Haemopoiesis and immune status during BNU-induced leukaemogenesis in mice

J Cancer Res Clin Onco198, 31-39 (1980) ~um~l of

~Cancer Research Clinical Oncology �9 Springer-Verlag 1980

Haemopoiesis and Immune Status During BNU-induced Leukaemogenesis in Mice***

H.J. Seidel

Abteilung Klinische Physiologic und Arbeitsmedizin, Universitfit Ulna Oberer Eselsberg, D-7900 Ulm/Donau, Federal Republic of Germany

Summary. Female BDF 1 mice were exposed to 0.04% BNU in the drinking water for 15 weeks. All animals except one observed for more than 3 weeks died ofleukaemia. After 1, 2, 3, 5, 7, 10, and 15 weeks of exposure marrow smears, pluripotent (CFU-S) and ganuloid committed (CFU-C) stern cells from bone marrow and spleen, and the lymphocyte response to different T- and B-cell mitogens and to alloantigens were determined. There was a progressive fall of the CFU-S and CFU-C content of the femur and even more in the spleen, and a progressive decrease in the number of normal marrow lymphocytes. After 10 and 15 weeks new "lymphoid" cells, presumably leukaemic cells, were seen in marrow. The lymphocyte stimulation assays showed a depressed reactivity, rather uniform in the thymus after more than 1 week and also, with a partial recovery after 3 weeks, in the spleen. The implications of these results for the chemically induced leukaemogenesis are discussed.

Key words: Chemical leukaemogenesis - Haemopoietic stem cells - Immune status

Leukaemogenesis is still poorly understood and much effort has gone to experimental models for the disease. In addition to C-type viruses, such as the Gross virus, and radiation several chemicals regularly induce leukaemias in mice and rats. Among these methylnitrosourea (MNU) and butylnitrosourea (BNU) have been of special interest to us, since both substances induce leukaemias in susceptible strains of mice in rather high frequencies and within acceptable latency periods (Yokoro et al. 1973; Dexter et al. 1974; Shisa and Matsudaira 1975). It may well be that, as demonstrated for other models (Ball and McCarter 1971; Nexo and Ul- rich 1978), viruses are also associated with these models of leukaemogenesis.

In these studies we were interested to see which changes in the haemopoietic cell system precede the clinical onset of leukaemia and whether a specific preleu-

* Supported by the Deutsche Forschungsgemeinsehaft SFB 112 and Ehlep contract with Euratom ** With the technical assistance of E. Barthel and K. Steinhoff

0171-5216/80/0098/0031/$1.80

32 H.J. Seidel

k a e m i c c o n d i t i o n o f m i c e e x p o s e d to B N U c o u l d be iden t i f i ed w h i c h is n o t on ly

cha rac t e r i s t i c fo r this m o d e l o f l e u k a e m o g e n e s i s b u t a lso re la ted to changes ob -

se rved af te r a l e u k a e m o g e n i c dose o f M N U (Seidel 1977 a) o r a l e u k a e m o g e n i c re-

g i m e n o f d i m e t h y l b e n z a n t h r a z e n e (Seidel 1977 b). T h e a i m o f these s tudies is to de-

f ine a c o m m o n p a t h w a y o f l e u k a e m o g e n e s i s , i f exis t ing, a n d to desc r ibe a h a e m o -

po ie t i c s ta tus t h a t r egu la r ly leads to the d e v e l o p m e n t o f ove r t l eukaemia . Th is

p a p e r desc r ibes p r e l e u k a e m i c c h a n g e s in the h a e m o p o i e t i c sys tem i n c l u d i n g the

h a e m o p o i e t i c s t em cells a n d the i m m u n e s ta tus o f mice c h r o n i c a l l y e x p o s e d to B N U .

Material and Methods

M i c e

Female BDF, mice (GI. Bomholtgard, Ry, Denmark) and CBA/Ca mice (Zentrale Tierversuchsanlage, University of Ulm) of 20-23 g weight were used. Ten animals per cage were kept in artificial light 12 h daily. They were fed commerciall pellets. Twenty mice had BNU in the drinking water and their spontaneous deaths were recorded. In all other studies with mice exposed to BNU and the respective controls, requiring the killing of the animals, four to six mice were used for each experimental point. After 15 weeks of BNU exposure 10 animals were studied.

Experimental Procedures

Animals were killed by cervical dislocation. The spleen weight was determined and a single cell suspen- sion of the spleen and the bone marrow of a femur was prepared in ct medium (Flow Laboratories) containing 270 fetal calf serum (Seromed). Appropriate cell concentrations were obtained by dilution after counting in a Coulter Counter. The ceils from the other femur were taken for bone marrow smears and stained with May-Griinwald-Giemsa solution. Differential counts were made under oil immersion (x 100); 500 cells/animal were classfied as previously described (Seidel 1972). In short: Erythropoiesis E1-E s, E1 is the largest cell with dark cytoplasm (proerythroblast), E~ the smallest nucleated cell with polychromatic cytoplasm. Granulocytopoiesis M~-M 8. M~ is the myeloblast, M~ the promyelocyte, M 3 and M, are myelocytes, M s is the metamyelocyte, M 6 and M 7 are juvenile and band forms, M 8 is the mature neutrophil granulocyte. M1-M . are combined as proliferative granulopoietic cells, M~-M~ as nonproliferating cells.

CFU~ Experiments

The procedure described by Till and McCuUoch (1961) was used to assay marrow and spleen colony forming units (CFU~). The pooled marrow or spleen cells were injected i.v. (1-6 x 104 marrow cells and 1-6 x 105 spleen cells in 0.25 ml) into irradiated recipients (10 animals per group). Radiation consisted of 800 rads exposure, 280 KV, 12 mA, 1.6 mm Cu and 1 mm A1 filter, focal distance 50 era, rate 30 R/ rain. Nine days after injection of cells the recipient animals were killed, their spleens were removed and placed in Bouin's solution, in preparation for counting macroscopic colonies after 24 h in the fixative.

CFU c Experiments

Cells from bone marrow or spleen were incubated in 3.5 cm plastic dishes containing 1 ml of~ medium (Flow) with 2070 horse serum (Seromed) and 0.370 agar. Colony growth was stimulated by the use of heat inactivated serum obtained from NMRI mice 3 h after i.v. injection of 50 ~g endotoxin (salmonella abortus equi, Difco). Optimal stimulation was obtained by adding 12.5 or 25 ~tl of endotoxin activated serum. The cell number per plate was 2-0.5 x l0 s for bone marrow cells and 2-0.5 x 106 for spleen cells. The cultures were done in triplicate and incubated in 570 CO2 at 37 ~ in a humidified atmosphere. After 7 days of incubation the colonies (> 50 cells being a colony) were counted with 40-fold magnification. At each experimental point, control cultures with normal bone marrow and spleen cells were included. The CFU c number was calculated from the pooled results of two different cell numbers plated. The number of colonies per dish varied about 10% of the mean.

Chemical Leukacmogenesis in Mice 33

Table 1. Bone marrow differentials and femur cellularity of BDF~ mice exposed to 0.04% BNU in the drinking water

Duration of Cells per Lymphoc. MI_ 4 Ms- s E1-5 Others exposure femur

no BNU 20 • 106 38% 4.9% 36.9% 18.4% 1.8% 1 week 13.9 36.0 5.2 38.0 20.0 0.8 2 weeks 14.2 32.7 5.2 42.4 17.7 2.0 3 weeks 15.6 22.4 6.2 37.9 31.4 2.1

Table 2. Bone marrow differentials and weight of thymus and spleen of BDF1 mice exposed for 15 weeks to BNU. The mice are listed with increasing thymus weight. Cells from mice with* were taken for the tests described in the text and in Fig. 1 and 2 and Tables 1-3. For controls see Table 1

Mouse no. Thymus Spleen Bone marrow differential mg mg

"lymphoid" lymphoc. M1-4 Ms-s E1-s

7* 22.7 38.8 6.6% 17.1 5.3 26.9 40.9 9 30.8 46.3 4.3 11.0 6.7 25.4 49.3 6* 32.7 46.3 2.2 3.2 4.0 38.5 50.9 4* 35.5 58.4 n.d. n.d. n.d. n.d. n.d. 3* 75.0 55.6 16.1 7.4 2.1 20.2 51.5 5 129.9 129.9 91.0 5.3 0 0 0.3 2* 169.9 63.8 7.0 2.5 2.9 24.0 61.2 1 185.7 440.0 86.9 0 0.3 6.2 1.8

10 442.7 69.8 52.2 2.5 4.4 11.0 27.4 8 607.3 190.6 63.5 6.5 1.2 23.3 2.2

Butylnitrosourea (BNU)

BNU from Serva (Heidelberg, FRG) was dissolved in tap water at pH = 5, 400 rag/1. This was freshly prepared three times per week and given as drinking water to the experimental groups.

Lymphocyte Stimulation

Spleen and thymus cells were cultured in c~ medium with 10~ fetal calf serum (FCS) and antibiotics in microtiter plates (Greiner, Niirtingen, FRG) in an end volume of 0.2 ml. 5 x l0 s cells were stimulated by: Concanavalin A (Con A, Pharmacia Fine Chemicals, Uppsala, Sweden) 4 7/ml, phytohemag- glutinin P (PHA, Difco, Detroit, USA) l:500, lipopolysaccaride B (LPS, E coli 026:B6, Difco) 50 ~/ml, pokeweed mitogen (PW, Gibco, Grand Island, USA) 1:80. Cultures were terminated after 48 hrs using a multiple cell culture harvester (Skantron, Lyerbee, Norway), after addition of 0.2 gCi 3H-thymidine (specific activity 2 Ci/mmol, Amersham, England) per well 16 h previously. Radioactivity on the filter paper disks was determined in a Searle Mark II scintillation counter in Rotisziut 11 (Roth, Karlsruhe, FRG).

Mixed lymphocyte cultures (MLC) were prepared under the same conditions but with 5~ FCS. Stimulator cells were 5 x 105 CBA/Ca spleen cells after irradiation with 1,200 rad, the total duration of the culture was 3 days. The stimulation was determined by subtraction of the counts in unstimulated cultures or, in case of the MLC, by subtraction of the counts after isogeneic stimulation with irradiated control cells and related to the counts obtained with cells from control animals. In a typical experiment the following counts were measured in stimulated control cells: Spleen cells stimulated by Con A: 57,000 cpm, by PHA 12,000 cpm, by LPS 54,000 cpm, by PW 7,000 cpm; thymus cells stimulated by

34 H.J. Seidel

Con A 14,000 cpm, by PHA 1,000 cpm, by PW 4,000 cpm. MLC with spleen cells 5,000 cpm, with thymic cells 1,000 cpm. In the results section the cpm of experimental groups is indicated by symbols: )" re- presenting values above 120~ of controls, I 80~-120~ of controls, ; 50~0-79~ and 1~ below 50~o of controls. All cultures were made in triplicate.

Results

Leukaemogenesis

Twenty BDF 1 mice were fed 400 mg/1 BNU in the drinking water for 15 weeks and the time and cause of death (macroscopically) was recorded. Only 14 animals could be followed after more than 3 weeks of exposure, all except 1 of these died with leukaemia or were killed when severely sick. The mean induction time was 125 days.

At 15 weeks of exposure another group of 10 animals was killed for the studies presented below. Six out of these 10 had macroscopically leukaemia and, as described later in more detail, the remaining four had large lymphoid cells in the marrow, presumably leukaemic cells.

Bone Marrow Smears

Bone marrow meats were analysed at 1, 2, 3, 5, 7, 10, and 15 weeks of exposure to BNU. The results are presented in Tables 1 and 2. Smears from animals 1-3 weeks after beginning of the experiments could easily be summarized since no ma- jor differences were observed between individual animals of one group. There was no change in the percentage of immature or more mature granulocytic cells, the absolute number of cells per femur however was slightly reduced. The number of erythroblasts was relatively increased after 3 weeks of BNU exposure and this increase seemed to be correlated with a relative decrease in the number of bone marrow lymphocytes. After 5, 7, and 10 weeks granulocytopoiesis remained unaf- fected, i.e., M t - 4 and M s - 8 were present in about normal percentages. The relative number of lymphocytes was however, reduced in three of five animals at 5 weeks, two of five at 7 weeks, two of four at 10 weeks to 8 ~ - 1 6 ~ in the smears.

The results of 10 mice after 15 weeks of BNU are presented in detail in Table 2. The mice were listed with increasing thymus weight. Macroscopically, the six mice with a thymus weight of 75 mg or more were considered leukaemic, but enlarged lymph nodes were not found. Bone marrow and spleen cells from mice nos. 7, 6, 4, 3, and 2, and thymic cells from mice nos. 7, 6, 4, and 3 were assayed in the tests described below. The bone marrow smears show a cell type of "lymphoid cells" which is characterized by a spongy chromatin and some chromatin condensations, visible nucleoli, and dingy blue cytoplasm with vacuoles. These cells varied in size. In all animals only few classical bone mar row lymphocytes were found. When the relative number of lymphoid cells, possibly the leukaemic cells, exceeded 16~, there was a decrease in the erythroblasts and also the granulocytic cells were reduced. This analysis, however, is somewhat lacking since the cell number per femur could not be presented for each animal. When the smears of mice after 10 weeks were reevaluated, some lymphoid cells were also found in those mice with low number of normal bone marrow lymphocytes.

Chemical Leukaemogenesis in Mice 35

% lOO

50

0

BNU CFU-S

, "K~. / / ~ �9 per spleen

. �9 " ~ _ ~ . . . . 4 t_ - , . . . . . . e. I I "I. 0 1 2 3 5 7 10 15 weeks

Fig. 1. Number ofpluripotent stem cells (CFU-S) in one femur and the spleen of BDFI mice chronically exposed to 0.04~ BNU in the drinking water in ~ of control values, determined also at the intervals indicated. The data of individual runs are connected by lines. The shaded area represents the control values +SEM, rising in the femur from ~4,400 CFU-S at the beginning to 5,200 at the end of the experiments, in the spleen from 800 to 1,000

% 10o

50,

8NU CPU -C

t ~ . . per femur / / I ~ �9 per spleen /

. .Oh �9

0 1 2 3 5 7 10 15 weeks

Fig. ~. Number of granuIoid determined stem ceils (CFU-C) in one femur and the spleen of BDFI mice chronically exposed to 0.04% BN U in the drinking water as Fig. 1. Control numbers were 15,000-18,000 CFU-C per femur and 700 to 1,300 CFU-C per spleen

Pluripotent ( CFU-S) and Granulocytie Committed (CFU-C) Stem Cells

C F U - S and C F U - C stem cells were analysed in bone m a r r o w and spleen at the intervals indicated. The results are presented in Figs. 1 and 2. There was an immedi - ate fall o f C F U - S in the spleen with C F U - S numbers be low 2 0 ~ o f cont ro l s t h roughou t the exper iment . The C F U - S o f the femur fell to values between 3 0 ~ - 5 0 ~ at 5 weeks and fur ther decl ined dur ing the next weeks. The kinetics o f the C F U - C were ra ther s imilar with a slight recovery in the spleen between the sec- ond and the th i rd week o f exposure and a (single) nea r no rma l value a t 15 weeks, whereas they remained low in the bone mar row. I t seems wor th ment ion ing that , in con t ras t to the C F U - C number , recognisable g ranu lo id precursors in the mar - row smears r emained no rma l in relat ive numbers up to 10 weeks o f B N U exposure and even after 15 weeks (Table 2). F o u r o f 10 animals had a b o u t no rma l numbers o f prol i fera t ive M 1 _ 4.

36 H.J. Seidel

Table 3. Stimulation of splenic lymphocytes by different mitogens of mice exposed to BNU. Further explanation see text

Duration Con A PHA PW LPS of exposure

lweek LLII TIll& IgUl~ $ ~ g 2 weeks ]. $ II) ITI ~ II!UU &~U 3 weeks t111 liT J, ~'11 ~ ,LI 5 weeks $ ~ $ ~ ~ J~

10 weeks ~ ~l ~ 15 weeks ~ 1) ~ J.

1=80%-120% of controls, "~= > 120%, ~=50%-79%, = < 50%

Table 4. Stimulation of thymic lymphocytes by different mitogens of mice exposed to BNU

Duration Con A PHA PW of exposure

lweek TST~ T~[ ~ T]'~ 2 weeks tl It 1~ 1} ~ UIt 1~ ~gl) 3 weeks ]" ~I It lI g l~ II II $ lI 5 weeks ~11) ~ ~ It

10 weeks 1) ~) ~) 15 weeks I/ 1~ lI

1=80%-120% of controls, $= >120%, $=50%-79%, ~) = <50%

Table 5. Mixed lymphocyte reaction of spleen and thymic cells of mice exposed to BNU, stimulated by 1200 rad irradiated lymphocytes from normal CBA/Ca mice

Duration of exposure Origin of lymphocytes

Thymus Spleen

1 week II] 1" T 1~ It J. 2 weeks 1} tl ~I ~ ~ g II 3 weeks 1~ I~ 1~ J. lIII 5 weeks ~l ~ .L

10 weeks n.d. l) 15 weeks n.d.

1=80%-120% of controls, $= >120%, ,L=50%-79%, It = <50%

Immune Reactivity

Cells f r o m the spleen a n d the t h y m u s o f B N U - t r e a t e d a n i m a l s were assayed by s t i m u l a t i o n u s i n g T- a n d B-cell m i t o g e n s a n d in the m ixed l y m p h o c y t e cu l tu re (MLC) . T h e resul ts o f several r u n s a re p r e sen t ed in s u m m a r y in Tab le s 3-5, The o r g a n weights o f t h y m u s a n d spleen are g iven in Fig. 3.


mg

40

2O

BNU thymus weight

..... ~ ,~ . : ~ e ~ ~ ~ - ~ ~ ~:~1 ~ .~ ~. ;...~,~;~ ~ . - ~ ~ e ~ ~ : : - : ~ : ~ t . ~ ~ s ~

; ' ' ' ' 'o " ' 1 2 3 5 1 15 weeks

mg

100,

50

BNU spleen weight

, , , , ; , i t , 0 1 2 3 10 15 weeks

Fig. 3. Thymus and spleen weight of BDF 1 mice exposed to 0.04% BNU in the drinking water. The shaded areas represent the mean weight of all control organs + S.D. as determined also at the intervals indicated for the experimental groups

After 5 or more weeks of BNU exposure a uniform depression of T- and B-cell reactivity was found. The response of thymic lymphocytes to Con A, PHA, and PW was below 50% of controls and splenic lymphocytes showed a reduced B cell response in addition. Also the MLC was reduced.

During the first weeks the results were rather heterogenous. In three of four experiments the Con A response of the thymus was enhanced after 1 week and also in some experiments the response to PHA and PW. Splenic lymphocytes showed in three of four experiments a very low stimulation by LPS and PW and were normal, depressed, or enhanced in their response to PHA and Con A. After 2 weeks of exposure the thymic response was very low throughout and the spleen remained almost unaltered. After 3 weeks a partial regeneration was observed in the spleen, but not in the thymus. The MLC followed the pattern of the Con A responses. The first arrow in each set of results represents the first experiment. The morphological data and the CFU-S and CFU-C numbers presented before are from the mice of this first series. In mice without macroscopic leukaemia after 15 weeks of BNU only marginal responses of T and B cells were seen.

Discussion

It was the aim of these studies to describe the effects of a highly potent leukaemo- gen, BNU, on the haemopoietic cell systems in the mouse before overt leukaemia

38 H.J. Seidel

had developed. In the experiments presented 13 of 14 mice, which were observed for a prolonged period, did develop leukaemias. In another group of 10 mice, killed at 15 weeks of exposure, six already had leukaemias as seen from the enlargement of the thymus, lymphoblasts in the marrow smears, and enlarged spleens. We know from studies in other laboratories that these leukaemic cells are 0-positive (Baines et al., pers. commun.).

The results of the different assay systems show that both pluripotent stem cells and immune cell functions are affected by the exposure to BNU. The granuloid committed stem cells did decline rather parallel to CFU-S and these alterations seem to be without effect on recognisable granulocytopoiesis in the marrow smears during the first 10 weeks. Pluripotent stem cells declined continuously to values below 25~o at 10 and 15 weeks. The spleen was much more sensitive in these assays than the bone marrow.

In other models of murine leukaemogenesis, especially the AKR leukaemia, such a depression of haemopoietic stem cells CFU-S and CFU-C in bone marrow and spleen was not seen. A slight increase in CFU-C has been reported in virus superinfected preleukaemic AKR mice in one group (Hays et al. 1976, 1978), a decrease of CFU-S from the bone marrow in already leukaemic mice was compen- sated by an increase of the CFU-S number in other organs in studies in another laboratory (Chevalier et al. 1974; Frindel and Chevalier 1975; Sainteny and Frindel 1979). Studies on stem cells in other models of chemical leukaemogenesis are not available, but our own reports on methylnitrosourea and dimethylbenzanthrazene (Seidel 1977 a, b) show that these compounds given in a leukaemogenic regimen, are also toxic to CFU-S and CFU-C. It will have to be clarified in the present mod- el whether this depression just reflects toxicity or is also related to final develop- ment of leukaemia. The toxicity might well be related to the activation of endog- enous viruses as shown to occur in models of chemical leukaemogenesis in mice by cell-free transmission (Ball and McCarter 1971) and more modern technology (Nexo and Ulrich 1978).

The lymphod system was analysed by stimulation of its T- and B-cell subpop- ulations by lectins and alloantigens, also by determination of the thymus and spleen weight and the number of bone marrow lymphocytes. The results of these assays seem somewhat heterogeneous at the beginning but certainly after 5, 10, and 15 weeks of exposure the responses in the stimulation assays were clearly reduced in the thymus and in the spleen, the number of normal bone marrow lymphocytes was reduced and - already after 1 week of BNU - thymus and spleen were reduced in weight. The better responses of T-cells in the thymus after 1 week in most experiments may be explained by a relative enrichment of reactive cells, possibly by a decreased influx of more immature non-reacting cells from the marrow, which, after 2 and more weeks, could explain the depression observed then. In the spleen the T-cell reactivity remained relatively stable for 3 weeks, the B-cell reactivity was very much reduced at 2 weeks.

More sophisticated methods should be applied to the appearance of "lymphoid cells" in the marrow after 10 and more weeks of BNU exposure. These cells were seen in small numbers even before the thymus was increased in weight, but from the presented data nothing can be said of the origin of these cells, whether they originate in the marrow and later migrate to the thymus or whether they originate


in the thymus and migra te to the bone m a r r o w and the spleen. Resul ts in o ther models suggest the bone m a r r o w origin o f leukaemic cells ( H a r a n G h e r a 1977,

1978). Obvious ly , the results of this s tudy do no t clarify the ma in ques t ions of mur ine

l eukaemogenes i s : the ques t ion of the target cell, the funct ion of the i m m u n e system, and the role o f viruses as aet iological factors. But fur ther studies in this and re la ted models should, e.g., show whether the depress ion o f the haemopoie t i c stem cell system over a p ro longed pe r iod is a pre leukaemic cond i t ion or jus t a toxic effect of the chemicals , pe rhaps necessary for the ac t iva t ion of viruses. The r educed immune funct ions m a y also represent a regular step in the process o f l eukaemogen- esis.

References Bathes P, Moore M, Schofield R (Pets. Cow_man.) Study of BNU-~nduced leukaemia in RF mice Ball JK, McCarter JA (1971) Repeated demonstration of a mouse leukemia virus after treatment with

chemical carcinogens. J Nat Cancer Inst 46:751-762 Chevalier C, Gaillord N, Frindel E (1974) The number of in vitro stem cells in AKR leukemic mice.

Blood 44:743-750 Dexter TM, Schofeld R, Lajtha LG, Moore M (1974) Studies on the mechanisms of chemical leukemo-

genesis. Br J Cancer 30:325-331 Frindel E, Chevalier C (1975) Measurement of the number of bone marrow multipotential stem cells

in AKR leukaemic mice. Biomedicine 23:166-167 Haran Ghera N (1978) Spontaneous and induced preleukemia cells in C57/B1/6 mice. J Natl Cancer

Inst 60:701-710 Haran Ghera N (1977) Target cells involved in radiation and radiation leukemia virus leukemogenesis.

In: Duplan JE (ed) INSERM Symp. No 4. Elsevier, pp 74-98 Hays EF, Craddock CG, Haskett D, Newell M (1976) In vitro coloy-forming cells in the marrow of

leukemic and preleukemic mice. Blood 47:603-610 Hays EF, Haskett DR, Kaspersky CJ, Craddock CG (1978) Alterations in bone marrow and thymus

lymphocytes in virus inoculated, inbred, preleukemic AKR/J mice. J Natl Cancer Inst 60:905-909 Nex~ BA, Ulrich K (1978) Activation of C-type virus during chemically induced leukemogenesis in

mice. Cancer Res 38:729-735 Sainteny F, Frindel E (1979) AKR leukemogenesis and the hemopoietic stem cell compartment. Exp He-

matol 7: [Suppl 6] 71 Seidel HJ (1972) Die Blutzellbildung bei der Rauscherleuk~imie (Mgusestamm Balb/c) und ihre Beein-

flussung durch Hypertransfusion. Z Krebsforsch 77:155-165 Seidel HJ (I 977 a) Hematologic effects of a single dose of methylnitrosourea. Exp Hematol 5:19-26 Seidel HJ (1977b) The effect of dimethylbenz(a)anthrazene on murine hematopoiesis. Z Krebsforsch

89:221-229 Shisa H, Matsudaira Y (1975) Origin of leukemic cells in mouse leukemia induced by N-butylni-

trosourea. Gann 66:3742 Yokoro K, Takizawa S, Kawamura Y, Nakano M, Kawase A (i973)Multicarcinogenicity of N-ni-

trosobutylurea in mice and rats as demonstrated by host conditioning. Gann 64:193-196

Received October 29, 1979/Accepted March 22, 1980

Documents

Haemopoiesis and immune status during BNU-induced leukaemogenesis in mice