zeit~cnr~fl

Blut 38, 349-360 (1979)

BlUr ~u o r ~

9 Springer-Verlag 1979

Effect of Actinomycin D and Busulphan on Stem Cells in Normal and Friend Virus Infected Mice H.-J. Seidel and U. Opitz Abteilung KlinischePhysiologieder Universitht Ulm, Oberer Eselsberg, M 24, D-7900 Ulm/Donau, Federal Republic of Germany

Die Wirkung von Actinomycin D und yon Busulphan auf die Stammzellen normaler und Friend-Virus infizierter M/iuse Zusammenfassung. Normale und Friend-Virus (FV-P) infizierte NMRI und

DBA/2-Mfiuse wurden mit Busulphan, Actinomycin D und einer Kombination beider Substanzen behandelt. Nach FV-P-Infektion entsteht ein neuer Zelltyp mit der F/ihigkeit, in vitro ohne Zugabe yon Erythropoetin (Ep) erythropoetische Kolonien (CFU~) zu bilden, die die normalen Ep-abhfingigen vollst/indig ersetzen. Nach der Chemotherapie wurden diese und normale CFUz, sowie CFU~ (pluripotente Stammzellen) und CFUe (granulopoetisch determinierte Stammzellen) untersucht. Busulphan (30 mg/kg oral) reduzierte CFUs und CFUc in normalen und FV-P-infizierten M/iusen in gleichem Ausmal3. Einige normale, Ep abh/ingige CFUE, die vor Therapie nicht vorhanden waren, wurden nach 6 Tagen im Knochenmark, nicht jedoch in der Milz, gefunden. Actinomycin D (2 • 120 #g/kg s.c.) reduzierte das CFU~-Wachstum bei normalen M/iusen in der Milz stfirker als im Knochenmark, bei den FV-P-infizierten Tieren war das Wachstum in beiden Organen vollst/indig unterdr/ickt. W/ihrend der nachfolgenden Regeneration wurde kein Ep-abh/ingiges Wachstum beobachtet. Die Kombination beider Substanzen unterdrfickte das CFU~Wachstum ffir mindestens 4 Tage im Knochenmark, ftir 9 Tage in der Milz. Die Ergebnisse werden diskutiert im Hinblick auf den Ursprung der Ep-unabhfingigen CFUE in den Stammzellkompartments und die M6glichkeiten der Chemotherapie. Sehliisselw6rter: Friend-Leuk/imie - Stammzellen - Chemotherapie Summary. Busulphan, Actinomycin D, and a combination of both were used

to treat normal NMRI and DBA/2 mice and mice with Friend virus induced polycythemia. In FV-P infected mice a new cell type is found after virus in* Supported by the Deutsche Forschungsgemeinschaft,Sonderforschungsbereich112, Project B1 0006-5242/79/0038/0349/$ 2.40

350

H.-J. Seidel and U. Opitz

fection, which gives rise to erythropoietic colonies (CFUE) in vitro without addition of Erythropoietin (Ep), which completely replace normal Ep dependent CFUE. After treatment, CFUs, CFUo, and CFU~, were studied. Busulphan (30 mg/kg p.o.) did reduce CFU~ and CFUe growth in virus infected and control mice to the same extent. Six days after Busulphan in the bone marrow, but not in the spleen, some Ep dependent CFU~ were observed in the virus infected animals, not seen before treatment. Actinomycin D (2 • 120 #g/kg s.c.) suppressed the CFU~ growth in normal mice more effectively in the spleen than in the bone marrow. In FV-P infected mice Act D induced a total suppression of CFUE colony growth in both organs. During regeneration no normal Ep-dependent CFUE growth was observed. The combination of both drugs completely suppressed CFUE growth for at least 4 days in the marrow and for 9 days in the spleen. During the regeneration all CFUE growth was Ep independent. The results are discussed with respect to the origin of the Ep independent colonies within the stem cell compartments and the possibilities of chemotherapy. Key words: Friend leukemia - Stem cells - Chemotherapy.

Murine leukemia models can be used to study the interaction of leukemic cell development with normal hemopoiesis, especially the stem cell compartments, and also the action of cytotoxic drugs on normal and leukemic hemoP0iesis. In chemotherapy studies it is of great importance to know how leukemic cell clones and normal stem cells are influenced by the drugs, since stem cell toxicity can be a limiting factor for successful treatment. Ideally this should be studied in the same individual. We here report studies using the Friend leukemia (induced by the polycythemic strain of the Friend virus, FV-P) as an experimental model with several advantages. After infection with the FV-P an erythropoietin (Ep) independent erythropoiesis develops which can selectively be measured in vitro by the CFU-E technique [2, 4, 6]. This new cell population originates in the stem cell compartments from a target cell between early (BFU-E) and late erythropoietic (CFU-E) precursor cells and can be considered an autonomous or tumor cell population [7]. With time after infection this Ep-independent erythropoiesis replaces completely the normal Ep-dependent one. The other stem cell compartments CFU-S (pluripotent stem cell) and CFU-C (granulocytopoietic committed stem cell) are also influenced by the virus, but autonomy of growth could not be demonstrated [1,121. In the peripheral blood, continuing reticulocytosis leads to an increase of the hematocrit. We tried to answer 2 questions using Actinomycin D (Act D) and Busulphan (BU) as cytotoxic agents. First, whether a specific action of these drugs on the "leukemic" versus the normal erythropoiesis - as tested in infected and uninfected animals - would be observed. Second, whether these drugs would reinduce the growth of normal, i.e., Ep-dependent erythropoiesis in FV-P infected mice. Act. D was chosen in a dose which almost selectively suppresses erythropoiesis [9,10], BU has a rather opposite action, since it mainly effects CFU-S and CFU-C [5, 8] and has a much smaller effect on erythropoiesis.

Effect of Actinomycin D and Busulphan on Stem Cells

351

Material and Methods 1. Virus The Friend virus, polycythemic strain (FV-P) was kindly provided by Prof. W. Sch~fer (Ttibingen/ Germany). It was serially passaged in N M R I mice. The mice were infected i.p. with 0.25 ml of a 10% cell-free homogenate of leukemic spleens (2-3 g in weight) in saline. 2. Mice Female N M R I mice (S~idd. Versuchstierfarm, Tuttlingen, Germany) and in a single experiment DBA/2 mice (Zentralinstitut for Versuchstiere, Hannover, Germany) of 19-23 g weight were used. Ten animals per cage were kept in artificial light 12 hours daily. They were fed commercial pellets and water ad libitum. 3. Experimental Procedures Four to six mice were used for each experimental point. They were killed by cervical dislocation, the spleen weight was determined and a single cell suspension of the spleen and the bone marrow of a femur was prepared in ~ medium (Flow Laboratories) containing 2 % fetal calf serum (Seroreed). Appropriate cell concentrations were obtained by dilution after counting in a Coulter Counter. Blood for hematological studies was taken from the retroorbital sinus and studied by standard procedures. 4. CFUs Experiments The procedure described by Till and McCulloch [11] was used. The pooled marrow or spleen cells were injected intravenously (2 • 104 marrow cells up to 1~ of a femur cellularity in the drug treated groups and 2-20 • 105 spleen cells in 0.25 ml) into irradiated recipients (10 animals per group). Radiation consisted of 800 rads exposure, 280 KV, 12 mA, 1.5 mm Cu and 1 mm A1 filter, focal distance 50 cm, rate 30 R/min. Nine days after injection of cells the recipient animals were killed, their spleens were removed, fixed in Bouin's solution and macroscopic colonies were counted. From these data the mean CFUs content per femur was calculated. No endogenously formed colonies were found in mice exposed to radiation and not engrafted with cells. 5. CFUc Experiments Cells from bone marrow (0.5-2.0 • 10a) or spleen (0.5 - - 2.0 • 100 were incubated in 3.5 cm plastic dishes containing 1 ml of g medium (Flow) with 20 % horse serum (Seromed) and 0.3 %00 agar. Colony growth was stimulated by the use of heat inactivated serum obtained from N M R [ mice 3 hours after i.v. injection of 50 #g endotoxin (salmonella abortus equi, Difco). Optimal stimulation was obtained by adding 12.5 or 25 #1 of endotoxin activated serum. The cultures were done in triplicate and incubated in 5 % CO2 at 37 ~ C 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, a control culture with normal bone marrow and spleen cells was included. The number of colonies per dish varied about 10% of the mean. 6. CFU~ Culture Procedure The method described by Iscove [3] was used. 0 . 8 ~ methylcellulose, 3 0 ~ fetal calf serum, Ep step III (Connaught Laboratories) 0.2 or 0.3 U/ml, depending on the batch, cr thioglycerol at an end concentration of 10-4 M and bone marrow or spleen cells in c~ medium were mixed in a final volume of 2.5 ml. 1 ml was plated in a 3.5 cm petri plastic dish (Greiner). For each experimental point 4 parallel cultures were set up. Colonies were counted after 48 h incubation at 37 ~ C in a humidified atmosphere containing 5 ~ CO2. Erythroid colonies with more than 8 small cells (erythroblasts) were scored without staining at a magnification of 80. As in controls day to day variations in the yield of erythroid colonies were found, predominantly dependent on the different batches of methylcellulose used and changes in pH during the preparation of the cell suspension and cultivation in the incubator, parallel cultures of controls were plated at each experimental point. The variation in the CFU~ numbers of parallel plates was mostly in the range of 10% In order to compare results more easily, CFUE numbers of mice after treatment were expressed in per cent of controls.

352

H.-J. Seidel and U. Opitz

7. Drug Treatment Busulphan was dissolved in 20% Tween 20 and 80% saline. A mouse of 20 g weight received

0.5 ml of an appropriate dilution by stomach tube. Actinomycin D was diluted in destilled water and 0.2 ml of the appropriate dilution, containing 120 #g/kg body weight, was given to a 20 g mouse subcutaneously.

Results

I. Development of Ep-independent Colony Growth after FV-P Infection As seen in Figs. 1 to 3 and described in detail previously [6] 12 to 15 days after FV-P infection the normal Ep-dependent CFU~ population was totally replaced by the Ep-independent (CFUI~I), the "leukemic" cell population. In these figures the number of CFUE grown in the presence of Ep was taken as 100 % line. When the same number of colonies grew with and without Ep, the CFU~ compartment was considered to be Ep-independent, i.e. "leukemic". II. Chemotherapy by BU (30 mg/kg p.o.) Peripheral blood (Table 1). Thirteen days after FV-P infection untreated mice had an increased hematocrit, elevated numbers of reticulocytes and a thrombocytopenia, persisting to days 18 and 26. BU treatment of infected mice had only a slight effect on the number of reticulocytes, which were still higher than in controls. Uninfected, BU-treated mice showed only a slight reduction of reticulocytes at day 18, i.e., 6 days after BU, and a thrombocytopenia at day 26. The thrombocytopenia of FV-P infected mice was increased also at day 26 by BU. Bone marrow and spleen (Table 2 and Figs. 1 a and b). The action of BU was striking in the reduction of the spleen weight of FV-P infected mice from 1945 mg at day 13 to 523 mg at day 18. Later the spleen weight had increased again. In the bone marrow and the spleen there was the expected fall of CFUs and CFUc to very low values one and six days after treatment. The same reduction was present in FV-P infected mice. The regeneration of CFUs seemed to be more rapid in FV-P infected than in control mice. In the CFUE concentration of the bone marrow and the spleen only a minor reduction was present in normal, BU treated

Table 1. Friend-Leukemia. Busulfan 30 mg/kg p.o., day 12 after FV-P infection Hct. (%)

Retic. (~,)

Throabocftes/m~ 3 x 103

51• I

58~ 11

day 13

FV-P FV-P + Bus. Bus.

57 • 1 55 • 1 51 • 2

205 • 50 128 • 70 68 • 13

day 18

FV-P FV-P + Bus. Bus.

57 • 4 57 • 8 48•

270 • 17 119 • 86 33• 7

167 • 196 • 852•

day 26

FV-P FV-P + Bus. Bus.

62 • 5 64 • 4 52 • 2

211 • 171 • 40 46 • 12

456 • 384 68 ~ 28 428 Z 250

controls (all)

950•

80

230 • 44 230 • 46 1134 • 98 59 73

Effect of Actinomycin D and Busulphan on Stem Cells

353

;~B -

~BB -

Z

n

-I-

I h

Ld

I LIkJ

1 IN

I 12

IB

I

I

I

16

IB

2~

DRYS RFTER FV-P

*

0

FV-P

B

El=

t 22

INFEs

~, FV-P + BU la EP

v FV-P + BU + EP

21;1~1

Z

IEB

El_ bA -i13_ 1.1_ v

BB-

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I 12

I Ill

I 15

f IB

I 2B

I 22

D,FIY5 RFTER gV-F INFEs b

e FV-F B EP

a. FV-P + BU ~ EP

v

FV-P + t]U + EP

Fig. 1. a (bone marrow) and b (spleen). CFUE concentration in bone marrow and spleen of FV-P infected NMRI mice with and without addition of Ep. Busulphan was given at day 12. The growth of cultures of infected but not treated mice in the presence of Ep is given as 100% line. Further explanation see text

mice. The regeneration of C F U s preceded that o f C F U s and CFUo. The action of B U on the C F U E c o m p a r t m e n t in FV-P infected mice is presented in figures 1 a and b. In the bone marrow, at days 13, 18, and 20 about 8 0 - 1 2 0 ~ of the C F U E were found to be Ep-independent. After treatment the number of CFUE in cultures plus Ep went up to 1 7 0 ~ at day 18 and in those without Ep up to 1 1 0 ~ . This indicates that during the regeneration o f the CFUE c o m p a r t m e n t s o m e C F U ~

354

H.-J. Seidel and U. Opitz

Table 2. Busulfan 30 mg/kg p.o., day 12 after FV-P infection bone marrow

cells/femur

control Busulfan

.~ FV-P

10.8 x 106 7.2 5.2

CFUs/femur CFUc/106 3040t220 200-300 275+-100

5.0

12~ 10

14.5

326~280

11.1 6.5 FV-P + Bus. 4.0

90t 10 90t20

17.2 15.8 7.0

6060d:430 80[;L~200 5400t240

6.3

2320t300

FV-P + Bus. control ~o Busulfan

FV-P

control Busutfan

FV-P FV-P + Bus.

weight

CFU-E/IO 5

483 = 100% 278 • 44 = 100~ 14% 90%

53%

143 mg 465~400 122 ca 200 1045 1100t400 1940

13% 400 = 100% 566 • 34 = 100% 12% 230% 11%

100%

460 = 100% 518 • 2B = 100% 14% 107% 170I 19%

spleen CFUs/108

ca 40

117 92 2570 532

420~180 ca 10

128 97 2510

2880t450 550t120 4820t430

1820

2500~630

ca 10

OFBc/107 CFU-E/IO 5 472 = 100% 44 • 0 = 100% 19% 53~ 11% 9% 510 = 100~ 171 • 20 = 100% 7% 42% 75% 12% 93 • 18 = 100% 62%

Table 3. Friend-Leukemia. Actinomycin D 120/~g/kg s.c., days 12 and 13 after FV-P infection Hct

(%)

Retic.

(%,)

Thrombocytes/mm3 x 103 972 -+ 152

c0ntr01s (all)

50 -+ 1

42 +

9

>,

FV-P" FV-P+AcLD Act.O

46• 59• 50 • 1

52• 137• 9 •

64 74 5

425• 92 278~ 83 1312 • 344

>,

FV• FV-P+Act.D ActO

47• 54• 44 • 5

97• 38• 3•

50 43 2

523• 35 468• 177 1116 • 208

FV-P FV-P + Acid Acid

51• 53 • 2 48 • 1

54• 5• 22 •

34 3 7

387• 107 333 • 51 1194 • 164

FV-P FV-P + Act.D Auto

55 59 • 2 50•

205 449 • 66•

126 17

910 288 • 122 816•

again were sensitive to Ep, i.e., they required the hormone for growth. Only 2 days later, no normal Ep dependent CFU~ colonies were present. In the spleen, the CFUE concentration was very much reduced and there was no difference in the colony counts with or without addition of Ep. III. Chemotherapy by Actinomycin D (120 #g/kg s.c. day 12 and 13 after FV-P infection) Peripheral blood (Table 3). No effect on the hematocrit was seen in control and FV-P infected mice. The reticulocyte numbers were very much reduced one and two days after the last dose in controls and regenerated at day 4. In FV-P infected mice the reduction was delayed, the minimum was observed at day 4, i.e., day 17 after infection. At day 23 a tremendous overshoot occurred. Thrombocytes were not influenced by the drug in controls or FV-P infected mice.

Effect of Actinomycin D and Busulphan on Stem Cells

355

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n

380

b_ "~

280

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112

14

DFIY5 FIFTER F V - P ~' FV-P 0 EP

I

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IG

IB

2B

INFECTION

A FV-P + FICT D B EP

v FV-P + FICT D + EP

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I

I

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I]5

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INFECTION

a FV-P + lqCT D 0 EP

v FV-P + B~T I) + EP

F i g . 2 a b. C F U ~ c o n c e n t r a t i o n i n b o n e m a r r o w (a) a n d s p l e e n (b) o f E V - P i n f e c t e d N M R I C o n t r o l s a n d m i c e a f t e r t r e a t m e n t w i t h A c t i n o m y c i n D a t d a y s 12 a n d 13

mice.

Spleen and bone marrow (Table 4 and Figs. 2a and b). The action of Act. D on the spleen weight was most prominent at day 15, i.e. 2 days after the last dose. The CFUs compartment was enlarged in the spleen at day 17 in normal and even more in FV-P infected animals. The CFUo concentrations were increased in the femora of normal mice at days 14 and 15 and also in the spleen at day 15. A reduction of the CFU-E compartment to 45 ~ in the marrow and 2 ~ in the spleen was

356

H.-J. Seidel and U. Opitz

Table 4. Chemotherapy of Friend-Leukemia. Actinomycin D 120 #g/kg s.c., days 12 and 13 after FV-P infection ~leen

bone marro~

control

.8 ~

:ells/femur CFgs/femur

CFUc/IO6

CFU-E/IO 5

14.1 x 106 2890:L610

400 = 100~

468 • 35 = 100~ 45g

weight

CFIJs/IO8

100mg 262~130

CFLIc/107

Act. B

9.3

288~120

198~

72

2530!150

FV-P

5,1

1215.t100

20~

338

1700t390

11%

Fv-P +Act.O

4,3

1720t100

175~

226

5150~260

30~ 540 =100~

72

224~

80~

830

114~

195~

160

13,3

600 = 100~

Act. D

12.1

1587,

4.7 7.1

FV-P FV-P +Act.O control

14.2

Act. g

13.4

FV-P

6.1

FV-P +Act.O

8.4

520 = lOO~ 1990t260 2030!160

48%

116

control

73~

175 • 29 = 100~ 280~

166 • 22 = 100~ 39~

-

109

-

110

4820~390

58~

700

38~

334

CFU-E/IO5

932= 10~ 81+- 16 = 10~

20 +- 15 = 100~ 174g

11~ 39 + 10 = 101~ 19~

10100i'900

seen one day after the last dose of Act. D, followed by an overshoot in both organs. At day 4, the CFU~ concentration had fallen again in the marrow and was increased further in the spleen. In infected animals, Act. D treatment resulted in the complete absence of colony growth one day after the last dose in the bone marrow and spleen. One day later, a few CFU~. colonies developed in the bone marrow cell cultures with and without Ep. After 2 additional days (17 days after FV-P and 4 days after the last dose of Act. D), an overshooting regeneration was seen in the bone marrow and spleen. The absolute number of the CFUE concentration rose from 50/105 at day 15 to 1600/105 in the marrow, and from 0/105 to 1800/105 in the spleen within 2 days. During the regeneration of the CFU~ compartment after Act. D treatment, no normal Ep dependent CFUE colony growth could be observed. IV. Combined Chemotherapy by BU and Actinomycin D The combined treatment with both drugs in even higher doses was used in order to eradicate more efficiently "FV-P transformed" cells and to study, if under these conditions normal Ep-dependent erythropoiesis would reoccur during the regeneration, arising from normal precursor cells. In this experiment, DBA/2 mice were treated with 60 mg/kg BU at day 13 and 120/~g/kg Act. D at days 12, 13, and 14 after FV-P infection. Peripheral blood (Table 5). In control animals the reticulocytes were almost completely absent at days 16 and 19 (4 and 7 days after the first drug treatment). A regeneration with numbers well above controls was seen at day 24 and more pronounced at day 32. In FV-P infected mice the hematocrit was reduced to normal levels by the treatment. This was the result of an almost total suppression of reticulocytes as seen at days 16 and 19. Thereafter a regeneration took place. The thrombocytes were reduced late after treatment in normal and also in infected mice. Spleen and Bone Marrow (Table 6, Figs. 3a and b)

Effect of Actinomycin D and Busulphan on Stem Cells

357

Table 5. Friend-Leukemia. Busulfan 60 mg/kg day 13 and Actinomycin D 120/~g/kg days 12, 13, and 14 after FV-P infection Hct.

{~)

Retie.

Thrombocytes/mm 3 x 103 1060• 126

44•

10

FV-P

50•

127•

15

FV-P FV-P + ~rugs Drugs

65t T 55t B 54• Z

224• 25 2 4 t 18 12 1

610• 85 62~t453 993•

FV-P ~, FV-P + Drugs Drugs

67 • 7 52• 48•

216 • 142 1• 2 1• 2

365 2 35 322• 586•

FV-P =-. FV-P + Oruqs

~rugs

65 • 7 48 • 4 45 t 1

69 • 126 • ~1 t

7 36 15

328~ 53 152 • 95 245•

FV-P FV-P + Drugs Drugs

69• 62 • g 50•

294• 60 309 • 110 120• 67

523• 112 338 • 293 256• 90

c~ controls (all)

51 • 1

(~)

667•

55

Table 6, Friend-Leukemia. Busulfan 60 mg/kg day 13 and Aclinomycin D 120 #g/kg days 12, 13,

and 14 after FV-P infection spleen

bone marrow cells/femur cgntrol

13,3 x 105 14.4

control drugs

10,5 6.5 10,4

FV-P FV-P + dr.

CFUJfemur 4380~220 /20~23~

0FUG/106 CFU-E/105 1320 = 10(]% 4(11 -+ 26 116% 970 = ION 72n -~ 114 = 100~ ~ Ig

145% 0%

7.I

weight

.CFUJIO8

112 1315 119

47~470

40

10% O%

13.5 6.9 9.7 5.6

46402 10 602 10 7700t4(]0 24~ 10

970 = 10~ 037 :~ 54 = 100% 0%o 13~ 192% 0%

108 38 2447

52002260 10210 4850t450

79

13~10

~onlro] drugs

13.3 10,1 8.6 7.6

-720~ ID

860 = 100% 501 ~ 58 = 100% 2% 105% 172% 2%

93 87 2070 295

control drugs FV-P FV-P + dr.

7350~700 8402 80

13.3 5,5

9,0 9.6

-

1240 = 100% 418 • 82 = ION 89% 243~ 133% 68%

110 147 2418 1517

CFU-E/I@ 5

O%

1800 120

control drugs FV-P FV-P + dr.

FV-P FV-P + dr,

CFUn/I07

2310 = 100%, 80 • 18 51% 2280 = 101~ 340 • 21 = 100%

118Nt500 3750~310 16602 80

3200 = 100% 198 • 25 = 100% 1% 33%

0% 2230 = 101~ 228• 33= 10~ 240 193% 29% 38% 2250 = 100% 72% 73% 37%

83 • 4 = 100% 600%

The CFUs and CFUo data mainly confirm the results obtained by BU alone. A regeneration was seen in control and infected mice in bone marrow and spleen at day 24 and was almost compleled in the CFU~ compartment at day 32. Erythroid colony growth in normal mice was completely suppressed 4 days after treatment (day 16) in the bone marrow and spleen. Regeneration began in the marrow at day 7 (day 19). Twenty days after drug treatment (day 32) a tremendous overshoot in the marrow and the spleen indicated an active regeneration of the

H.-J. Seidel and U. Opitz

358

258

-

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158-

--I12. t

u.

188

v Ld

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1,

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8-

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18

28

15

35

38

3E

DRY5 RFTER INFECTLON e FV-P I~ EP ~ FV-P -I- RCT D $ BU 0 EP v FV-P $ RCT D $ 6U + EP 258"-

288. z

Q.

Ld

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n I

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38

3E

DRY5 RFTER INFECTION ~' FV-P ~I EP ~. FV-P § FICT D § I~U Ol EP v FV-P + ACT D + 6U § EP

Fig. 3 a b. CFU~ concentration in bone marrow(a) and spleen (b) of FV-P infected DBA/2 mice. Controls and mice after treatment with Busulfan (day 13) and Actinomycin D (days 12, 13, 14)

erythroid cell system. In FV-P infected animals the complete suppression of erythroid colony growth was seen at day 16 in the marrow and in the spleen. Regeneration started in the marrow at day 19, control values were reached at day 24. In the spleen no growth was obtained at this stage. At day 26 an overshoot was present in the marrow and the spleen. All colony growth was Ep independent.

Effect of Actinomycin D and Busulphan on Stem Cells

359

Discussion

Chemotherapy of virus induced disorders can only be successful if the drug used definitively blocks the activity of the virus and prohibits a reinfection. From the results in this FV-P model it can be seen, that neither BU nor Act. D alone nor in combination were able to induce a long-term remission. The reocurrence of the FV-P induced polycythemia was obvious in all groups by the increase of the spleen weight, reticulocytosis, and increase of hematocrit. The stem cell compartments seemed to be equally sensitive to the drug action in controls and in FV-P infected animals. In both groups BU did suppress CFUs and CFUc to a much higher degree than the CFUE compartment, and Act. D had the opposite effect. Compared to controls, Act. D seemed to suppress the CFU~ growth in FV-P infected mice somewhat more effectively but the reticulocytes fell 1-2 days later than in controls (see Table 3). The reason for this remains unclear. The main question, however, was whether the regeneration after drug-induced aplasia would produce a normal hemopoiesis first, which later would be transformed again or if the regenerating cells would be "leukemic" from the very beginning of the regenerative process, as indicated by an Ep-independency. One would assume that normal Ep-dependent CFUE would be generated by normal precursors, most probably CFUs, whereas the immediate appearance of Ep-independent CFUE would have many interpretations, such as the descent from infected CFUs or any other transformed immediate precursor compartment or a very rapid reinfection, at least more rapid than during the induction of the disease where at least 10 days are required for a full Ep-independency of the CFU~ compartment. After BU-treatment indeed normal, i.e., Ep-dependent CFU~ growth was observed during the regenerative process in the marrow 6 days after the drug (text Fig. l a). Although it was only during one day in the experiments reported here, it could be repeated several-fold in the laboratory. In the spleen the regeneration was delayed about 4 days and all CFUE growth was Ep-independent (text Fig. 1 b). The resum6 would be that indeed during the regeneration some normal erythroid precursors are generated in the bone marrow, but they are completely replaced within a few days. Act. D was much more effective in the suppression of the CFUE growth without affecting CFU~ or CFUe. Within 2 days, however, the regeneration went from almost zero to 450 ~ of untreated controls in the marrow and 250 ~ in the spleen. All CFUE growth was Ep-independent. It must be assumed that a cell, which is already transformed, gives rise to this tremendous increase, and that many cells more or less synchronously reach a stage where they can form colonies again. Act. D would have prevented the passing of cells to this stage and their further maturation to reticulocytes as seen from the peripheral blood data. The idea that such a immature cell exists and may be the target cell for the virus, is supported by studies using Act. D treatment prior to virus infection [7]. The combination of Busulphan and Actinomycin D was chosen in an attempt for a more profound and more prolonged suppression of erythropoiesis. In the combination, both drugs were used in a higher dosage than in the previous experiments, which resulted in a complete suppression of the erythropoiesis seen in

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m a r r o w a n d spleen 4 days after drug treatment. The onset o f regeneration was delayed to days 8 (bone m a r r o w ) a n d 13 (spleen). T h e p r o l o n g e d suppression o f erythropoiesis a n d the simultaneous a t t a c k on C F U s a n d C F U E b y the drugs used did n o t reinduce n o r m a l erythropoiesis in F V - P infected mice. A s earlier results [7] s u p p o r t the hypothesis that the target cell for F V - P might be a cell intermediate between B F U - E a n d C F U - E , drug induced changes in the early e r y t h r o i d p r e c u r s o r cells are o f interest. Act. D in the dose used seemed to have no effect on B F U - E colony g r o w t h [10]. The effect o f B U on the B F U - E c o m p a r t m e n t has n o t yet been analyzed. W h e t h e r reinfection o f cells by surviving F V - P is the cause for the i m m e d i a t e occurrence o f Ep i n d e p e n d e n t C F U E colony g r o w t h during regeneration or cell p r o l i f e r a t i o n f r o m " t r a n s f o r m e d " p r e c u r s o r cells, c a n n o t be decided b y o u r experiments. The results with BU, however, indicate that at least a few n o r m a l p r e c u r s o r cells m u s t exist, giving rise to Ep d e p e n d e n t C F U - E colonies during regeneration after B U treatment. It w o u l d be o p t i m a l to find a d r u g which kills only the " t r a n s f o r m e d " cells w i t h o u t affecting the r e p o p u l a t i o n c a p a c i t y of the n o r m a l stem cells. In the present studies these were killed also b y BU. I f they w o u l d survive the treatment, they w o u l d be triggered into p r o l i f e r a t i o n a n d differentiation a n d possibly feed a n o r m a l hemopoiesis.

References 1. Golde, D.W., Faille, A., Sullivan, A., Friend, C. : Granulocytic stem cells in Friend leukemia. Cancer Res. 36, 115-119 (1976) 2. Horoszewicz, J.S., Leong, S.S., Carter, W.A. : Friend leukemia: rapid development of erythropoietin-independent hematopoietic precursors. J. Natl. Cancer Inst. 54, 265-267 (1975) 3. Iscove, N.N., Sieber, F. : Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture. Exp. Hematol. 3, 32-43 (1975) 4. Liao, S.K., Axelrad, A.A. : Erythropoietin-independent erythroid colony formation in vitro by hemopoietic cells of mice infected with Friend virus. Int. J. Cancer 15, 467-482 (1975) 5. Marsh, J.C. : The effects of cancer chemotherapeutic agents on normal hematopoietic precursor cells: A review. Cancer Res. 36, 1853-1882 (1976) 6. Opitz, U., Seidel, H.J., Bertoncello, I. : Erythroid stem cell in Friend virus infected mice. J. Cell Physiol. 96, 95-104 (1978) 7. Opitz, U., Seidel, H. J. : Studies on the target cell for the Friend virus (FV-P strain) using the CFU~-technique. Blut 37, 183-192 (1978) 8. Reissmann, K.R., Samorapoompichit, S.: Effect of erythropoietin on proliferation of erythroid stern cells in the absence of transplantable colony-forming units. Blood 36, 287-296 (1970) 9. Reissmann, K.R., Ito, K. : Selective eradication of erythropoiesis by Actinomycin D as the result of interference with hormonally controlled effector pathway of cell differentiation. Blood 28, 201-212 (1966) 10. Rich, I., Heit, W., Kubanek, I3.: The long and short term effect of Actinomycin D on erythropoiesis. Exp. Hematol. 5, Suppl. 2, 78 (1977) 11. Till, J.E., McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Res. 14, 213-222 (1961) 12. Wendling, F., Tambourin, P.E., Jullien, P. : Haematopoietic CFU in mice imfected by the polycythemia-inducing Friend virus. I Number of CFU, and differentiation pattern in the spleen colonies. Int. J. Cancer 9, 554-566 (1972) Received June 13, 1978 / Accepted September 5, 1978