot
0
'
2;o
'
~
~
8oo
,~
DAYS AFTER FIRADIATION
FIG. 2. Survival curves for male (A) and female (B) mice of the various treatment groups. ( - - ) : control mice, ( ~ ) : C A M , ( - - - - - - ) : 2.84 Gy, (- . . . . ): C A M plus 2.84 Gy, ( - - - - - - ) : 2.84 Gy plus CAM. The ratio of the survival of the exposed and unirradiated groups expressed as a percentage is plotted as a function of time i n d a ~ s after irradiation.
I
440
K. YOSHIDAet al.
male and female mice are illustrated in Fig. 1, and the mean survival times are listed in Table 1. In both male and female mice, the CAM insertion only groups did not display significant life shortening compared with control groups. However, in the three kinds of irradiated groups of both male and female mice, statistically significant life shortening was observed in comparison to the non-irradiated groups.
DISCUSSION Our present report clearly demonstrated that CAM insertion immediately after radiation promoted the development of myeloid leukemia compared with the radiation only group. CAM insertion alone did not have any effect on leukemogenesis. From these results, CAM insertion is thought to be involved in the promotion of radiation-induced myeloid leukemia. In fact, the dose of 2.84 Gy was sufficient to cause DNA damage in hematopoietic progenitor cells, but was not enough to induce the development of pre-leukemic cells [1]. The mice carry the inserted CAM throughout their life-span, and therefore they will suffer moderate inflammatory response constantly. Accordingly, such chronic inflammatory response may also eventually lead to the development of myeloid leukemia; however, the mice with CAM inserted 7 days before radiation did not show any exacerbating effect. The condition of hematopoiesis within a week after irradiation may be crucial in determining whether DNA-damaged hematopoietic progenitor cells develop into pre-leukemic cells or not. In the case of CAM insertion, the mice must first suffer stress from the surgical injury, and next the inflammatory response. If it is true that the tissue injury may be important, an exacerbating factor in radiation-induced myeloid leukemogenesis rather than the chronic inflammatory response. To resolve this problem, several experiments are now in progress. The spontaneous incidence of myeloid leukemia in female mice is slightly higher than male mice, whereas the radiation-induced incidence is significantly lower. It is hardly likely that there is a difference in radiation sensitivity of hematopoietic progenitor cells between female and male mice. Therefore, sex hormones may be involved in the development of leukemia. Androgens can stimulate
hematopoiesis [6-8], but beyond that the effects of sex hormones on leukemogenesis are not clearly understood. Whatever the mechanisms may be, it is clear that physiological conditions such as tissue injury and/ or inflammatory response and sex difference play important roles in the development of myeloid leukemia. CAM-inserted mice lived for a normal life-span. However, irradiated mice had significant life shortening compared with non-irradiated groups. Such shortened survival in irradiated mice is probably related to the incidence of myeloid leukemia, because this was the major lethal disease of C3H/He mice in the early phase. Acknowledgements--This work was supported by a special project grant for carcinogenesis studies from the Science and Technology agency.
REFERENCES 1. Seki M., Yoshida K., Nishimura M. & Nemoto K. (1991) Radiation-induced myeloid leukemia in C3H/He mice and the effect of prednisolone acetate on leukemogenesis. Radiat. Res. 127, 146. 2. Walburg H. E. Jr, Cosgrove G. E. & Upton A. C. (1968) Influence of microbial environment on development of myeloid leukemia in X-irradiated RFM mice. Int. J. Cancer 3, 150. 3. Zalman F., Maloney M. A. & Patt H. M. (1979) Differential response of erythropoietic and granulopoietic progenitors to dexamethasone. J. exp. Med. 149, 67. 4. Culpepper J. A. & Lee F. (1985) Regulation of IL 3 expression by glucocorticoids in cloned murine Tlymphocytes. J. lrnmun. 135, 3191. 5. Seki M. (1973) Hematopoietic colony formation in a macrophage layer provided by intraperitoneal insertion of cellulose acetate membrane. Transplantation 16, 544. 6. Jack W. & Adamson J. W. (1976) Steroids and hematopoiesis. III. The response of granulocytic and erythroid colony-formingcells to steroids of different classes. Blood 48, 855. 7. Alan L., Rosenblum L. & Carbone P. P. (1974) Androgenic hormones and human granulopoiesis in vitro. Blood 43, 351. 8. Kamamoto T., Sakoda H., Yoshida Y., Uchino H., Imai N. & Mori K. J. (1985) Effects of androgen on transient endogenous spleen colonies and other hematopoietic stem cells in mice. Expl Hemat. 13, 616. 9. Bennet J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A. G., Gralnick H. R. & Sultan C. (1976) Proposal for the classification of the acute leukaemias. Br. J. Haemat. 33, 451.
0145-2126/93 $6.00 + .00 © 1993 Pergamon Press Ltd
E X A C E R B A T I N G FACTORS OF R A D I A T I O N - I N D U C E D M Y E L O I D LEUKEMOGENESIS KAZUKO YOSHIDA, KUMIE NEMOTO, MAYUMI NISHIMURAand MASATOSHI SEKI Division of Physiology and Pathology, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku Chiba-shi Chiba, 263 Japan (Received 6 August 1992. Revision accepted 19 December 1992)
Abstract--The spontaneous incidence of myeloid leukemia in female mice was slightly higher than in male mice, whereas the radiation-induced incidence was significantlylower than in male mice. We also examined whether the incidence of myeloid leukemia was related to inflammatory response. Mice had a piece of cellulose acetate membrane inserted into the peritoneal cavity to cause inflammation. This did not affect the incidence of myeloid leukemia in unirradiated mice at all, but in 2.84 Gy irradiated mice the incidence (35.9% in male, 26.0% in female mice) increased significantlycompared with irradiated-only mice (23.9% and 12.0%, respectively). From these results, the physiological fluctuation of humoral factors by means of inflammatory response is considered to increase the development of radiation-induced myeloid leukemia. Key words: Myeloid leukemia, inflammatory response, tissue injury, sex difference, exacerbating factors, radiation.
not inflammatory reaction would act as a promoter of radiation-induced leukemogenesis. When a piece of cellulose acetate membrane (CAM) was inserted into the peritoneal cavity of mice, CAM became covered by macrophage-fibroblast cell layers because of inflammatory reaction [5]. Such cell layers could act as an artificial hematopoietic microenvironment. Colonies consisting of hematopoietic progenitor cells were formed on the macrophage-fibroblast cell layers when bone marrow cells were transplanted into the peritoneal cavity of CAM-inserted mice [5, 6]. We used this system to induce inflammatory reaction, and the mice were then exposed to about 3 Gy of irradiation, the most effective dose for leukemogenesis. It is well known that androgens can stimulate hematopoiesis. In addition, they can also increase the number of hematopoietic stem cells [6-8]. We therefore tried to determine whether the incidence of radiation-induced myeloid leukemia differed between female and male mice.
INTRODUCTION WE HAVEreported that the spontaneous incidence of myeloid leukemia in C3H/He male mice was less than 1%, but that it could be increased considerably by whole body irradiation [1]. The incidence of myeloid leukemia increased in proportion to the radiation dose, and the most effective dose was seen to be about 3 Gy, with an incidence of 23.9%. The incidence of myeloid leukemia was influenced by the animal facility conditions, i.e. germ free mice rarely developed leukemia, or the incidence of myeloid leukemia was observed to decrease with improvements of the animal facility conditions [2]. Our previous report clearly demonstrated that the administration of prednisolone acetate (which synthesizes glucocorticoids) after irradiation resulted in a significant increase in the incidence of myeloid leukemia [1]. The mechanism of this promoting effect is not clearly understood, but it may be related to the recovery of hematopoiesis after irradiation, because it has been reported that glucocorticoids suppress the production of growth factor (IL-3, CSF) in mRNA as well as in serum [3, 4]. These results suggest that the stimulation or suppression of hematopoiesis may play a role in the development of myeloid leukemia. Then, we examined whether or
MATERIALS AND METHODS Animals Male and female C3H/He mice, bred at our institute, 8-10 weeks old, were used. They were maintained under clean conventional conditions. Irradiation Mice were exposed to 2.84Gy of whole body X-
Abbreviations: CAM, cellulose acetate membrane; FSD, focus surface distance. 437
438
K. YOSHIDA et al. -7d
GROUPS
i
Od i
CONTROL SACRIF~ED
>
C.A.M.
HEMATOLOGICAL AND HISTOPATHOLOGICAL
EXAMINATION 2.84GY
C.A.M.+2.84GY DIED
HISTOPATUOLOGICAi.
EXAMINATION 2.84GY+C.A.M.
FIG. 1. Experimental groups and procedures. Control: nonirradiated; CAM: CAM insertion only; 2.84 Gy: 2.84 Gy irradiation only; CAM + 2.84 Gy: CAM insertion 7 days before irradiation; 2.84 G y + CAM: CAM insertion immediately after irradiation.
irradiation at a dose rate of 0.614 Gy/min with 200 KV, 20 mA, 0.5 mm AI + 0.5 mm Cu filters and an FSD (focus surface distance) of 56 cm.
Experimental schedule Experimental groups and schedules are illustrated in Fig. 1. A 2 × 2 cm sheet of CAM (Joko Co. Ltd) was inserted into the peritoneal cavity of mice. This laparotomy was done under pentobarbitone anesthesia, and the mice lost little blood because it was performed along the media line of the abdomen. The experimental groups consisted of nontreated mice (control), CAM only inserted mice (CAM), 2.84 Gy irradiation only mice (2.84 Gy), CAM insertion at 7 days before irradiation mice (CAM + 2.84Gy), and CAM insertion immediately after 2.84 Gy irradiation mice (2.84 Gy + CAM). All experimental groups consisted separately of male and female mice. All mice were monitored throughout their life time except for those showing symptoms of advanced leukemia such as anemia and palpable splenomegaly, which were sacrificed at the agonal period and then examined hematologically. All mice were examined by standard histological procedures. The criteria of myeloid leukemia were published elsewhere [9]. RESULTS
The effects on inflammatory response on myeloid leukemogenesis The results of all experiments are shown in Table 1. In male mice, the spontaneous incidence of myeloid leukemia was less than 1% and the insertion of CAM alone did not affect this rate. However, the incidence was significantly increased in the three kinds of irradiated groups. A m o n g these, the insertion of CAM 7 days before irradiation did not increase the incidence of myeloid leukemia compared with the radiation only group (22.4 and 23.9%, respectively). On the
other hand, the incidence was significantly increased by the insertion of C A M immediately after irradiation compared with the other two irradiated groups (36.5%). In female mice, the incidence of myeloid leukemia of both the non-treated and CAM insertion groups was about 4%. The incidence of myeloid leukemia was also increased by radiation only, but the difference was not statistically significant by the Z2 test (12.0%) in this sample size. However, the incidence of myeloid leukemia was increased significantly compared with the control groups when the mice had the CAM inserted into the peritoneum 7 days before or right after irradiation (16.0% p < 0.05, 26.0% p < 0 . 0 1 , respectively). In particular, the development of myeloid leukemia in the C A M inserted mice after irradiation showed a significant difference compared with the irradiation only group ( p < 0.05).
Sex difference on myeloid leukemogenesis The spontaneous incidence of myeloid leukemia of female mice was slightly higher than male mice, but the difference was not statistically significant. In spite of the fact that development of myeloid leukemia in unirradiated female mice was slightly higher than in male mice, the incidence did not increase to the same level as that of male mice when the mice were irradiated at 2.87 Gy, or with radiation plus CAM insertion. The difference between male and female irradiated only groups was statistically significant by the ~ test ( p < 0.1).
Life shortening The survival curves for all experimental groups of
Modifying factors of radiation-induced myeloid leukemogenesis
439
TABLE 1. INCIDENCE OF MYELO1D LEUKEMIA AFTER 2.84 Gy IRRADIATIONOR 2.84 Gy IRRADIATION PLUS C A M INSERTION
Exp. groups
No. of leukemic mice
No. of mice
Incidence of leukemia (% S.E.)
Mean survival days (S.E.)
Male Control CAM 2.84 Gy C A M + 2.84 Gy 2.84 Gy + C A M
110 49 109 49 104
1 0 26 *ae 11 a 38 a~
0.9 _+ 0.9 0 23.9 _+ 4.1 a~ 22.4 - 6.0 a 36.5 -+ 4.7 a~
657.8 690.5 565.4 526.5 528.7
-+ 12.8 -+ 18.9 +- 14.5 **f +- 27.5 f +- 12.9 f
Female Control CAM 2.84 Gy C A M + 2.84 Gy 2.84 Gy + C A M
49 49 50 50 50
2 2 6e 8° 13ad
4.1 4.1 12.0 16.0 26.0
672.9 638.9 504.4 451.4 484.0
+- 23.3 -+ 22.8 _+ 18.0 f +_ 18.0 f - 22.1 f
_+ 2.8 _+ 2.8 +_ 4.6 c +_ 5.2 b +- 6.2 ad
*abcde: ~(2 test for the incidence of myeloid leukemia. a p < 0.01, b p < 0.05: statistically significant differences between non-irradiated groups and irradiated groups, c p < 0.05, a p < 0.1: statistically significant differences between 2.84 Gy and C A M plus 2.84 Gy groups. **~: generalized Wilcoxon test for survival curve, p < 0.001: statistically significant differences between non-irradiated and irradiated groups.
100
~:2~q.~ ~_~'t~
A c~
200
400
600
800
DAYS AFTER IRRADIATION
100
B ?
ot
0
'
2;o
'
~
~
8oo
,~
DAYS AFTER FIRADIATION
FIG. 2. Survival curves for male (A) and female (B) mice of the various treatment groups. ( - - ) : control mice, ( ~ ) : C A M , ( - - - - - - ) : 2.84 Gy, (- . . . . ): C A M plus 2.84 Gy, ( - - - - - - ) : 2.84 Gy plus CAM. The ratio of the survival of the exposed and unirradiated groups expressed as a percentage is plotted as a function of time i n d a ~ s after irradiation.
I
440
K. YOSHIDAet al.
male and female mice are illustrated in Fig. 1, and the mean survival times are listed in Table 1. In both male and female mice, the CAM insertion only groups did not display significant life shortening compared with control groups. However, in the three kinds of irradiated groups of both male and female mice, statistically significant life shortening was observed in comparison to the non-irradiated groups.
DISCUSSION Our present report clearly demonstrated that CAM insertion immediately after radiation promoted the development of myeloid leukemia compared with the radiation only group. CAM insertion alone did not have any effect on leukemogenesis. From these results, CAM insertion is thought to be involved in the promotion of radiation-induced myeloid leukemia. In fact, the dose of 2.84 Gy was sufficient to cause DNA damage in hematopoietic progenitor cells, but was not enough to induce the development of pre-leukemic cells [1]. The mice carry the inserted CAM throughout their life-span, and therefore they will suffer moderate inflammatory response constantly. Accordingly, such chronic inflammatory response may also eventually lead to the development of myeloid leukemia; however, the mice with CAM inserted 7 days before radiation did not show any exacerbating effect. The condition of hematopoiesis within a week after irradiation may be crucial in determining whether DNA-damaged hematopoietic progenitor cells develop into pre-leukemic cells or not. In the case of CAM insertion, the mice must first suffer stress from the surgical injury, and next the inflammatory response. If it is true that the tissue injury may be important, an exacerbating factor in radiation-induced myeloid leukemogenesis rather than the chronic inflammatory response. To resolve this problem, several experiments are now in progress. The spontaneous incidence of myeloid leukemia in female mice is slightly higher than male mice, whereas the radiation-induced incidence is significantly lower. It is hardly likely that there is a difference in radiation sensitivity of hematopoietic progenitor cells between female and male mice. Therefore, sex hormones may be involved in the development of leukemia. Androgens can stimulate
hematopoiesis [6-8], but beyond that the effects of sex hormones on leukemogenesis are not clearly understood. Whatever the mechanisms may be, it is clear that physiological conditions such as tissue injury and/ or inflammatory response and sex difference play important roles in the development of myeloid leukemia. CAM-inserted mice lived for a normal life-span. However, irradiated mice had significant life shortening compared with non-irradiated groups. Such shortened survival in irradiated mice is probably related to the incidence of myeloid leukemia, because this was the major lethal disease of C3H/He mice in the early phase. Acknowledgements--This work was supported by a special project grant for carcinogenesis studies from the Science and Technology agency.
REFERENCES 1. Seki M., Yoshida K., Nishimura M. & Nemoto K. (1991) Radiation-induced myeloid leukemia in C3H/He mice and the effect of prednisolone acetate on leukemogenesis. Radiat. Res. 127, 146. 2. Walburg H. E. Jr, Cosgrove G. E. & Upton A. C. (1968) Influence of microbial environment on development of myeloid leukemia in X-irradiated RFM mice. Int. J. Cancer 3, 150. 3. Zalman F., Maloney M. A. & Patt H. M. (1979) Differential response of erythropoietic and granulopoietic progenitors to dexamethasone. J. exp. Med. 149, 67. 4. Culpepper J. A. & Lee F. (1985) Regulation of IL 3 expression by glucocorticoids in cloned murine Tlymphocytes. J. lrnmun. 135, 3191. 5. Seki M. (1973) Hematopoietic colony formation in a macrophage layer provided by intraperitoneal insertion of cellulose acetate membrane. Transplantation 16, 544. 6. Jack W. & Adamson J. W. (1976) Steroids and hematopoiesis. III. The response of granulocytic and erythroid colony-formingcells to steroids of different classes. Blood 48, 855. 7. Alan L., Rosenblum L. & Carbone P. P. (1974) Androgenic hormones and human granulopoiesis in vitro. Blood 43, 351. 8. Kamamoto T., Sakoda H., Yoshida Y., Uchino H., Imai N. & Mori K. J. (1985) Effects of androgen on transient endogenous spleen colonies and other hematopoietic stem cells in mice. Expl Hemat. 13, 616. 9. Bennet J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A. G., Gralnick H. R. & Sultan C. (1976) Proposal for the classification of the acute leukaemias. Br. J. Haemat. 33, 451.