Action of Isologous Bone Marrow From Urethan-Treated C57BL/6 Mice on Radiation Leukemogenesis 1. 2

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1 Actin f Islgus Bne Marrw Frm Urethan-Treated C57BL/6 Mice n Radiatin Leukemgenesis 1. 2 I. BERENBLUM, LOUISA BOIATO, L. FIORE DONATI,3 and N. TRAININ,. Department f E%perilnental Bilgy, Isaac Wlfsn Building, The We1nann Institute f Science, Rehvth, Israel SUMMARY Bne-lDarrw cell suspensins frid islgusldice pretreated with urethan were as effective in inhibiting radiatin leukem.genesis in C57BL/6 m.ice as bne ldarrw frid nnnal ldice f the sam.e strain. The results supprt earlier findings, by a different m.ethd f testing, frm. which it was cncluded that the prldting effect f urethan in leukem.genesis did nt depend n inactivatin f the bne marrw. Fem.ales appeared t develp leukemia 1D0re l'eadily than ldales in 2 grups f irradiated ldice that were subsequently treated with bne ldarrw. The pssible significance f this finding is discussed.-j Nat Cancer Inst 32: , A DISTINCTIVE feature f leukemia (lymphma) inductin in mice by repeated ttal-bdy X irradiatin is that pstirradiatin injectin f islgus bne-marrw cell suspensin inhibits the leukemgenic effect (1-3). [A similar inhibitin is prduced by pstirradiatin injectin f spleen cell suspensin (3).J By the tw-stage technique fr leukemia inductin (4), with a single expsure t X rays as initiatr and multiple injectins f urethan as prmter, it was fund that bne marrw injectins subsequent t the urethan treatment failed t antagnize the augmentatin f the leukemgenic effect by the urethan (5). The result. was interpreted t mean 1) that the actin f urethan in the tw-stage prcess des nt functin via the bne marrw, and 2) that inhibitin f leukemgenesis by bne marrw nrmally perates at a very early stage. It was felt, hwever, that the experiment was nt altgether decisive, since the bne marrw, administered 24 hurs after each urethan injectin, might have cme t late each time t exert its ptential inhibitry actin. On the ther hand, had the bne marrw been given immediately after each urethan injectin, the equally valid criticism culd have been made that the urethan was destrying the injected bne marrw cells befre they had had a chance t act. 1 Received September 3, Tbe wrk was supprted in part by researcb grant CA-{)5455, tbe Natinal Cancer Institute, Natinal Insti tutes f Healtb, Public Healtb Service. NATO Researcb Fellw. Present address: Istitut di Anatmia e Istlgia Patlgica, Universita di Bar!, Italy. We wish t tbank Mr. Y. Sarnsi Cr bls valuable tecbnical assistance. 723

724 BERENBLUM, BOIATO, FIORE-DONATI, AND TRAININ In the present experiment, a different testing system was adpted which avided bth these pssible cmplicatins: Mice were given multiple injectins f

2 724 BERENBLUM, BOIATO, FIORE-DONATI, AND TRAININ In the present experiment, a different testing system was adpted which avided bth these pssible cmplicatins: Mice were given multiple injectins f large dses f urethan, and 48 hurs after the last injectin, by which time the urethan had disappeared frm the bdy while recvery f the bne marrw frm the actin f the urethan culd nt yet have taken place (6, 7), the animals were killed and their bne marrw was cllected fr injectin int irradiated mice, as a test jr its capacity t inhibit leukemgenesis. Bne marrw frm untreated mice, injected int irradiated animals, was used fr cmparisn, and irradiated mice withut subsequent bne marrw injectin served as cntrls. MATERIALS AND METHODS The mice were C57BL/6, riginally btained frm The Jacksn Labratry, Bar Harbr, Maine, and subsequently bred in this labratry by brther X sister matings. Abut equal numbers f males and females were used in each grup. Three grups f mice served as dnrs f bne marrw: 1) mice given 5 intraperitneal injectins, at intervals f 5 t 6 days, f 0.2 milo percent aqueus urethan (representing 20 mg f urethan/injectin: 100 mg in all). These mice were 7 t 8 weeks ld at the time f the first injectin, and 10 t 11 weeks ld when killed, 48 hurs after the last injectin. 2) Mice given a single intraperitneal injectin f 20 mg urethan. These were 10 t 11 weeks ld at the time f injectin and were killed 48 hurs after the injectin. 3) Nrmal mice, 10 t 11 weeks ld. The bne marrw preparatins were btained by aspiratin f the lng bnes (2 femurs and 2 tibiae frm each animal) and suspended in Tyrde's slutin, t prvide apprximately 20 X 10 6 cells per 0.4 ml. This amunt was injected int the tail vein f the irradiated mice f grups I, II, and III, as described later. Five grups f mice were irradiated with 150 r when 7 t 8 weeks ld, and 3 further times with the same dse at weekly intervals, ttaling 600 r. The cnditins f ttal-bdy radiatin were: 200 kv, 15 ma, with 1 mm Al and 0.5 mm Cu filters; dse rate: 31 t 35 r per minute. One hur after the last irradiatin, grup I received the bne marrw injectin frm the dnrs pretreated with 100 mg f urethan, grup II was given the bne marrw injectin frm the dnrs pretreated with 20 mg f urethan, and grup III received the bne marrw injectin frm nrmal cntrl mice. Grups IV and V served as parallel cntrls (receiving X irradiatin, as stated, but withut subsequent bne marrw injectins). Dnrs and recipients were always f the same sex. The animals were kept in an air-cnditined rm at 21 t 24 C, fed Purina Labratry Chw, ccasinally supplemented with barley and sunflwer seeds, and prvided with tap water ad libitum. They were examined peridically and ki1led when mribund, with the thymus and varius ther rgans kept fr histlgical examinatin. The remaining survivrs were ki1led 78 weeks after the first irradiatin and examined similarly. RESULTS As shwn in table 1, 69 percent f the irradiated cntrl mice develped leukemia. (The spntaneus incidence f lymphatic leukemia in ur unirradiated stck f C57BLj6 mice is abut 0.7 %.) In the 3 experimental grups that received bne marrw after the irradiatin, whether frm JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 < r '" Z N> 0 i. ""... i N> '" <":l ::t:... <0 0> 01>. TABLE I.-Incidence f leukemia in C57BL/6 mice given injectins f islgus bne marrw frm nrmal and urethan-treated mice X irradiatin: 4 expsures f 150 r each at weekly intervals (ttaling 600 r) Bne marrw suspensins: injected intravenusly 1 hur after the last irradiatin Number Pt Incidence f leukemia/effective ttal* f mice Grup Surce f bne marrw used In males In females (P)t Ttal I Frm mice pretreated with 100 mg urethan 62 2/31 = 6% 5/18 = 28% <0.05 7/49 = 14% <0.001 II Frm mice pretreated with 20 mg urethan 58 3/25 = 12% 2/23 = 9% 0.9 5/48 = 10% <0.001 III Frm untreated mice 70 2/24 = 8% 10/36 = 28% < /60 = 20% <0.001 IV Cntrl 1 (withut bne marrw injectin) 73 23/32 = 72% 28/37 = 75% 51/69 = 74% V Cntrl 2 (withut bne marrw injectin) 69 26/40 = 65% 17/28 = 60% 43/68 = 63% Cntrls (ttal) /72 = 68% 45/65 = 69% 95/137 = 69% *Elfectlve ttal - number f survivrs at time f appearance f first leukemia in that f(p) = significance f sex di1ference, with Xl test. grup. tp = significance f di1ference frm the cntrl. c:j ::t: t::j td Z a:: =<1 z t' c:j... Is:: --l I>J C1

4 726 BERENBLUM, BOIATO, FIORE-DONATI, AND TRAININ nrmal r frm urethan-treated animals, the incidence f leukemia was lwered t 10 t 20 percent. There was a tendency fr a higher incidence f leukemia in females than in males in grups I and III, in which the irradiated mice were treated with bne marrw, but nt in grup II, nr in the cntrl grups IV and V. The time f appearance f leukemia fr each individual muse is recrded in table 2. N striking differences, either in relatin t the type f treatment r t the sex f animals, are discernible. Table 3 classifies the data accrding t whether the leukemic lesins were cnfined t the thymus r disseminated t ther rgans in additin t invlvement f the thymus. (Nnthymic leukemias did nt develp in this series.) While there appears t be n difference in the prprtin f "lcalized" t "generalized" leukemia amng the 3 experimental grups receiving bne marrw fllwing irradiatin, it wuld seem that the "generalized" variety is mre cmmn amng the irradiated cntrls than amng the 3 experimental grups. DISCUSSION The results shw that, under the cnditins f the experiment, bne marrw frm mice pretreated with urethan is as effective in inhibiting radiatin leukemgenesis as bne marrw frm nrmal mice. This agrees with ur earlier findings, with a different methd f testing (5), frm which tw cnclusins were reached: 1) The prmting effect f urethan in leukemgenesis did nt depend n inactivatin f the bne marrw, and 2) the inhibitry effect f bne marrw was primarily cncerned with the early stages f leukemgenesis. The present results are nt incmpatible with these cnclusins. That the prmting effect f urethan in leukemgenesis might invlve a mechanism nt perating via the bne marrw des nt necessarily cnflict with the knwn damaging actin f urethan n bne marrw (8) r the fact that bne marrw frm urethan-treated mice fails t prmte thymic regeneratin in irradiated mice, while bne marrw frm nrmal mice is effective (9). Urethan undubtedly has many different effects n the bdy, each pssibly invlving a different mechanism and a different pint f attack. [It has been shwn, fr instance, that the carcingenic actin f urethan n the lung and its initiating actin n the skin are bth independent f its anesthetic actin (10).] Furthermre, it is mre than likely that bne marrw plays a different rle in relatin t (a) the acute (lethal) actin, (b) the subacute (thymus invlutin) actin, and (c) the chrnic (leukemgenic) actin f ttal-bdy X irradiatin. The higher incidence f leukemia in females, bserved in tw f the grups f irradiated mice treated with bne marrw, may pssibly be cnnected with the knwn leukemgenic actin f estrgens in certain strains f mice (11, 12). It is cnceivable that, in the prcess f radiatin leukemgenesis, the estrgens nrmally present in females cntribute t JOURNAL OF THE NATIONAL CANCER INSTITUTE

5 2: S' :> Ii!: II a i:q TABLE 2.-Analysis f times f death accrding t grups Individual weeks f death r killing* With leukemia Withut leukemia Grups 0' ' 0' ' "" I 28,35 16,24,31(2),37 19, 20, 21, 24, 37(2), 39, 32, 53, 54, 61, 67 41,43(2), 54(2), 56, 58, 60, 62, 64, 65(2), 67(2), 68(2),74,77 II 43(2),76 29(2) 43, 52, 56, 58(2), 61, 63(3) 37, 40, 51, 52(3), 62, 64(2),66(2), 69, 72(3), 75 III 19, 20 13,18(2),23,24(2),27, 37, 54, 75 20, 37, 59, 62, 65, 66, 67(4),68,69,71(2), 72(3), 74 30,37,54(5),60,61,63, 67, 68, 69(2), 75 IV 17,19,22,23(5),24, 18,22(2), 23(2), 24(2), 25(3), 27(2), 29, 31, 25(5), 26, 27(8), 31, 34(3),37(2), 50, 64 32(2), 37, 39, 45, 58 23, 46, 61, 65, 71, 77 27, 32(2), 55, 64, 66, 69 V 18(2), 23(7), 24, 27(4),28(2),30, 32(2),33(2),34,35, 36(2), 47 18(2), 22(2), 23(5), 26, 27(4),35,40,45 32,34,40 23, 27 (2), 28, 34, 47 (2), 65 <Figures In parentheses represent numher f mice where mre than 1 died In the same week. Number f survivrs at end f experiment (78th week) With leukemia Withut leukemia 0' ' 0' ' c::l II- 1'! II t;:j t 2: a:: II ::l... Z >,l II t:< c::l... a:: II- -l I\:) -l -<

6 728 BERENBLUM, BOIATO, FIORE-DONATI, AND TRAININ TABLE 3.-Analysis f leukemia incidence accrding t whether lesins were lcalized t thymus r invlved ther rgans Leukemic lesin cnfined t thymus Number f mice in which Leukemic infiltratin present in lymph ndes, spleen, liver, etc., as well as in thymus Grups 0' 9 Ttal 0' 9 Ttal I II III Ttal I t III IV V Ttal IV and V the leukemgenic actin, the effect being, hwever, t slight t be nrmally bserved. One might then expect the effect t becme demnstrable when the radiatin cmpnent is artificially repressed by injectin f bne marrw, as in the present experiment, n the assumptin that the inhibitry influence f bne marrw perates nly against radiatin leukemgenesis and nt against estrgen leukemgenesis. (This pssibility is, at present, being investigated by us.) An alternative interpretatin f the apparent sex difference is that the ptency, r effectiveness, f bne marrw frm female dnrs is weaker than that frm males. Since, fr "cmpatibility" reasns, irradiated females received bne marrw nly frm females and irradiated males nly frm males, there was n way t tell whether their relative effectiveness was the same. That relatively large dses f bne marrw (each cntaining abut 20 X 10 6 cells) were used in these experiments makes it unlikely, hwever, that such an explanatin culd accunt fr the apparent sex difference bserved. REFERENCES (1) KAPLAN, H. S., BROWN, M. B., and PAULL, J.: Influence f bne-marrw injectins n invlutin and neplasia f muse thymus after systemic irradiatin. J Nat Cancer Inst 14: , (2) KAPLAN, H. S., N AGAREDA, C. S., ROSSTON, B. B., and BROWN, M. B.: Cmparative activity f islgus versus hmlgus muse bne-marrw n lymphid tumr prductin fllwing irradiatin. Prc Sc Exp BiI Med 98: , (3) LORENZ, E., LAW, L. W., and CONGDON, C. C.: The rle f bne-marrw and spleen in induced and spntaneus lymphatic leukemia. In Ciba Symps "Leukaemia Research." Lndn, J. & A. Churchill, 1954, pp (4) BERENBLUM, 1., and TRAININ, N.: Pssible tw-stage mechanism in experimental leukemgenesis. Science 132: 4{}-41, JOURNAL OF THE NATIONAL CANCER INSTITUTE

7 URETHAN-TREATED BONE MARROW IN X-RAY LEUKEMIA 729 (5) BERENBLUM, I., REWALD, F. E., and TRAININ, N.: Failure f bne marrw t interfere with the augmentatin f X-ray leukemgenesis by urethan. J Nat Cancer lnst 27: , (6) BRYAN, C. E., SKIPPER, H. E., and WHITE, L., JR.: Carbamates in the chemtherapy f leucemia. IV. The distributin f radiactivity in tissues f mice fllwing injectin f carbnyl-labeled urethane. J Bii Chem 177: , (7) BERENBLUM, I., HARAN-GHERA, N., WINNICK, R., and WINNICK, T.: Distributin f CB-Iabeled urethans in tissues f the muse and subcellular lcalizatin in lung and liver. Cancer Res 18: , (8) ROSIN, A., and GOLDHABER, G.: The effect f repeated dses f urethane (ethyl carbamate) n the mittic activity and cellular cmpsitin f the bne marrw f nrmal mice. Bld 11: , (9) BERMAN, I., and KAPLAN, H. S.: The differential actin f irradiated and chemically treated marrw n survival and n thymic regeneratin in irradiated mice. Radiat Res 11: , (10) BERENBLUM, I., BLUM, B., and TRAININ, N.: Failure f the urethane antagnistlysergic acid-diethylamide (LSD-25)-t inhibit lung carcingenesis r the initiating phase f skin carcingenesis in mice. Bichem Pharmacl 2: , (11) LACASSAGNE, A.: Sarcmes lymphldes apparus chez des suris lnguement traitees par des hrmnes estrgenes. C R Sc Bii (Paris) 126: , (12) GARDNER, W. U., KIRSCHBAUM, A., and STRONG, L. C.: Lymphid tumrs in mice receiving estrgens. Arch Path 29: 1-7, VOL. 32, NO.3, MARCH 1964

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