7
J Cancer Res Clin Oncoi (1984) 108:214-220 C eer esearch Clinical 9 9 Springer-Verlag 1984 Leukemia Induction by Methylnitrosourea (MNU) in Selected Mouse Strains Effects of MNU on Hemopoietic Stem Cells, the Immune System and Natural Killer Cells Hans Joachim Seidel and Ludwika Kreja Department of Clinical Physiology and Occupational Medicine University of Ulm, Oberer Eselsberg, D-7900 Ulm, Federal Republic of Germany Summary. T cell leukemias were induced by a single dose ofmethylnitrosourea (MNU) in DBA/2, C57/B1/ 6, NMRI, BDF1, and CBA mice. The latency period in the CBA strain was much longer than in the others. Studies on the pluripotent stem cells in the bone mar- row and T cell reactions of thymus and spleen cells showed a toxicity of MNU for these parameters but no significant differences between the strains. The ac- tivity of the natural killer (NK) cells in the spleen and peritoneal exudate cells, studied also after additional stimulation by injection of Corynebacterium parvum, was influenced by MNU, but again no relation to leu- kemogenesis could be established. The first leukemic (transplantable) cells were found in the thymus. The presence of leukemic cells could be responsible for low NK cell activities found in BDF1 and DBA/2 mice late after MNU. Key words: Chemical leukemogenesis - Mouse strain Stem cells - NK cells Introduction Methylnitrosourea (MNU) is a potent leukemogen in adult mice, and the development of leukemias with T cell characteristics following MNU has been described by several groups (Joshi and Frei 1970a, b; Dexter et al. 1974; Frei and Lawley 1980; Baines et al. 1979; Frei 1980). We are concerned with the study of cellular events in the hematopoietic cell system which precede the development of leukemia and which should in- clude the essential changes during the latency period. With butylnitrosourea as leukemogen, which has to be given in a prolonged exposure in the drinking water, we were able to describe reductions in the number of Offprint requests to: Dr. H.J. Seidel (address see above) hematopoietic stem cells and the immune functions; the relation of these changes to the development of the first detectable leukemic cells, however, remained un- certain (Seidel 1980; Seidel et al. 1983). In this report we describe leukemia induction in four strains of mice and one hybrid strain and the effects of the leukemo- genic dose of MNU on hematopoietic parameters such as the number of pluripotent stem cells (CFU-S), T cell reactions, and also the natural killer (NK) cells. The results of the study show that CBA mice have a much longer latency period for leukemogenesis than C57/B1/6, DBA/2, NMRI, and BDF1 mice. The early toxicity of MNU for CFU-S and T cell parameters, however, does not show a specific reaction of CBA mice although minor strain differences in various pa- rameters were observed. Methods Mice Female mice aged 8-10 weeks and with 2~25 g body weight at the beginning of the experiments were used. DBA/2, C57/B1/6, and BDF a (C57/B1/6 • DBA/2 hybrids) were obtained from G1. Bom- holtgard, Ry, Denmark; NMRI mice from SiJddeutsche Versuchs~ tierfarm, Tuttlingen, Germany; and CBA mice from the Central Ani- mal Facility of the University of Ulm. They were kept under the stan- dard conditions of the Central Animal Facility with artificial light for 12 h daily, and were fed commercial pellets and water ad libi- turn. Methylnitrosourea Methylnitrosourea (MNU) was obtained from Serva, Heidelberg; it was dissolved in saline and 50 mg/kg was injected IV; 0.2 ml of the solution for a mouse weighing 20 g. CFU-S Pluripotent hematopoietic stem cells were determined according to Till and McClulloch (1961), without reference to the recent publica- tion by Magh et al. (1982). Cells (4 x 104 or 4 x 10s; 1 day and 1 week after MNU) were injected IV into lethally irradiated recipients (10 animals per group). Irradiation consisted of an 8-Gy exposure; 280 kV; 12 mA, a filter of 1 mm Cu and 1 mm A1; focal distance,

Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

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Page 1: Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

J Cancer Res Clin Oncoi (1984) 108:214-220 C eer esearch Clinical �9 �9 Springer-Verlag 1984

Leukemia Induction by Methylnitrosourea (MNU) in Selected Mouse Strains

Effects of MNU on Hemopoietic Stem Cells, the Immune System and Natural Killer Cells

Hans Joachim Seidel and Ludwika Kreja Department of Clinical Physiology and Occupational Medicine University of Ulm, Oberer Eselsberg, D-7900 Ulm, Federal Republic of Germany

Summary. T cell leukemias were induced by a single dose ofmethylnitrosourea (MNU) in DBA/2, C57/B1/ 6, NMRI, BDF1, and CBA mice. The latency period in the CBA strain was much longer than in the others. Studies on the pluripotent stem cells in the bone mar- row and T cell reactions of thymus and spleen cells showed a toxicity of MNU for these parameters but no significant differences between the strains. The ac- tivity of the natural killer (NK) cells in the spleen and peritoneal exudate cells, studied also after additional stimulation by injection of Corynebacterium parvum, was influenced by MNU, but again no relation to leu- kemogenesis could be established. The first leukemic (transplantable) cells were found in the thymus. The presence of leukemic cells could be responsible for low NK cell activities found in BDF1 and DBA/2 mice late after MNU.

Key words: Chemical leukemogenesis - Mouse strain Stem cells - NK cells

Introduction

Methylnitrosourea (MNU) is a potent leukemogen in adult mice, and the development of leukemias with T cell characteristics following MNU has been described by several groups (Joshi and Frei 1970a, b; Dexter et al. 1974; Frei and Lawley 1980; Baines et al. 1979; Frei 1980). We are concerned with the study of cellular events in the hematopoietic cell system which precede the development of leukemia and which should in- clude the essential changes during the latency period. With butylnitrosourea as leukemogen, which has to be given in a prolonged exposure in the drinking water, we were able to describe reductions in the number of

Offprint requests to: Dr. H.J. Seidel (address see above)

hematopoietic stem cells and the immune functions; the relation of these changes to the development of the first detectable leukemic cells, however, remained un- certain (Seidel 1980; Seidel et al. 1983). In this report we describe leukemia induction in four strains of mice and one hybrid strain and the effects of the leukemo- genic dose of MNU on hematopoietic parameters such as the number of pluripotent stem cells (CFU-S), T cell reactions, and also the natural killer (NK) cells. The results of the study show that CBA mice have a much longer latency period for leukemogenesis than C57/B1/6, DBA/2, NMRI, and BDF1 mice. The early toxicity of MNU for CFU-S and T cell parameters, however, does not show a specific reaction of CBA mice although minor strain differences in various pa- rameters were observed.

Methods

Mice

Female mice aged 8-10 weeks and with 2~25 g body weight at the beginning of the experiments were used. DBA/2, C57/B1/6, and BDF a (C57/B1/6 • DBA/2 hybrids) were obtained from G1. Bom- holtgard, Ry, Denmark; NMRI mice from SiJddeutsche Versuchs~ tierfarm, Tuttlingen, Germany; and CBA mice from the Central Ani- mal Facility of the University of Ulm. They were kept under the stan- dard conditions of the Central Animal Facility with artificial light for 12 h daily, and were fed commercial pellets and water ad libi- turn.

Methylnitrosourea

Methylnitrosourea (MNU) was obtained from Serva, Heidelberg; it was dissolved in saline and 50 mg/kg was injected IV; 0.2 ml of the solution for a mouse weighing 20 g.

CFU-S

Pluripotent hematopoietic stem cells were determined according to Till and McClulloch (1961), without reference to the recent publica- tion by Magh et al. (1982). Cells (4 x 104 or 4 x 10s; 1 day and 1 week after MNU) were injected IV into lethally irradiated recipients (10 animals per group). Irradiation consisted of an 8-Gy exposure; 280 kV; 12 mA, a filter of 1 mm Cu and 1 mm A1; focal distance,

Page 2: Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

H.J. Seidel and L. Kreja: Leukemia Induction by Methylnitrosourea (MNU) 215

MNU leukemogenesis, strain differences

MNU single injection, 50mg/kg i.v.

100 t - - DBA/2

% ~ , 7 . . . . .~ . l i ] i i - - - - - C 5 7 B I / 6

[ ~ L ' " ' I ......... B D F ,

-~i I i ........ C B A / C a

'

' I

i l I !

50- ~.-7 k - 7 ................ = ] L.~ i

i :,

. . . . . . L 1 L . . . . . . . . - : , . ~

1 - - ~ : : j : I_ i ! ...... "7, . . . . i

LL .............................................. L_, ,.r _......':.:: , ..................... = L L - - - - : ~ . ~ . ~ ............... L.,..~ ] - i ~ . :

, ! i i 0 16 2 0 36 4 0 56 (30 7'0

w e e k s a f te r M N U

Fig. 1. Mortality pattern of 5 different mouse strains following injection in early adulthood of 50 mg/kg methylnitrosourea (MNU) IV 20 animals per group

50 cm; and rate, 30 cGy/min. The recipient animals were killed 9 days after injection of cells, and their spleens were removed and placed in Bouin's solution in preparation for the counting of macro- scopic colonies after 24 h in the fixative. From these data the mean CFU-S content per femur was calculated. No endogenously formed colonies were found in mice exposed to radiation and not engrafted with cells.

Lymphocyte Stimulation

Cells from the thymus or the spleen were cultured as described before (Seidel et al. 1983). Stimulator cells for mixed lymphocyte cultures (MLC) were 5 • 105 CBA/Ca mouse spleen cells for DBA/2, C57/B1/ 6, NMRI and BDF 1 spleen cells, and BDF 1 spleen cells for CBA/Ca spleen cells after irradiation with 12 Gy. The cultures were termi- nated after 48 h, or in the case of MLC 72 h; a multiple cell culture harvester (Scatron, Lyerbee, Norway) was used after addition of 0.2 IxCi 3H-thymidine (specific activity 2 Ci/mmol, Amersham, Eng- land) per well 16 h previously. The following counts were obtained in controls: Con A stimulation 50000-70000 cpm, MLC 10000 ~ 1 5 000 cpm.

NK Cell Assay

Experimerital groups consisted of three to seven mice. Their spleens were taken and finely minced to single-cell suspensions, which were washed twice and used for the assay systems. Peritoneal exudate cells (PEC) were harvested from groups of seven mice by washing the peritoneal cavity with 2 ml c~-medium with heparin. Two groups of mice were used at each experimental point, in one of which the ani- mals had received Corynebacterium parvum (CP), 175 ~tg per mouse by injection 4 days previously to enhance the NK-specific lysis (Ojo et al. 1978). The method used was that described by Kiessling et al. (1975): 20 x 106 YAC-I cells per ml were incubated with 500 ~tCi 51Cr as sodium chromate NazCrO 4 (5 mCi/ml specific activity > 350 Ci/mmol NEN, Frankfurt, FRG) for 30 min at 37 ~ in 5% CO2 atmosphere, and subsequently washed three times with cold me- dium. Effector cells, either from the spleen cell suspensions or PECs, were incubated at an effector-to-target cell ratio of 100:1 (spleen cells) or 50:1 (PECs) for 6 h in 0.15 ml, containing 0.05 ml labeled YAC-1 cells, 8 x 10S/ml, 0.1 ml effector cells, 4 or 2 x 107/ml, with shaking. Controls included samples without effector cells, one repre- senting the spontaneous release and the second, after addition of Za- ponin, the maximum release. All assays were done in triplicate. After the incubation period 1 ml cold medium was added, the samples were

centrifuged, and 0.575 ml supernatant and the remaining volume in- cluding the pellet (sediment) were counted in a Berthold BF 5300 7- counter. The percent SlCr release was calculated according to the formula:

2 x Supernatant (0.575 ml) % 51Cr release=

Supernatant + Sediment x 100.

The results are expressed as

% Releaseexp.- % Releasespo,t ' % Specific lysis • 100.

% Release . . . . - % Releasespont.

Results

I. Leukemia Induction by M N U in Various Mouse Strains

As seen in Fig. 1, the mortality pattern of DBA/2, C57/B1/6, and BDF1 mice after the single injection of 50 mg/kg MNU is identical: the first mice die of leu- kemia at about 13 weeks and the median survival time is 19-20 weeks. The mortality pattern of CBA/Ca mice was completely different. The first leukemias were seen at 25 weeks, and the median survival time was more than 40 weeks. All DBA/2, C57/B1/6, BDF1, and NMRI mice had leukemia seen in enlargement of the thymus and the spleen, and in most cases also vari- ous lumph nodes. Occasionally, histological controls were performed. There were, however, some mice, about 10-20% in these strains, in which no thymoma was found or in which no diagnosis could be made due to autolysis. In CBA/Ca mice the percentage of mice without thymoma was about 30%.

II. Detection of Transplantable Cells (BDF1 Mice)

Cells, 3 x 106 from the thymus or all cells of one femur of individual mice were injected IV at 3, 9, and 12

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216 H.J. Seidel and L. Kreja: Leukemia Induction by Methylnitrosourea (MNU)

Table 1. Detection of transplantable (leukemic) cells in the thymus and bone marrow of BDF 1 mice after the injection of 50 mg/kg M N U following IV injections of 3 x 106 thymus cells or all cells from one femur into normal ( ~ ) or 4 Gy-c0nditioned (~ ) syngeneic recipients

9 weeks after M N U 12 weeks after M N U

Organ Bone marrow Thymus

Mouse No. ~ ~ ~

Bone marrow Thymus

1 - - 65 a 45 34 27 78 61 2 - - - 125 45 43 38 34 3 - - 48 27 - - 127 77 4 . . . . . . 125 86 5 34 a 20 140 140 . . . . 6 14 23 18 34 - 65 b - 7 20 20 44 21 - - 43 35

3 weeks after MNU no leukemias were observed in the recipients. Figures give time of death o f recipient animals in days a The numbers give the survival time of recipient animals after transplantation of 3 • 106 thymus cells

(median value of 5 recipients) or the cells of one femur (1 recipient per femur) b No postmortem diagnosis possible, all other recipient animals had leukemia

weeks after MNU exposure into normal or 4-Gy-con- ditioned syngeneic recipients. By 3 weeks after MNU there were no transplantable cells in either organ (Table 1). At 9 weeks after MNU the femurs of three of seven mice and the thymuses of the same three plus three further mice contained leukemic cells; in one of these transplantation was only successfull with 4-Gy- conditioned recipients. At 12 weeks two femora and five thymuses of the seven mice contained leukemic cells, which were detected both in normal and in con- ditioned recipients. One additional femur contained cells leading to the deaths of a 4-Gy-conditioned ani- mal only. The total observation time of the recipients was 6 months. The leukemia-positive recipient ani- mals died 14-140 days after the cell inoculum, most deaths occuring within the range of 20-50 days. In al- most all instances where normal and irradiated recip- ients died of leukemia, the irradiated recipient died earlier than the normal one and the time difference was greater, with longer intervals from transplanta- tion to death. At 5 months after the end of the study, i.e. 12 weeks after MNU and additional 5 months, all animals treated with MNU in the experiment, but not used as donors, had died of leukemia.

IlL Studies During the Latency Period

1. Thymus Weight and Number of CFU-S. The thymus weight was very much reduced 1 day and 1 week after MNU administration (Fig. 2). Regeneration started between 1 and 3 weeks after MNU and was complete, in C57/B1/6 mice only at 9 weeks. Regeneration was faster in NMRI mice than in all other strains, as seen 3 weeks after MNU. All thymuses studied at 9 weeks had the normal appearance for the organ, with two

distinct equally sized lobes and a white smooth sur- face. Animals with one smaller lobe and a grayish ap- pearance of the organ - suggestive for the develop- ment of leukemia, as seen in the studies with cell trans- plantations - were not taken. During the 9-week-pe- riod of this experiment no change in controls was seen in all strains except C57/B1/6, and the mean values for control animals obtained at the beginning and during the experiment are given in Fig. 2. In C57/B1/6 control animals the thymus weight had decreased from

o/~ BDF 1 DBA/2 CBA

0 1day I 3 9wks

NMRI C57BL6 /o

0 1day1 3 9wks

Thymus weight after 50mg/kg MNU i.v.

Fig. 2. Thymus weight of five different mouse strains after the injec- tion of 50 mg/kg M N U . Control values _ 1 SD at the beginning o f the study are given in absolute numbers and set as 100%; they did not change significantly during the experiment, except in C57/B1/6 mice where the thymus weight was about 40 mg at the end of the ex- periment. Four MNU-trea ted animals were studied; at 9 weeks after M N U only thymuses with normal macroscopic appearance were taken

Page 4: Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

H.J. Seidel and L. Kreja: Leukemia Induction by Methylnitrosourea (MNU) 217

Table 2. NK-specific lysis of YAC-1 target cells by spleen and peritoneal exudate cells (PEC) of control animals at 3 and 9 weeks after the beginning of an experiment, without ( - C P ) and with (+CP) stimulation of NK cells by Corynebacterium parvum

3 weeks 12 weeks

Spleen PEC + CP Spleen PEC + CP

- C P +CP - C P +CP

DBA/2 5.0• 11.7• 31.1• 1.7• 13.4• 28.5• CBA 7.0• 29.1• 48.9• 2.6• 20.2• 58.7• BDF 1 10.1• 24.2• 69.2• 9.7• 21.9• 59.9•

70_+ 1 mg to about 40 mg at the end of the experi- ment.

The numbers of CFU-S per femur (Fig. 3) were very much reduced after MNU and regenerated thereafter. There were significant strain differences in the CFU-S numbers of the control animals at the be- ginning of the experiments. The numbers showed a slight increase after additional 9 weeks (not shown). CFU-S regeneration started after more than 1 week and was completed after 9 weeks in DBA/2, CBA, and C57/B1/6 mice, but not in BDF1, and NMRI mice.

2. Mitogenie Response of T Cells. The results are not presented in detail, since no significant strain differ- ences could be detected in nonleukemic mice studied up to 12 weeks after MNU administration. The MLC reactivity of spleen cells was reduced in all strains to a minimum of less than 50% of controls (cpm) at 3 weeks and regenerated to subnormal levels in most ex- periments at 9 and 12 weeks. The Con A responsive-

O/o BDF1 lOO 63o0 t 6(x)

O- r-~-~*--~--~--~-~,'--~ 1day I 3 9wks

0/0 NMRI 100] ,~ 4'aom soo

0 1day 1 3 9wks

oB, / , / .

C57BL6

CFU-S PER FEMUR after 50rag kg/MNU i.v.

Fig. 3. Number of pluripotent stem cells (CFU-S) per femur after in- jection of 50 mg/kg MNU. Control values _+ 1 SEM at the beginning of the study are Nven in absolute numbers and set as 100%; there was only a slight increase during the experiment (not indicated). Bone marrow cells from four MNU-treated mice were pooled

hess of cells from the thymus and the spleen was less markedly affected and varied around control levels at 9 and at 12 weeks.

3. NK Cell Activity. In control animals of the strains DBA/2, BDFt, and CBA the spontaneous NK cell ac- tivity against YAC-1 target cells is rather low in the spleen cell suspensions (Table 2). This is due to the age of the animals and the duration of the experiment. The specific lysis by control spleen cells is given in Table 2 at two experimental intervals, without and also 4 days after injection of CP, which led to a very pronounced increase of the specific lysis. For PECs the sponta- neous lytic activity is below 5%, and is not indicated; after CP injection, however, lytic values between 28.5% and 69% can be observed. There is a clear strain difference, DBA/2 having the lowest, CBA an intermediate, and BDF 1 the highest NK cell activity. The values shown in Table 2 were recorded in a typical experiment. These controls were studied in parallel to all experimental groups.

After MNU application the NK cell activity was changed. Results of repeated experiments are given in Table 3. There is considerable variation. In DBA mice the depression of NK cell activity of PECs at 3 weeks was a consistent finding, but this activity later normal- ized. Spleen cells had an enhanced activity after CP stimulation at 6 weeks, followed by a depression at 9 weeks and also at 12 weeks, then with and without CP stimulation. In CBA mice a very consistent depression of NK activity was seen after CP stimulation in the spleen and the PECs at 3 weeks. In fact, CP was not followed by the usual increase at all. At 6 weeks, the spontaneous spleen cells activity was enhanced, at later time points no significant finding was obtained. In BDF1 mice in most experiments a depression of the CP-stimulated specific lysis was seen, which was more pronounced with spleen cells than with PECs. Two ex- periments with spleen cells and PECs of DBA/2 and CBA mice are given in detail in Fig. 4. Administration of CP 4 days before the assay was followed by the usual increase in the spleen in control animals and also

Page 5: Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

218 H.J . Seidel and L. Kreja: Leukemia Induction by Methylnitrosourea (MNU)

Table 3. NK-specific lysis ~ of YAC-1 target cells by spleen cells and PECS of mice at various intervals after 50 mg/kg of M N U

Time 1 week 3 weeks 6 weeks 8 weeks 9 weeks 12 weeks after M N U Spleen Spleen PEC Spleen PEC Spleen Spleen PEC Spleen PEC

- C P + C P - + + - + + - + - + + - + +

> 150% of controls,~126%-149%, IJ 75%-125%, ~,50%-74%, ~, 20%-49%, 1 0-10% a Results obtained in repeated experiments are given as percentages of values in the appropriate control groups with cells from non-MNU-

treated animals. Specific lysis in controls is shown in Table 2

MNU-treated DBA/2 mice 3 and 9 weeks after MNU, although to a reduced extent in experiment 2. CBA mice, however, did not respond to CP stimulation in either experiment 3 weeks after MNU. The NK cell activity of PECs was studied only after CP stimula- tion. At 3 weeks a reduction was seen in both mouse strains compared to control animals, to a greater ex- tent in CBA mice.

Discussion

The aim of these experiments was to study leukemia development after a single exposure to MNU 50 mg/ kg in various mouse strains and relate findings during the latency period to leukemogenesis. This would be facilitated by strain differences in the leukemia induc- tion. In fact, the median induction time in CBA mice was significantly longer than in all other strains.

The hematopoietic system after the administration of MNU reflects an early toxicity of MNU. It is inter- esting that in a comparison of the effects of sublethal doses of four different compounds only the leukemo- genic N-ethyl-N-nitrosourea was followed by a de- crease in bone marrow and thymus cellularity (Frei and Maitra 1974). In these studies bone marrow and thymus regeneration, which was observed within 3 weeks, was considered an essential step in thymoma development. Irrespective of these early events, which are also seen in leukemogenic protocols based on ionizing radiation (van Bekkum et al. 1981), we were more interested in hematopoietic function during the latency period and wanted to see whether any changes would persist or become apparent before leukemic cells could be detected. These events should logically be present in DBA/2 and BDF1 mice in a very similar way and be quite different in CBA mice. The results demonstrate that the numbers of CFU-S, the size of the thymus, and also the response of thymus and spleen cells to mitogenic stimulation were no different.

Specific changes in the response of preleukemic thy- mocytes, such as have been reported for AKR mice and also found in mice after infection with Moloney virus for example, could not be found (Nagaya 1973; Zatz 1975; Mertens and Krueger 1976).

Recent descriptions of the NK cell system and spe- cific studies of the NK cells during tumorigenesis and leukemogenesis have laid great emphasis on this cell system (Roder et al. 1981; Herberman and Ortaldo 1981). There are, however, only very few data on NK

NK cell activity after MNU injection

spleen and PEC

% Exp. 1 lysis

20

E xp . 2

- - 3 w e e k s - -

D contr. MNU

s p ~ i m , by c. p,

- 9 weeks -- PEC - 4 5 - 2 7 64415 45 418 52 - 9 4 0 - 3 7 45-41

DBA CBA DBA CBA DBA CBA

Fig. 4. Detailed presentation of the NK cell activity in the spleen and the peritoneal exudate ceils 0PECs) of DBA/2 and CBA mice 3 weeks (two experiments) and 9 weeks after the injection of 50 mg/kg MNU. Control values for spleen cells are given in open bars'; spontaneous NK cell activities are presented in the left two bars' (e); NK cell ac- tivities after the in vivo injection of 175 gg CP 4 days before the assay on the right (A). PECs were studied after CP stimulation only. The numbers in the lower part give the % specific lysis of PECs of con- trols and, connected by a small arrow, of mice of the specific strain at 3 or 9 weeks after the M N U injection. In all groups the higher number (left) represents the control values. The large arrows indicate complete loss of the stimulating effects o f CP on the splenic NK cell activity in CBA mice 3 weeks after the M N U injection. For standard deviations see results in Table I

Page 6: Leukemia induction by methylnitrosourea (MNU) in selected mouse strains

H.J. Seidel and L. Kreja: Leukemia Induction by Methylnitrosourea (MNU) 219

cells during the latency period o f tumor induction. These include a reduct ion o f N K specific lysis o f target cells after exposure to ure thane (Gorel ik and Herber- man 1981), to d imethylbenzanthracene (Ehrlich et al. 1980), and also to leukemogenic radia t ion (Parkinson et al. 1981; Gorel ik and Herbe rman 1982). We there- fore studied the N K cell funct ion in our experiments to see whether there would be changes at all, and fur- ther, whether there were strain differences. The N K - specific lysis by cells f rom the control animals showed, as expected, that the spontaneous activity is rather low in animals o f this age. After CP stimulation, however, the values are high, with small s tandard deviations, and are therefore more useful for a comparison. There is no doub t tha t reduced N K activities were found in m a n y groups o f M N U - t r e a t e d animals in the spleen and in PECs. The reduct ion in the spleen, when seen, canno t be explained by simple dilution o f N K cells due to an increase in organ size, since this was no t ob- served 3-12 weeks after M N U . Nevertheless, the N K cell studies remain difficult to discuss. The very low activity 3 weeks after M N U , especially in C B A mice, after CP st imulat ion will be studied further. The da ta fur ther suggest a recovery o f the N K cell funct ion in C B A mice and a depression in BDF1 and D B A / 2 mice 9 and 12 weeks after M N U .

This finding is o f great importance, since the trans- p lanta t ion studies in B D F j mice show that this is the time when the first animals have leukemic cells. The results in Table 1 show that these cells are first de- tected in the thymus, since six animals had transplant- able cells only in that organ. The bone mar row seems to be seeded f rom the thymus. The differences in sur- vival times between condi t ioned and uncondi t ioned recipients suggest tha t regulatory influences and /or defence mechanisms are active in this system.

I t was no t possible to tell in this experimental set- up for any individual mouse whether, for example, the N K cell reduct ion precedes the presence o f leukemic cells, for two reasons: a certain number o f leukemic cells mus t be present for successful t ransplantat ion, and when present, leukemic cells m a y act as cold tar- gets in the N K cell assay, as has been recently found with a BNU- induced leukemia, L 40 (Seidel et al. 1983).

The results o f our s tudy indicate that the acute tox- icity o f M N U varies little a m o n g the strains, a l though there are marked differences in susceptibility to the leukemogenic effect - in terms o f the dura t ion o f the latency period. Fur ther studies will have to consider the fact that mouse leukemia viruses can be induced by M N U (Fey et al. 1980) and their possible influence on parameters in this study. They will fur ther have to in- clude the analysis o f T cell subsets in the thymus, and the N K cell system in individual mice, in dependence

on the presence or absence o f leukemic cells, which could be useful for a fur ther analysis o f the pa thways o f T cell leukemogenesis.

Acknowledgements. The authors appreciate the excellent assistance of E. Barthel and K. Steinhoff. The study was supported by the Deut- sche Forsehungsgemeinschaft (SFB 112) and the Bundesministerium des Inneren.

References

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Received September 30, 1983/Accepted April 26, 1984