463.Full.Pdf

The Histopathologic Study of the Development of the Irradiation-induced Leukemia in C57BL Mice and of Its Inhibition by Testosterone*

PRAWASE WASI,~ AND MATTHEW BLOCK

(Department of Medicine, University of Colorado Medical Center, Denver, Colo.)

SUMMARY It is probable that the coexistence of two factors is necessary for the development of leukemia in the C57BL mouse exposed to total-body irradiation. The first factor is destruction, followed by regeneration, of thymic lymphocytes. The second factor is the postirradiation environment which provides circumstances such that the regeneration of the thymic lymphocytes becomes uncontrolled, leading to the development of leukemia. Testosterone, by inhibiting the regeneration of thymic lymphocytes after radiation injury, removes one of the two essential factors, thereby reducing the incidence of leukemia in irradiated C57BL mice.

Leukemia may be readily induced in various and kept in identical laboratory conditions. The strains of mice either by ionizing radiation or cages were cleaned with boiling water once a week. chemical carcinogens (~6), estrogens (16, 18), or The animals were checked at least once a day. by combinations of these agents (39, 56). Treat- In total, 340 animals were distributed into four ment of mice with testosterone decreases the in- groups (Table 1). Group I was kept as a control, cidence of spontaneous (52) and irradiation-in- Group II was treated with testosterone, Group duced leukemia (17, 3~). III received total-body x-radiation, and Group IV The present investigation was undertaken with received total-body x-radiation and testosterone. two purposes in mind: (a) to investigate the histo- Testosterone was given as testosterone propio- genesis of leukemia induced in C57BL mice by hate (10 mg/cc in sesame oil), 0.05 cc. intramus- irradiation and (b) to determine the mechanism by which testosterone inhibits or decreases the inci- TABLE 1 dence of leukemia after irradiation. EFFECT OT TESTOSTERONE INJECTIONS ON THE DE- VELOPMENT OF RADIATION-INDUCED LEUKEMIA MATERIALS AND METHODS IN C57BL FEMALE MICE Five- to 6-week-old female C57BL/6 mice were obtained from the Cancer Research Genetics DIED OR DIED OR SACRIFICED Laboratory, University of California, and from the SA CRIFICED WITHIN 12 BETWEEN 12 AND 27 Roscoe B. Jackson Memorial Laboratory. WK. AFTER X-RAY TOTAL WE. AFTER GROUP No more than six animals were placed in identi- NO. X-RAY cal stainless steel cages, 10 X 7 X 4.5 inches in (No leukemia oh- With Without size. They were maintained on Purina Laboratory served) leukemia leukemia Chow, given water ad libitum by water bottles, I. Control 66 39 27 * Supported by an Institutional cancer grant of the Ameri- II. Testosterone- can Cancer Society to the University of Colorado and a grant treated 63 42 21 from the Hematology Research Foundation, Chicago. lll. X-radiated 104 71 22 11 ~f Supported by H. M. King Bhumipol Adulyadet of Thai- IV. X-radiated and land. testosterone- treated 107 79 26 Received for publication July 8, 1960. 463

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. 464 Cancer Research Vol. ~1, May 1961 cularly in a single injection, starting from the first phatic leukemia, lymphogenous leukemia, lymph- day of irradiation and continuing twice weekly for oid leukemia, lymphadenoma, malignant lympho- 10 weeks, or until the experiment was terminated. ma, round-cell sarcoma, lymphosarcoma of Kund- Kaplan's method of fractionating total-body rat, and thymoma (12). When a neoplastic pro- irradiation into four equal doses given at 4-day liferation of abnormal lymphocytes was found the intervals (80) was followed. In a preliminary ex- animal was classified as having leukemia. periment, a 952-r total dose (238 r • 4) resulted m a 90 per cent mortality. In subsequent experi- RESULTS ments the total dose was reduced to 500 and 812 r, TissuEs given in four equal doses. Physical factors of irra- Group I (Control) diation for the x-ray unit were: 220 kvP, 15.0 ma., Leukemia was not observed in the animals in 0.5 ram. Cu and 1.0 mm. A1, master cone only, per- Group I during the period of 7 months following forated plastic plate above treatment box, ~ inch the initiation of the experiments (see Table 1). in thickness, 50 cm. to mid-thickness of mice. Specimens from these animals were used as con- HVL-I.3 ram. Cu, 50 r/rain in air at 50 cm. Six trois for the other three groups. animals held in a 6.5 X 6.5 • 7.5-inch cardboard box were exposed to irradiation at a time. Group II (Testosterone-treated) Two animals from each of Groups III and IV Following continuous testosterone treatment were sacrificed 24 hours and 4 days after each dose gradual involution of the thymus occurred. By the of irradiation. Beginning 1 week after the last end of 2 weeks it showed a definite decrease in size. dose of irradiation, two to four animals from each By 10 weeks the thymus became markedly atroph- of the four groups were sacrificed almost on a ic and occasionally was not visible macroscopi- weekly basis, until obvious leukemia developed in cally. When testosterone treatment was discon- Group III. These animals were selected at random. tinued at the end of 10 weeks the thymus regained In addition, all moribund animals were sacrificed, its normal size within the following 3 or 4 weeks. and autopsies were also performed on all animals The thymus underwent further atrophy and usual- found dead except those which were too autolyzed. ly could not be seen macroscopically when the tes- Prior to sacrifice, tail blood was obtained for the tosterone was continued beyond 10 weeks. determination of hemoglobin and of red blood cell, The number of small lymphocytes in the thymic white blood cell, platelet, differential, and reticulo- cortex was decreased within 2 weeks after the be- eyte counts. These were performed by standard ginning of testosterone administration. This be- methods, except that mieropipettes were used for came more marked at 5 weeks (Fig. 2 vs. Fig. 1). the red and white blood cell counts. A direct meth- The large lymphocytes and the reticulum cells be- od was used for the platelet count (54). The size came more prominent because of a marked de- of the thymus, liver, and spleen was measured. crease in the number of small lymphocytes. By 18 The intrathoracie organs (thymus, heart, lungs, weeks the thymus was almost depleted of all types and mediastinal lymph nodes) were removed en of lymphocytes (Fig. :3). Nevertheless, at no stage bloc. Representative tissue was obtained from was there an increase in karyorrhexis or decrease liver, spleen, kidney, adrenal gland, aortic or in mitoses compared with those in the controls. mesenteric lymph nodes, and Peyer's patches. The Following continuous testosterone administra- right femur was removed and cut open so that the tion no changes were observed in the myeloid tis- marrow was exposed to the fixative. All the tissues sue of the bone marrow or of the spleen. The were fixed in Zenker formol solution (9:1 dilution) lymphatic tissue of the spleen and the lymph for 1-2 hours. After fixation the femur was decal- nodes appeared to undergo a slight involution cified in 5 per cent HNO3 for 20 minutes to an which was difficult to differentiate from the normal hour, depending on the age of the animals. After variaton seen in control animals. this the tissues were processed by a modified These findings, the involution of the thymus Maximow's technic (sectioning at 6 g in nitrocellu- and lack of effect upon other lymphopoietic organs, lose, and staining by hematoxylin, eosin-azure II) confirm the observations made by Kaplan et al. (~). When the morphologic changes were minimal (s8). the specimens were re-examined as unknowns to To study further the more immediate effect of avoid bias. testosterone, an additional 21 animals were treated Nomenclature.raThe term leukemia as used here with 5 rag. of testosterone propionate intramus- refers to the disease variously called lymphoid tu- cularly and sacrificed at 2, 4, 9, 12, 24, 36, and 48 mor, lymphocytic neoplasm, lymphoma, lympho- hours after treatment. Histopathologic study did sarcoma of the thymus, lymphoblastoma, lyre- not demonstrate any change which might explain

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. WASI AND BLocK--Irradiation-induced Leukemia and Testosterone 465 the mechanism whereby testosterone caused thy- following dose was given, the hematopoietic tis- mic involution. sues became increasingly atrophic. Twenty-four hours after the last dose of irradiation the thymus Group III (Total-Body Irradiation) was reduced to about one-third to one-fourth of the Radiation injury was not studied in detail. This normal size. Its capsule became much thickened. had been done previously (3). Animals which re- The cortex could not be differentiated from the ceived 812 r had slightly more damage to hemato- medulla (Fig. 4A). The number of lymphocytes in poietic tissue with slower regeneration than did the cortex was decreased (Fig. 4B). animals receiving 500 r. Animals subjected to these The marrow was more bloody than normal ow- two doses will be described together. ing to an expansion of the sinuses. A few myelo- Twenty-four hours after first dose of irradiation. cytes, erythroblasts, and megakaryocytes re- --In the animals sacrificed 24 hours after the first mained. The spleen was reduced to about a third dose of irradiation the thymus was slightly de- of normal size. The red pulp wag almost complete- creased in size. Microscopic examination revealed ly depleted of myeloid and lymphatic cells. The karyorrhexis and a decrease in the number of small white pulp was reduced in size, containing only a lymphocytes more marked in the cortex than in few lymphocytes. The lymph node was also very the medulla. Mitoses were also decreased in num- atrophic. Reticular and plasma cells were the domi- ber. However, the small lymphocytes immediately nant cell types. under the capsule were hardly decreased in num- Regenerative phase (24 hours to 6 weeks after ber. The lymphocytes in the medulla were in- fourth dose of irradiation).--Thymus: One week creased in number. Eosinophils were slightly in- after irradiation the thymuses had increased in creased in number in the cortex. size to approximately half the size of the controls. Changes in other hematopoietic tissues were not The thymic cortex had been densely repopulated as striking as in the thymus. The bone marrow with lymphocytes (Fig. 5A). These cells were showed a decrease in cellularity, more marked in somewhat larger, and the nuclear chromatin was the erythroblastic series than in the granulocytic more delicate than that of the small lymphocytes and megakaryocytic series. There was a compensa- which were the dominant cells in control animals tory expansion of the blood sinuses. Only a small (Fig. 5B. vs. Fig. 1B). However, the cortical amount of cellular debris was observed in the bone lymphocytes seen 1 week after irradiation ap- marrow. In the lymph nodes there was a slight re- peared to be smaller than the lymphocytes seen 4 duction in the number of lymphocytes and a mod- days after each dose of irradiation. Mitotic figures erate amount of karyorrhexis. The spleen showed were still frequent. reduced lymphocytic and myeloid elements. The Two weeks after irradiation the thymus was reticulum cells of the red pulp were more prominent two-thirds of normal in size. The number of small than usual because of a marked reduction in the lymphocytes in the thymic cortex had increased number of erythroblasts. These hematopoietic tis- (Fig. 6). The majority of these lymphocytes, how- sues had a lesser amount of nuclear debris than did ever, were still somewhat larger than those in the the thymus. normal thymus (Fig. 6B vs. 1B). Four days after first dose of irradiation.--The Three weeks after irradiation the thymus was thymuses of the animals sacrificed 4 days after the decreased slightly in size owing to a moderate re- first dose of irradiation and immediately prior to duction in the number of lymphocytes in the cor- the second dose were approximately two-thirds of tex (Fig. 7). A similar temporary involution had normal size. The cortex was repopulated by large been reported by Kaplan and Brown (33). Mitotic lymphocytes, and mitoses were increased in num- figures were seen in approximately normal number. ber. The normal architecture, with cortex more There was no increase in karyorrhexis. cellular than medulla, had also been regained. Re- After this there was a gradual increase in size generation in the other hematopoietic tissues ap- of the thymus and in number of thymic lympho- peared to be somewhat less advanced than in the cytes. The thymus regained normal size and struc- thymus. In the spleen and marrow regeneration of ture in 5-6 weeks after irradiation (Fig. 8). erythroblasts was more advanced than that of Spleen: One week after irradiation the spleen granulocytes and megakaryocytes. had almost regained normal size owing to an ex- From second dose to 24 hours after fourth dose.- tremely active erythroblastic regeneration in the Following each dose of irradiation, the hemato- red pulp. These erythroblasts occurred in large poietic tissues underwent damage and regenera- clumps, occupying more than half of the red pulp. tion. Because the recovery after each dose of ir- The white pulp had increased in size because of an radiation was far from complete at the time the accumulation of medium-sized lymphocytes. How-

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. 466 Cancer Research Vol. 21, May 1961 ever, this regeneration was much less active than The cytoplasm was scanty, and the nuclei were that of the erythroid series. Regeneration of the more delicate than those of normal lymphocytes. granulocytes and megakaryocytes was not yet Mitotic figures were increased in number. This observed. proliferative change usually involved the entire Two weeks after irradiation the spleen became cortex of the thymus. In accordance with the ob- even larger than the control. Grossly visible nod- servations of Carnes, there was no evidence to in- ules composed of dense aggregations of erythro- dicate that the proliferative changes originate any- blasts were seen in the red pulp. The regeneration where except in the thymic cortex (6). However, in of the white pulp was less than that of the red some cases it was localized to a small area or to pulp. For the first time megakaryocytes and gran- one lobe of the gland. During this period there was ulocyte precursors were found among erythro- no alteration in the lymph node, spleen, liver, bone blasts. marrow, and peripheral blood. During the following weeks there was a decrease It was not possible to decide exactly when the in numbers of erythroblasts and myelocytes in the leukemia began, because the change from normal red pulp; the white pulp increased in size due to a was so gradual. The first unequivocal histologic reaccumulation of lymphocytes. By 5-6 weeks evidence of leukemia was found in the thymus of after irradiation the spleen again appeared normal. an animal sacrificed 1~ weeks after irradiation, Bone marrow: The bone marrow at the comple- similar to what had been observed by Kaplan on tion of irradiation was very atrophic as described the basis of serial biopsies of the thymus in similar above. However, in contrast to the splenic red experimental material (~7). The peripheral blood pulp, some myeloid elements were demonstrable of this animal was still completely normal, and in the marrow r hours after the last dose of ir- there was no enlargement of any organs at autop- radiation. One week after irradiation there was a sy. On microscopic examination a striking abnor- remarkable regeneration of all myeloid cells. The mality was found in the thymus. The medulla was blood sinuses were decreased in size. The cellulari- greatly reduced in size, with correponding hyper- ty of the bone marrow was approximately 50-60 trophy of the cortex (Fig. 10A). The latter con- per cent that of normal. Approximately 80 per sisted of dense sheets of stem cells (Fig. 10B). cent of the marrow elements at this stage were These cells were large in size, with scanty cyto- erythroblasts, as compared with ~0-~5 per cent in plasm and pale-staining nuclei containing one or control animals. The successive changes consisted two small nucleoli. Other organs were normal in of increasing cellularity with further decrease in microscopic appearance. size of blood sinuses. Granulocytes and precursors In the following 15-week period after this first were increased so that in ~-3 weeks their number observation of the malignant change, ~ out of 38 was greater than that of the erythroblasts. The animals in this group were found to have leukemia. bone marrow attained normal appearance in 5-6 In eight of these ~ animals the leukemic process weeks. was confined to the thymus without involvement Lymph node: Analysis of lymph nodes was dif- of any other organ, and in two of these it was ficult because of their small size and variation in noted only in a part of the thymus. histologic appearance. A gradual accumulation of Those animals which had widespread leukemia lymphocytes was observed, and lymphoid follicles could be recognized in most cases by their inac- were reformed in the cortex of the lymph node so tivity, weight loss, and lusterless tails. Most of that a normal appearance was attained within 5-6 them had respiratory distress owing to an enlarged weeks after irradiation. thymus, frequently associated with a straw-col- In summary, after 500 r or 81e r of fractionated ored or serosanguinous pleural effusion. Liver, total-body irradiation, all the hematopoietic tis- spleen, and lymph nodes were always markedly sues, after an initial phase of necrosis and atrophy, enlarged. Microscopic examination revealed ex- had returned to normal in 5-6 weeks. tensive infiltration of these organs with medium Over-regeneration of the thymus and the develop- or large lymphocytes (Fig. 11, A-F). In addition, ment of leukemia.--Six weeks after the last dose of the bone marrow and renal cortex were similarly irradiation, the thymus began to increase in size involved. Leukemic tissue was not found in the due to cortical hypertrophy. In those animals in renal medulla. which the size of the thymus still remained normal the cortex had expanded at the expense of the Group IV (Irradiation plus Testosterone) medulla, due to a proliferation of large lympho- Thymus: Twenty-four hours after the first dose cytes (Fig. 9). These cells were twice as large as of irradiation, the hematopoietic tissues were normal small lymphocytes (Fig. 9B vs. Fig. 1B). similar to those in animals which received irradia-

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. WASI AND BLocK--Irradiation-induced Leukemia and Testosterone 467 tion alone. In contrast, at 4 days after the first globin and red count were not significantly differ- dose of irradiation, the thymus had fewer lympho- ent from those of the controls. The white blood cytes than did corresponding animals in Group cells fluctuated around the lower limit of normal. III. Blood counts were obtained from an additional 30 At 24 hours after the last dose of irradiation animals prior to and after treatment with testos- fewer lymphocytes were found in the thymus of terone in sesame oil or sesame oil alone. There was Group IV than of Group III animals. Other hema- a drop in white count in both groups similar to topoietic organs showed a degree of atrophy simi- that described for Group II animals. Therefore, lar to that seen in animals in Group III. the decreased white count in the testosterone- One week after irradiation the thymus was treated animals was apparently not due to a spe- about one-third to one-half the size of the thymus cific action of testosterone. The platelet count was of corresponding animals in Group III. Microscop- not altered by treatment with testosterone. ic examination revealed an increase in number of IS- large lymphocytes in the thymic cortex of animals X-RAY

in Group IV. However, the number of lympho- ...... cytes and of mitotic figures was much less than 16- :: ::"~...~:.:.:.:.:.:-:.:.:'.:-:.:.:.:-:-:. :.:.:.:.:-:,:.:.:,:. :.::..:.:,:::::.:/~,:,:,:, :-:"":.:,:':.:.::..:.:,:.:" ".:,:-: .3'..:-:,:.:.:.:.:.Y::.:-.: .:.:,:-v...::-.: ,:.:.:.:.:.:.:--.:,...... ~.~.!,::.:.~.!.!--"::,. that seen at a corresponding time in the thymuses HI.:.:.:.~.~.~.~.~.~-~.~.~.~.....~.~.~..:.~.~.:.~:~.~.~`~.:.~.~.~.:.~.~..:..~..~..:...~.:.~.:.~.~.~.~.~.~.:.~....~ii.~.~.~`~`.~.~.:~-~.~.~.~.:~."...~..~.::. of Group III (Fig. 12 vs. Fig. 5). I4- In the following weeks the thymus rapidly decreased in size to such an extent that frequently 12- it was not visible macroscopically. The thymus consistently showed a marked reduction in the I0- number of lymphocytes (Figs. 13 and 14). It was composed primarily of cords of reticular cells with S- only a few lymphocytes. The thymus remained MEAN VALUE FROM CONTROL ANIMALS atrophic as long as the testosterone treatment was WITH S.D. OF .9 continued. The thymus fully regenerated within : --" 5001" 3-4 weeks after cessation of 10 weeks cf testos- e- .... -4 500 r -I- TESTOSTERONE o ~ 819 r terone treatment. During the following 1~ weeks T (> ..... -o812 r -I- TESTOSTERONE except in two animals, there was no evidence of the hyperproliferation seen in the thymus of I ! I I ! I I t Group III animals. However, in two animals of i 2 3 4 5 6 7 8 Group IV at sacrifice the thymic cortex contained WEEKS AFTER STARTING X-IRRAOIATION large lymphocytes, although it was normal in size. CHART1.--Hemoglobin concentration in C57BL mice given Two other animals in Group IV died of leukemia, torsi-body x-radiation with and without testosterone treat- proved by post-mortem examination (Table 1) at ment (0.5 mg. testosterone propionate in sesame oil twice a 5 months after irradiation. week). Myeloid Tissue: Regeneration of the myeloid Group III and Group IV tissue of the bone marrow and red pulp of the There was little difference in the blood counts spleen in Group IV was not significantly different of the animals which received 500 r alone and from that in the animals which received x-radia- those which received 500 r and testosterone during tion alone (Group III). A moderate retardation of the first 6 weeks after treatment (Charts 1-4). the lympl~atic tissue of the spleen and lymph nodes However, the hemoglobin returned to normal was observed. However, this was rather difficult to faster in the animals which received 812 r and tes- interpret because of variation of the amount of tosterone than in the animals which received 812 r lymphatic tissue even in the controls. alone. The blood counts of animals with leukemia are presented in Tables ~B and ~C. The counts of PERIPHERAL BLOOD animals of both Group III and Group IV not de- Group I (Control) veloping leukemia were within normal range. Peripheral blood counts of 50 control animals The peripheral blood was normal in all but one are summarized in Table 2A. animal when leukemia was confined to the thymus (Table ~B). In this animal (No. 6) there was a Group II (Testosterone-treated) mild anemia. Peripheral blood counts were done in 40 animals Some animals (Table ~C) with widespread leu- from the testosterone-treated group. The hemo- kemia had a mild anemia and thrombopenia and

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. 468 Cancer Research Vol. 21, May 1961 a moderate lymphocytosis and reticulocytosis. The differences in reaction to irradiation of Numerous smudge cells were usually present in Group III and Group IV animals were: blood smears. Most of the white blood cells were 1. Repopulation by lymphocytes of the thymus small to large atypical lymphocytes. A few reticu- of Group III and not of Group IV in the first 6 lar cells were also seen in the blood smear of one weeks after irradiation. animal. ~. Uncontrolled proliferation of medium and MISCELLANEOUS OBSERVATIONS large lymphocytes beginning at 6 weeks after irradiation in the thymus of Group III, in contrast Loss of hair was observed following prolonged to failure of regeneration of lymphocytes in Group testosterone treatment (Groups II and IV). These IV as long as treatment with testosterone was animals also showed a markedly atrophic X zone continued. of the adrenal cortex similar to that in the previous 3. Development of leukemia in Group III and observation (9, 10, 49, 55). After testosterone was lack of development of leukemia in all but two ani- discontinued, this zone regenerated very rapidly. mals in Group IV.

DISCUSSION INCIDENCE OF LEUKEMIA IN C57BL MICE Strain C57BL mice has a 5-8 per cent incidence of spontaneous leukemia occurring primarily after 1 year of age (18, 30). In the present study no animal lived longer than 9 months so, as anticipated, leukemia did not develop in control animals (Group I). Kaplan and Brown (30) reported an incidence of lymphoid tumors in up to 98 per cent of C57BL female mice exposed to 951 r fraction- ;/ ated total-body x-radiation begun at the age of 6] 83 _ 3 days. In this study a large number of ir- MEAN VALUE FROM CONTROL radiated animals were sacrificed before they had ANIMALS WITH S OOF lOT MILLION lived long enough to develop leukemia. Conse- - :. 500 r =el e- ...... 9500 r + TESTOSTERONE quently, the incidence of leukemia is lower than z__ol v o812 P that reported by Kaplan and Brown (30). m Om ~1 ~ ...... ~812 I" 4" TESTOSTERONE x,, I PATHOGENESIS OF LEUKEMIA These studies and those of Kaplan, Nagareda, WEEKS AFTER STARTING X-IRRADIATION and Brown (88) suggest that the development of CHART ~.--Red blood cell counts in C57BL mice given leukemia in C57BL mice exposed to irradiation total-body x-radiation with and without testosterone treat- depends on the coexistence of two factors: (a) a ment (0.5 mg. testosterone propionate in sesame oil twice a thymus in which the lymphocytes undergo degen- week). eration, atrophy, and regeneration, and (b) factor(s) other than the thymus. Kidney infection and urinary calculi were ob- The thymus.--The special role of the thymus in served in a few animals treated with testosterone. the development of spontaneous and induced leu- The hair of the C57BL mouse, normally black kemia in mice has long been recognized. Enlarge- and shiny, turned grey and dull after exposure to ment of the thymus was always found in sponta- irradiation. Deposition of melanin at the posterior neous leukemia in Ak strain mice (14, 51). Thy- tip of the spleen was occasionally observed in the mectomy reduces the incidence of spontaneous leu- control animals, but more frequently and in larger kemia (14, 4~, 51) and of leukemias induced by amount in irradiated animals. When this pigment methylcholanthrene (43), irradiation (~5, 35), and was present in large amount, interference with by leukomogenic virus (~0, 46). Law (41), working regeneration of the myeloid elements in the red with the high-leukemia RIL and the low-leukemia pulp was noticed. C3Hb mice, observed a marked increase in the incidence of leukemia in (C3Hb X RIL)FI bearing SUMMARY OF OBSERVATIONS grafts of RIL thymic tissue as compared with The only significant effect of testosterone alone control (C3Hb X RIL)F1 mice. Our observations (Group II) was induction of a decrease in number and prior observations (~3, ~6, 3~) that the first of lymphocytes in the thymus. evidence of leukemia was found in the thymus and

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C,~RT 3.--White blood cell counts in C57BL mice given ment (0.5 mg. testosterone propionate in sesame oil twice a total-body x-radiation with and without testosterone treat- week).

XRAY MEAN VALUE FROM CONTROL that the thymus was involved in every animal ANIMALS WITH S.D. OF 338,000 which had leukemia further support the concept 1600 -- ":- : 500 r / ~ " ..... - 500r I TESTOSTERON8TERONE that the thymus plays a special role in the develop- / ,l (~ o 812 r ment of leukemia in these mice. 14oo-t ;: ~ ..... ~ sis r + TESTOSTERONOSTERONE J ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ! ...... :...... The thymus, similar to other hematopoietic tis- I~00 !iiiii,i.iiiiiiiiiiiiiiiii~i iiiiiiiiiiiiiii;ii~ii::~ii::iiiii::i ::::ii:: sues, undergoes a degeneration, atrophy, and regeneration following total-body irradiation. How- ever, unlike other tissues, the thymus later becomes ...... i~i~i~i~i~l~i~l~!~i~i~::~i~;~;~; .oo - iiii~ ~iiiiiiiiiiiiiiiiiiiiiii iiili iii iiiiiiiiiiiiiiiiii!iiiiiiiiiiiiii iiiiiii ii ii i~~i'~ii i~i~i~ii~ii '~',iii ! i!iithe site of an uncontrolled lymphocytic proliferation leading to the development of leukemia. The ii i~ ~ii i!iii iNi i ii iiii! importance of this cycle of degeneration, atrophy, .oo- ~! and regeneration in the thymus is supported by / /" / / prior experiments. Arnesen (1) noted that the thymus atrophied prior to the development of spontaneous leukemia in AKR/O mice. Shielding the /" 2~- .,," thymus in total-body irradiated mice, which pre- .,.,,, vents degeneration of the thymic lymphocytes, el l I I I l I also prevents the development of leukemia (~4). ~0w~O WEEKS AFTER STARTIN* X-IRRADIATION Leukemia develops when nonirradiated thymic grafts are implanted subcutaneously into thymectomized, irradiated mice, and, furthermore, the CHART 4.--Platelet counts in C57BL mice given total-body x-radiation with and without testosterone treatment (0.5 rag. leukemia arises from the transplanted thymus testosterone propionate in sesame oil twice a week). (37). These observations apparently contradict

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. 470 Cancer Research Vol. 21, May 1961 our hypothesis that the thymus must undergo de- Factor(s) other than the thymus.--Irradiation of generation, atrophy, and regeneration (repopula- the thymus alone does not cause leukemia (~4), tion by lymphocytes) in order for leukemia to de- even though the thymus goes through successive velop. However, thymic lymphocytes are almost en- phases of degeneration, atrophy, and regeneration. tirely destroyed and then regenerate when the Transplantation of a normal thymus into non- thymus is transplanted (5, 6, 7, 53). Thus, the se- irradiated mice, which also induces the same se- quence of events after irradiation (degeneration, quence of events, does not result in leukemia. atrophy, and regeneration) is duplicated regardless However, transplantation of a nonirradiated thy- of whether the newly formed lymphocytes are de- mus into a thymectomized irradiated mouse causes rived from the host (44, 45), from the nonirradi- leukemia (36), and an abnormal regeneration of ated thymic graft (37), or from both sources. the thymic lymphocytes occurs (5). Therefore, The thymus is unique in being the only lympho- there must be some factor(s) other than the thy- poietic organ in which endodermal tissue is inti- mus needed to produce leukemia in the total- mately mixed with mesenchymal tissue (50). The body-irradiated mouse. thymus reacts differently to irradiation from other What, then, is the nature of the factor(s) other lymphatic tissues as shown by the following ob- than the degeneration and regeneration of the servations: (a) thymic lymphocytes are more sen- thymic lymphocytes? Shielding the spleen (47, sitive to irradiation than other lymphocytes; 48), or leg (~8, 31), as well as intravenous injection (b) regeneration after irradiation is more rapid; of bone marrow of splenic cells (8, 34, 48), prevents and (c) at about 3 weeks after irradiation, as al- leukemia in irradiated mice. These procedures do ready reported by Kaplan and Brown (33), the re- not alter the initial degenerative effect of total- generation of the thymus is interrupted by a tran- body irradiation upon the thymic lymphocytes. sitory partial involution. It is not known whether However, regeneration of thymic lymphocytes is these differences account for the special role played more rapid when total-body irradiation is modified by the thymus in the development of leukemia. by leg shielding (29) or injection of bone marrow

TABLE 2 BLOOD COUNTS

DIFFERENTIA L

A~I~L~L Hm RBC WBC PLA TELETS RETICS. NO, GM. ~0 106/cv x4M 10Z/cv MM 10z/c'~ MM % % Neu- % Lymphocytes trophils A. Control animals Control* l 15.6__+0.9 I 10.6__+1.07 I 16.6+5.9 1982.0+338.0[ 3.8+1.7 1 70-90 I 10-80 B. Leukemia limited to thymus 1 14.2 9.60 13.20 944.0 4.6 84 16 2 13.9 9.5~ 7.70 889.0 3.6 83 17 3 14.8 8.72 10.10 1,145.0 2.2 94 6 4 14.3 10.74 12.45 1,457.0 2.8 88 12 5 15.2 10. O0 9.70 615.0 2.0 76 24 6 12.8 8.40 8.30 965.0 3.4 83 17 7 14.8 9.74 13.15 1,319.0 1.8 88 12 C. Wides)read leukemic infiltration 8 13.8 9.80 11.45 549.0 5.0 93 M, (10 Sin) 7 9 14.2 9.60 70.00 69.0 8.0 40 M, 52 L, (45 Sm) 8 10 10.08 13.45 7.2 82S, 8L 10 11 17.1 10.10 7.50 685.0 4.0 61 S 89 12 15.2 10.34 32.75 240.5 11.8 20 S, 46 L 34 13 17.4 9.32 113.20 55.0 17.0 83 S, 9 L, (230 Sm) 8 14 23.05 77.0 9.2 Atypical lymphocytes and reticular cells 15 13.7 8.34 19.35 79.0 8.0 57 S, (50 Sm) 43 16 12.5 47.30 95.7 15.0 46 S, 29 L ~5

* Mean and standard variation based on 50 controls. Hb= hemoglobin; RBC = red blood cells; WBC -- white blood cells; Retics. = reticulocytes; S = small lymphocytes; M = medium lymphoeytes (lymphoblasts); L = large lymphocytes (stem cells); Sm = smudge cells (ruptured lymphocytes) per 100 WBC.

(4, 34, 57). This would suggest that, for leukemia cidence of spontaneous leukemia. On the contrary, to develop in C57BL/6 mice, regeneration of thy- Murphy (5~), working with spontaneous leukemia mic lymphocytes must take place in a specific en- in Rockefeller Institute strain of mice, reported vironment such as that provided by total-body that orchiectomy resulted in an increase in the in- irradiation. Injection of hematopoietic cells or cidence of leukemia, whereas ovariectomy had no protection of these cells probably modifies the significant effect. However, these findings are not post-irradiation environment through cellular re- applicable to all strains of mice, because Kaplan population (13, 19) rather than by humoral fac- (~5) did not find that gonadectomy had a signifi- tor (~1). cant influence on the incidence of irradiation-in- Chart 5 illustrates the interrelationship of the duced leukemia in C57BL mice. two factors needed for the development of leuke- Daily injection of 500 gg. of testosterone into mia in C57BL mice exposed to total-body irradia- rats causes more reduction in the weight of the tion. thymus than of the lymph node and of the spleen (53). These results agree with our observation and MECHANISM OF THE PROTECTIVE EFFECT OF TES- that of Kaplan et al. (:38) that injection of testos- TOSTERONE AGAINST LEUKEMIA IN MICE terone causes a greater reduction in the number A relation between sex hormones and leukemia of lymphocytes in the thymus than of the spleen in mice has been long recognized. The incidence in or lymph nodes. This may be due to direct destruc- females is higher than in males in certain strains tion as found with glucocorticoids (11, 58) or to an (51, 5~); estrogens (15, 40) increase and testos- inhibition of production of the lymphocytes or a terone decreases (17, 3~) the incidence, and ovari- combination of these factors. An inability to dem- ectomy but not orchiectomy (51) reduces the in- onstrate destruction of lymphocytes after injection

| TOTALBODY l' X-RAY . ~ LEUKEMIA / "" .-- INCREASED NORMAL- DECREASED

~) LOCAL )('RAY OVER THE THYMUS INCREASED ; NO LEUKEMIA NORMAL" ~:~~~:~" DECREASED

TOTAL BODY X-RAY+ SHIELDED THYMUS INCREASED NO LEUKEMIA NORMAL ::~.~`~:~:....~..C~.~.!;.::~`~:~:~:~.:~.~:.~...~.~.~.~.~.~.~:~...... ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: DECREASED

(~) TOTAL BODY X-RAY | INJECTION OF HEMATOPOIETIC.CELLS NO LEUKEMIA INCREASED

NORMAL- ,..~,,-.:::..v.,~....~).;:~::...., ...... ~,,:~:~::.:.::,,- ...... ::..:...=, ...... :,:...... DECREASED ~

(~) TOTALBODY X-RAY TO THYMECTOMIZED ANIMAL INCREASED ~ ...... ~ " LEUKEMIA...-"/.~-'- NORMAL" ~!~ -! DECREASED THYMUS I __..~.-"- TRANSPLANTE~t...~'"""

( TESTOSTERONE r INCREASED (~) TOTALBO~Y X'RAY" NO LEUKEMIA

NORMAL T.:.."~..~;,.~:.,~..~:5"..:.::..'.~::.::~:~:--~" .~':i.-::..:::::.':::::'..Y..'.'.;':.::.'::::.'.:.~.":~:.:.;:'::::~.'..::.... ~:::::.'::~,.:~:::~.::.':~:..'.'.':::.':::::...""'.'.'" DECREASED .... , "'1 6 i ~' ;5 ~1. 5 6 7 '8 ~) I()I'1 ~ i'3 14. 15 WEEKS :::.."~;::;::::'::-::;::::':::::'~::::::::::::NORMAL RANGE THYMUS ...... MYELOIDTISSUE CHART 5.--Mechanism of the development of leukemia in mice exposed to irradiation

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. 47~ Cancer Research Vol. s May 1961 of testosterone does not exclude this possibility, 3. BLOOM, W. (ed.). Histopathology of Irradiation from Ex- because under the influence of low-grade but pro- ternal and Internal Sources. National Nuclear Energy Se- ries, Division IV, Vol. 221. New York: McGraw-Hill Book longed irradiation one may completeIy deplete a Co., Inc., 1948. hematopoietic organ without any demonstrable in- 4. BROWN, M. B.; KAPLAN, H. S.; WEYMOUTH, P. P.; and crease in karyorrhexis or decrease in mitotic ac- PAULL, J. Effect of Intravenously Injected Bone Marrow tivity (~). Cell Suspensions on Thymic Regeneration in Irradiated Testosterone had a relatively insignificant ef- C57 Black Mice. Science, 117:693-95, 1953. 5. CARNES, W., and KAP~N, H. S. Relation of Lymphoma fect upon the degeneration of thymic lymphocytes Induction to Cortical Differentiation in Thymic Grafts. induced by total irradiation. In contrast, tes- Fed. Prec., 15:510, 1956. tosterone (a) inhibited the regeneration of lympho- 6. ~. Histogenesis of Lymphomas in Irradiated C57BL cytes in the thymus which occurred during the 6 Mice Bearing Nonirradiated Thymic Grafts. Prec. Am. Assoc. Cancer Research, 9.:99, 1956. weeks after irradiation and (b) inhibited the malig- 7. CARNES, W. H.; KAPLAN, H. S.; BROWN, M. B.; and nant proliferation of lymphocytes which began 6 HmscH, B. B. Indirect Induction of Lymphomas in Ir- weeks after irradiation. Testosterone did not sig- radiated Mice: III. Role of the Thymic Grafts. Cancer Re- nificantly modify the effect of irradiation on other search, 16: 429-38, 1956. hematopoietic tissues. 8. COLE, L. J.; HOWELL, P. C.; and ELLIS, M. E. Incidence of Neoplasms in Mice Protected against Lethal Doses of X- We, therefore, suggest the following mechanism rays by Spleen Homogenate. Prec. Am. Assoc. Cancer Re- to explain the inhibition of the development of search, 2:100, 1956. leukemia in C57BL mice exposed to irradiation 9. CRAMER,W., and HORNING, E. S. On the Relationship be- and injected with testosterone. We have already tween the Male Gonads and the Adrenal Gland. Lancet, indicated the probability that two factors (regen- 1:1330-31, 1937. 10. D~ANESr.Y, R., and P&RKES, A. S. Multiple Activities of eration of the thymus and the post-irradiation en- Androgenic Compounds. Quart. J. Exper. Physiol., 9.6: vironment) must be present in order for leukemia 393--402, 1937. to develop. Testosterone, by preventing the regen- 11. DOUOHERTY,T. F., and WroTE, A. Functional Alterations eration of the thymus, removes one of the two es- in Lymphoid Tissue Induced by Adrenal Cortical Secre- tion. Am. J. Anat., 77: 81-105, 1945. sential factors (see Chart 5F). Furthermore, when 12. DUNN, T. B. Normal and Pathologic Anatomy of the testosterone was discontinued after 10 weeks the Reticular Tissue in Laboratory Mice, with a Classification thymus regenerated and leukemia did not develop and Discussion of Neoplasm. J. Nat. Cancer Inst., 14: in animals followed for an additional 5 months. On 1281-1433, 1954. the other hand, the development of leukemia is 13. FORD, C. E.; HAMERTON, J. L.; BARNES, D. W. H.; and LouTIr, J. F. Cytological Identification of Radiation-Chi- not inhibited if treatment with testosterone is not maeras. Nature, 177: 452-54, 1956. begun until 3 weeks after irradiation (38). There- 14. FURTH, J. Prolongation of Life with Prevention of Leuke- fore, not only must the two factors be present, but mia by Thymectomy in Mice. J. Gerontol., 1: 46-54, 1946. they must coexist. 15. GARDNER, W. U. Influence of Estrogenic Hormones on Abnormal Growth. Occas. Publ. Am. Assoc. Adv. Sci. No. ACKNOWLEDGMENTS 4. Lancaster, Pa.: Science Press, 1937. 16. ~. Steroid Hormones in the Induction of Cancer. The authors are indebted to Mr. Glen Mills, Department of Cancer Research, 7: 37-38, 1947. Visual Education, for the microphotography. 17. --~. Ovarian and Lymphoid Tumors in Female Mice Subsequent to Roentgen-Ray Irradiation and Hormone REFERENCES Treatment. Prec. See. Exper. Biol. & Med., 75:434-86, 1. ARNESEr~, K. The Adrenothymic Constitution and Suscep- 1950. tibility to Leukemia in Mice, A Study of the ARK/O and 18. GARDNER, W. U.; DOUOHERT~, T. F.; and WI~a~s, WLO Strains and Their Hybrids. Acta Path. et Microbiol. W. L. Lymphoid Tumors in Mice Receiving Steroid Hor- Scandinav. (Suppl.), 109:1-95, 1956. mones. Cancer Research, 4: 78-87, 1944. 2. BLOCK, M.; SMALL~R, V.; and BROWN, J. An Adaptation 19. GENOOZIAN, N.; URSO, I. S.; CosovoN, C. C.; CONOER, of the Maximow Technique for Preparation of Sections of A. D.; and MXKINOD).N, T. Thymus Specificity in Lethally Hematopoietic Tissues. J. Lab. & Clin. Med., 42:145-51, Irradiated Mice Treated with Rat Bone Marrow. Prec. 1953. Soc. Exper. Biol. & Med., 96: 714-20, 1957.

~(~. 1.--Normal thymus. (A) Normal architecture; there lymphocytes and more prominent reticulum cells than in Fig. are more lymphocytes in the cortex than in the medulla, X 100. 1B, X1300. (B) Cortex of Fig. 1A composed of dense sheet of small lympho- FIe. 3.--Thymus after 18 weeks of testosterone treatment. cytes, X1300. (A) Marked reduction in size and no differentiation of cortex from medulla, X100. (B) Same thymus as in Figure 3A; even I~G. 2.--Thymus after 5 weeks of testosterone treatment. in the most cellular area there is a great decrease in concentra- (A) Decrease in width and in concentration of small lympho- tion of small lymphocytes and increased prominence of reticu- cytes in cortex, X100. (B) Cortex of Fig. ~A; fewer small lure cells, X 1300.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. FIG. 4.--Thymus 34 hours after the last dose of total-body irradiation. (A) Decrease in the number of cortical lymphocytes, X100. (B) Cortex of Fig. 4A, )<1300. FIG. 5.--Thymus 1 week after irradiation. (A) The cortex has been repopulated with large lymphocytes, )< 100. (B) Note comparatively large lymphocytes in the cortex versus normal small lymphocytes of Fig. 1B, X1300. FIG. 6.--Thymus ~ weeks after irradiation. (A) Expansion of cortex due to increase in number of lymphocytes, )<100. (B) Lymphocytes are smaller than those seen at 1 week (Fig. 5B) after irradiation, • 1300.

~ 4 B

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. FIG. 7.--Thymus 3 weeks after irradiation. (A) Partial involution due to a decrease in number of cortical lymphocytes, X100. (B) Higher power of Fig. 7A; decrease in number of lymphocytes, X 1300. FIG. 8.--Thymus, 6 weeks after irradiation. (A) Normal- appearing, compare with Fig. 1A, X 100. (B) Higher power of Fig. 8A, normal appearing lymphocytes, X 1300. FIG. 9.--Thymus 8 weeks after irradiation. (A) Cortex has expanded at the expense of the medulla, X 100. (B) Prolifera- tion of large lymphocytes in the cortex, X 1300.

~~ ~~,~ ~ ~ ~i ~, ~ ~,,~ ~,,

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. FIa. 10.--Thymus 1~ weeks after irradiation with definite evidence of leukemia. (.4) Reduction in size of medulla due to cortical hypertrophy, X100. (B) Extensive proliferation of stem cells in cortex, X 1800. Fla. 11.--Leukemic infiltration in advanced leukemia. (A) Thymus, X1800. (B) Bone marrow, XS00. (C) Liver, X800. (D) Spleen, X800. (E) I,ymph node, X 100. (F) Renal cortex, X 100.

~ ~i,~84

t~ J

~ i, ~'

* f D

2B. 3. f

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. FIG. l~.--Thyluus 1 week after irradiation and testosterone. (.4) Lesser cellularity of cortex than in Group III at same time (Fig. 5A), X100. (B) Lesser number of lyinphocytes than in Fig. 5B, X 1300. FIG. 13.--Thymus ! weeks after irradiation and testosterone. Decrease in size due to a reduction in the number of lymphoeytes, X 100. FIG. 14.--Thymus 3 weeks after irradiation and testosterone. (A) Further atrophic change, X 100. (B) Lymphocytes are markedly decreased in ntmlber with increased prominence of retieulum cells, X ltl00.

ill -~= j ~., -I

9 S @

30. GRoss, L. Effect of Thymectomy on Development of Leu- search, 13: 262-68, 1953. kemia in C3H Mice Inoculated with Leukemic "Passage" 40. LACASSAGNE, A. Sarcomas lymphoides apparus chez de Virus. Proc. Soc. Expel Biol. &Med., 100: 325-28, 1959. souris longuement trait6es par des hormones cestrog~nes. 21. JACOBSON, L. O. Evidence for a Humoral Factor (or Fac- Comp. Rend. Soc. de Biol., 126:198-95, 1937. tors) Concerned in Recovery from Radiation Injury: A 41. LAw, L. W. Increase in Incidence of Leukemia in Hybrid Review. Cancer Research, 12: 315-25, 1953. Mice Bearing Thymic Transplants from a High Leukemia 22. JACOBSON, L. 0.; SIMMONS, E. L.; and BLOCK, M. The Ef- Strain. J. Nat. Cancer Inst., 12: 789-805, 1952. fect of Splenectomy on the Toxicity of Sr 89 to the Hemato- 42. LAW, L. W., and MILLER, J. H. Observations on the Effect poietic System of Mice. J. Lab. & Clin. Med., 34:1640-55, of Thymectomy on Spontaneous Leukemias in Mice of the 1949. High-Leukemic Strains, RIL and C58. J. Nat. Cancer 23. KAPLAN,H. S. Comparative Susceptibility of the Lymph- Inst., 11:253-62, 1950. oid Tissues of Strain C57 Black Mice to the Induction of 43. ~. The Influence of Thymectomy on the Incidence of Lymphoid Tumors by Irradiation. J. Nat. Cancer Inst., Carcinogen-Induced Leukemia in Strain DBA Mice. Ibid., 8:191-97, 1948. pp. 425-37. 34. ~. Preliminary Studies of Effectiveness of Local Ir- 44. LAw, L. W., and POTTER, M. The Behavior in Transplant radiation on the Induction of Lymphoid Tumors in Mice. of Lymphocytic Neoplasms Arising from Parental Thymic Ibid., 10: 267-70, 1949. Grafts in Irradiated, Thymectomized Hybrid Mice. Proc. 25. ~. Influence of Thymectomy, Splenectomy, and Nat. Acad. Sc., 42:160o71, 1956. Gonadectomy on Incidence of Radiation-Induced Lymph- 45. ~. Further Evidence of Indirect Induction by X- oid Tumors in Strain C57 Black Mice. Ibid., 11:83-90, Irradiation of Lymphocytic Neoplasms in Mice. J. Nat. 1950. Cancer Inst., 20: 489-93, 1958. 26. ~. On the Etiology and Pathogenesis of the Leuke- 46. LEVINTttAL, J. D.; BUFFET, R. F.; and FURTH, J. Preven- mias: A Review. Cancer Research, 14: 535-48, 1954. tion of Viral Lymphoid Leukemia of Mice by Thymec- 27. ~. Early Microscopic Diagnosis of Lymphosarcoma tomy. Proc. Soc. Exper. Biol. & Med., 100: 610-14, 1959. in situ in Thymus of Irradiated Mice. Fed. Proc., 19:399, 47. LORENZ, E.; CONGDON, C. C.; and UPHOFF, D. Prevention 1960. of Irradiation-Induced Lymphoid Tumors in C57BL Mice 28. KAPLAN,H. S., and BROWN, M. B. Further Observations on by Spleen Protection. J. Nat. Cancer Inst., 14:291-301, Inhibition of Lymphoid Tumor Development by Shielding 1953. and Partial-Body Irradiation of Mice. J. Nat. Cancer Inst., 48. LORENZ,E.; LAW, L. W.; and CONGDON, C. C. The Role of 12: 427-36, 1951. Bone Marrow and Spleen in Induced and Spontaneous 39. --. Effect of Peripheral Shielding on Lymphoid Tissue Lymphatic Leukemia. Leukemia Research, A Ciba Foun- Response to Irradiation in C57 Black Mice. Science, 116: dation Symposium, pp. 189-98. Boston: Little, Brown & 195-96, 1952. Co., 1954. 30. ----. A Quantitative Dose-Response Study of Lymphoid 49. MARTIN, S. J. Effect of Certain Endocrine Secretions on Tumor Development in Irradiated C57 Black Mice. J. Nat. the X Zone of the Adrenal Cortex of the Mouse. Proc. Cancer Inst., 13:185-208, 1952. Soc. Exper. Biol. & Med., 28:41-42, 1930. 31. ~. Protection against Radiation-induced Lymphoma 50. MAXIMOW, A., and BLOOM, W. A Textbook of Histology, Development by Shielding and Partial-Body Irradiation of 7th ed. Philadelphia: W. B. Saundcrs & Co., 1957. Mice. Cancer Research, 12: 441-44, 1952. 51. McENDY, D. P.; BOON, M. C.; and FURTH, J. On the Role 32.--. Testosterone Prevention of Post-Irradiation of Thymus, Spleen and Gonads in the Development of Lymphomas in C57 Black Mice. Ibid., pp. 445-47. Leukemia in a High-Leukemia Stock of Mice. Cancer Re- 33. ~. Radiation Injury and Regeneration in Lymphoid search, 4: 337-83, 1944. Tissues. J. REBUCK, F. BETHELL,and R. MONTO (eds.). In: 52. MURPHY, J. B. The Effect of Castration, Theelin, and The Leukemias: Etiology, Pathophysiology, and Treat- Testosterone on the Incidence of Leukemia in a Rockefeller ment, Chapter 9. New York: Academic Press, Inc., 1957. Institute Strain of Mice. Cancer Research, 4: 622-24, 1944. 34. KAPLAN,H. S. ; BROWN, M. B. ; and PAULL, J. Influence of 5S. REINHARDT,W. 0., and WAINMAN,P. Effect of Thyroidec- Bone Marrow Injections on Involution and Neoplasia of tomy, Castration, and Replacement Therapy on Thymus, Mouse Thymus after Systemic Irradiation. J. Nat. Cancer Lymph Nodes, Spleen in Male Rats. Proc. Soc. Exper. Inst., 14:306-16, 1953. Biol. & Med.,49:257-60, 1942. 35. --. Influence of Postirradiation Thymectomy and of 54. SKIRMONT, E.; MARKS, E. K.; and JACOBSON, L. O. A Thymic Implants on Lymphoid Tumor Incidence in C57 Modified Technique for Counting Blood Platelets. Am. J. Black Mice. Cancer Research, 13: 677-80, 1953. Med. Tech., 15:86-87, 1949. 36. KAPL~N, H. S.; CARNES, W. H.; BROWN, M. B.; and 55. STARKEY, W. F., and SCHMIDT, E. C. H. The Effect of HmSCH, B. B. Indirect Induction of Lymphomas in Irra- Testosterone-Propionate on the X-Zone of the Mouse diated Mice: I. Tumor Incidence and Morphology in Mice Adrenal. Endocrinology, 23: 339-44, 1988. Bearing Nonirradiated Thymic Grafts. Cancer Research, 56. WocH, P.; HIRSCH, B. B.; BROWN, M. B.; NAGAREDA,C. S.; 16:422-35, 1956. and KAPLAN, H. S. Lymphoid Tumor Incidence in Mice 37. KAP~N, H. S. ; HmSCH, B. B. ; and BROWN, M. B. Indirect Treated with Estrogen and X-Radiation. Cancer Research, Induction of Lymphomas in Irradiated Mice: IV. Genetic 16: 890093, 1956. Evidence of the Origin of the Tumor Cells from the Thymic 57. URSO, P., and CONGDON, C. C. The Effect of the Amount Grafts. Cancer Research, 16:434-36, 1956. of Isologous Bone Marrow Injected on the Recovery of 38. KAPLAN, H. S.; NAGAREDA,C. S.; and BROWN, M. B. En- Hematopoietic Organs, Survival and Body Weight after docrine Factors and Radiation-Induced Lymphoid Tumors Lethal Irradiation Injury in Mice. Blood, 12:251-60, 1957. of Mice. In: Recent Progress in Hormone Research, 10: 58. WRITE, A., and DOUGHERTY, T. F. The Role of Lympho- 393-332. New York: Academic Press, Inc., 1954. cytes in Normal and Immune Globulin Production, and the 39. K~r~SCHBAUM, A.; SHAPmO, J. R.; and MIXER, H. W. Mode of Release of Globulin from Lymphocytes. Ann. New Synergistic Action of Leukemogenic Agents. Cancer Re- York Acad. Sc., 46: 859-80, 1946.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1961 American Association for Cancer Research. The Histopathologic Study of the Development of the Irradiation-induced Leukemia in C57BL Mice and of Its Inhibition by Testosterone

Prawase Wasi and Matthew Block

Cancer Res 1961;21:463-473.

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