(Department of Pathology, Columbia University-Francis Delqfield Hospital, Xew York,
New York; and Boston Dispensary, Boston, Massachusetts)
SUMMARYTwo transplantable rat mammo-somatotropic tumor strains, MtT.F4 and MtT.W5,
were studied for AtH activity. MtT.F4 caused marked enlargement of adrenals, lym-phopenia, thymic atrophy, and moderate enlargement of most viscera. MtT.WS causedthymic enlargement, no lymphopenia, and moderate enlargement of most viscera, including the adrenals.
The findings suggested that both strains have growth-promoting and mammarygland-stimulating activities; MtT.F4 also has AtH activity, whereas MtT.WS does not.Similar findings suggested that one mouse MtT (Strain 50) had AtH activity and another (Strain 48) did not. Adrenalectomy further increased body and tumor weights inthe MtT.F4 hosts and had no such effect on MtT.WS hosts. In normal rats, adrenalec-tomy tripled thymic weights; in MtT.F4 rats adrenalectomy not only prevented thymic involution but caused enlargement of this organ to 6 times normal, thus disclosingthe somatotropic potency of its hormones. The increase in thymic weight, notably inadrenalectomized hosts, appears to be a good index of somatotropic activity and mayserve as a growth hormone assay.
Enhancement of tumor growth, and StH and MtH activities of strain MtT.F4 byadrenalectomy support the view that one cell produces one hormone with the threeactivities (MtH, StH, and AtH).
All pituitary tumors induced by estrogens, and tumor. This led to the supposition that these tu-most of those induced by ionizing radiation and mor cells produce either two hormones (growthoccurring spontaneously in rats, were found to hormone and prolactin) or a single hormone withhave marked mammary gland-stimulating and two functions. We favor the latter supposition—growth-promoting properties (4, 11, 14). These namely, that a single cell produces a large nativetumors appeared to be monomorphous and are hormone with the two activities (8).composed of cells that are probably related to In earlier studies, neither gonadal, thyrotropic,acidophils (4, 11, 14). The intensity of the two nor adrenotropic activities were detected in hostsfunctions of these tumor strains studied thus far of these tumors. The AtH content in blood andvaried, but the ratio between growth-promoting tumors was not elevated (2). The fact that theand mammary gland-stimulating properties was thymuses of the animals bearing these tumors wereabout the same, irrespective of the origin of the of normal size supported the opinion that they
*This work has been supported by Grants C-6215of the lacked AtEt activity. Enlargement of the adrenalsNational Cancer Institute and AT 80-1-2891of the Atomic was explained by somatotropism of the tumorEnergy Commission. cells an(j fatty degeneration.
Receivedfor publication January is, 1962. In the course of studies on the transplantable
mammo-somatotropic tumor1 (rat MtT.F4) it wasnoted that the thymuses of many tumor-bearinganimals underwent early and marked involutionand that the adrenals of these rats were usuallylarge. Similar observations, made with a mouseMtT variant, suggested some corticotropic activity of these tumors (5). More recent assays of thesetumors by Cohen et o/.2 disclosed a distinct AtHactivity, noted independently with MtT.F4 byGrindeland et cd. (9). Similarly, Lyons noted AtHactivity in MtT.F4 tumors, assayed in hypophy-sectomized rats.3 These findings led us to reinvestigate the features of several mammo-somatotropic tumors, including that of MtT.WS, which,according to recent data, still seemed to be devoidof AtH activity. The findings here reported indicate that such a difference between MtT strains isreal, with some possessing and others lacking AtHactivity.
In addition to the rat tumor strains, two mousemammo-somatotropic tumor strains were studied,one of which behaves as the rat MtT.F4 and theother as MtT.WS.
In the course of this work, the remarkable observation was made that adrenalectomy markedlyenhanced the growth-promoting effect of MtT.F4,which had adrenotropic activity, but did not influence that of the MtT.WS, which was devoid of it.
MATERIALS AND METHODSStrains of animals and of MtT.—Rats of the
highly inbred strains F/Fu and W/Fu (4, 11) andLAFi mice in which these tumors arose served asrecipients. Most rats were raised by us; the micewere obtained from the Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine.
F4 was induced by prolonged DES treatment ina female F/Fu rat (4). WS was induced in a female W/Fu rat by x-radiation of the head (14).
1A. I. Cohen and U. Kim, Adrenotropic Activity ofMammo-somatotropic Tumors. II. Adrenotropic HormoneContent of the Tumors and Functional Capacity of the HostAdrenals (in preparation).
3W. R. Lyons, personal communication.
The mouse MtT (Strains 48 and 50) were inducedin LAFi mice by head x-radiation and sustainedtreatment with DES (14).
Procedures.—Femalerats, about 3 months old,weighing about 170 gm., were given injections sub-cutaneously of mince of MtT in the upper back.The mice were also young adult females whengrafted in the same way with tumor mince.
Periodically, the animals were weighed, WBCand differential counts were made, and the size ofthe tumor was estimated.
About one-half of the experimental rats wereadrenalectomized bilaterally about 1 week beforethe MtT graft.
The adrenalectomized rats were given 2.5 mg.of Percorten (deoxycorticosterone trimethylace-tate)4 subcutaneously every 3 weeks and 0.5 percent sodium chloride in the drinking water adlibitum.
The animals were sacrificed when the MtTreached about 5 cm. across in the rats and 3 cm.in the mice. Body and organ weights were recorded. Representative tissues were examined microscopically and will be described in a succeedingreport.
RESULTSEFFECTSOF ADRENALECTOMYONBODY,TUMOR,
ANDORGANWEIGHTSIN F4-BEARINGRATS
In the first experiment, nine F/Fu rats wereadrenalectomized. Ten days later five were graftedwith F4, and four remained as controls. Five non-adrenalectomized animals received the same F4grafts, and five siblings were kept as controls.
This experiment was performed with the 35th-passage tumors. Thereafter, the strain was split,and consecutive passages were made separately innormal and adrenalectomized hosts in attempts toenhance adrenotropic activity of this tumor. Afterthree additional passages a second experiment wasperformed with each line as in the first experiment.Experiment 1 was terminated 70 days after theMtT graft and Experiment 2, 65 days later. Thefindings are summarized in Table 1, and P values5are indicated in Table 2.
Body and tumor weights.—Inthe tumor-bearingnormal rats the mean body weights increased from185 to 223 gm. (P < 0.01) and in adrenalectomized rats from 196 to 324 gm. (P < 0.01). Theincrement in weight in relation to time is shownin Chart 1.
0.01) ; there was a corresponding increase in tumorweight (12.1 vs. 6.4 gm., P > 0.1); Chart 2 showsthat tumors of the same size caused a much greaterincrease in body weight in the adrenalectomizedthan in the normal rats.
Adrenal and thymus weights.—The following
findings were most conspicuous: (a) enlargementof adrenals of F4 rats by about 9 times that of nor
mal controls; (b) complete atrophy of the thymusin all F4-bearing normal rats; (c) increase inweight of the thymus in tumor-bearing adrenalectomized hosts much above that of adrenalectomized controls (1062 vs. 434 mg., P < 0.01).
Evidently, removal of the adrenals disclosed thegrowth-promoting ability of F4.
the mammary glands approximately tripled. Thisis much less than that noted earlier with thisstrain (4), which in the course of years of subpas-sages lost much of its hormone-producing capacity. The breast of F4 hosts are full of milk cysts,some of which measured as much as 1 cm. in diameter. Adrenalectomy prevented milk secretion butnot stimulation of lóbulo-alveolar growth (1, 3).
Other viscera.—The weights of the other viscera
increased, with the exception of those of the ovaryand uterus (4). The enlargement of the kidneys
adrenotropic, growth-promoting, and mammarygland-stimulating activities.
EFFECTS OF ADRENALECTOMYON BODY, TUMOR,AND ORGAN WEIGHTS IN WO-BEARINGRATS
In W5 rats, intercurrent lobular pneumoniacaused a heavy loss in several experimental groups.Therefore, the experiment was repeated, and thecombined data are presented in Table 3. The dataare in agreement with those of earlier work (14),indicating that this MtT does not cause thymic
CHARTS1-5.—The figures on tumor-bearing rats are indicated by heavy lines; those on rats which were not tumor-bearing, by light lines; interrupted lines signify adrenalectomy.
The figures in parentheses indicate tumor size: 4 = 1.0 cm.in average diameter (2 = 0.5 cm.).
CHART1.—Effectof adrenalectomy on body weight of ratsbearing MtT.F4.
and the associated nephrotic changes have beendescribed earlier (4).
Lymphocyte levels.—Lymphocyte levels in the
four groups of animals are charted in relation totumor size (Chart 3). There was a gradual decrease in lymphocyte counts in the tumor-bearingrats. Adrenalectomy checked this decline. A gradual increase in granulocyte levels in the tumor-bearing rats is shown in Chart 4.
These findings, together with those of lympho-penia and disproportional adrenal enlargement inF4 rats, support the conclusion that Strain F4 has
18
16
14
12
IO
8
6
4
Z
200 250 300
Body Weight ( gm )
CHART2.—Relationship between body weight and tumorsize in MtT.F4-bearing adrenalectomized and normal rats. Seelegend to Chart 1.
involution. In fact, the weights of thymuses of W5hosts with large tumors were almost twice those ofnormal controls.
Adrenals. —The adrenals in W5-bearing normal
hosts were about 1.8 times as heavy as those ofnormal controls, and this can be regarded as evidence of growth hormonal activity.
Mammary glands. —The mammary glands of
rats with W5 tumor weighed 2.8 gm. as comparedwith 1.0 gm. of normal controls. Milk secretionwas much less than with F4.
Lymphocyte levels.—There was a slight reduction of lymphocyte levels in both W5-bearing and
CHART3.—Effect of MtT.F4 on lymphocyte levels in nor- CHART4.—Effectof MtT.F4 on granulocyte levels in normal and adrenalectomized host. See legend to Chart 1. mal and adrenalectomized host. See legend to Chart 1.
ADHENALECTOMIZEDHOSTSThe first experiment with F4 described above
was performed with the 35th-passage tumors. Itwas noted by us earlier that the activity of adreno-tropic tumor in mice was enhanced by consecutive
DOO
<0
Ã
22
18
W5 ON ADREX RATS,'-^ ADREX CONTROLS
10 -TUMOR
(2.8)
NORMAL CONTROLS""WSON NORMAL RATS
GRAFTAROExltl!1
1 l20 40 60 80
Days After Tumor Graft
CHART5.—Effectof MtT.WS on lymphocyte levels in normal and adrenalectomized host. See legend to Chart 1.
ADRENOTROPICACTIVITYOFMOUSESeveral autonomous MtT were induced in mice
by whole-body x-radiation (5). The common features of several of these tumors were: coarseacidophilic granules in the cytoplasm of manytumor cells, ability to stimulate the mammarygland, associated somatotropic effects, lack of distinct stimulation of endocrine organs, and somedependency on estrogens. Similar tumors wereinduced perhaps with greater efficiency by whole-body neutron irradiation (7). In a later study,estrogen (DES) alone induced pituitary tumors in51 per cent of 103 mice, x-radiation (550 r overhead and neck) in 7.7 per cent of 78 mice, and thetwo procedures combined in 69 per cent of 83mice (14). On the basis of transplantation tests, itwas concluded that most, if not all, of these tumorswere of MtT type.
One such tumor (Strain 50), studied extensively, produced changes in tumor hosts which suggested AtH activity, another (Strain 48) did not.Table 4 records some salient findings with thesestrains. Strain 50 caused marked obesity withmoderate lymphopenia and thymic involution;Strain 48 did not. Two tumors of each strain wereassayed for AtH activity (Table 5). AtH was notdetected in the two mice of Strain 48 ( <0.3 mil/mg), whereas the two mice of Strain 50 contained
»77 days and 335 days after grafts of Strains 50 and 48.
t Mean.
passages in adrenalectomized mice.6 Therefore, thestrain was "split," and consecutive passages were
made separately in normal and adrenalectomizedhosts. After three additional passages, the lymphocyte counts, body, tumor, and organ weights werecompared in the two lines.
These passages in adrenalectomized hostsfailed to increase the adrenotropic activity of thistumor. A sharp decrease in lymphocyte counts occurred in tumor-bearing rats in both lines. Adre-nalectomy abolished the lymphopenia in both andincreased the gain in body weight.
2.4 and 6.1 mll/mg of AtH. The marked increasein body weight in Strain 50-bearing mice was due,in part, to obesity (which is a characteristic feature of AtT [6,12]), in part to StH activity of thisstrain. In an assay by Dr. Meites, Strain 50 contained about 5 times as much prolactin (0.50 IU/100 mg MtT) as did Strain F4.7
COMMENTSSpecificity of AtH function.—None of the nu
merous TtT studied in hundreds of mice exhibitedadrenotropic activity, even in hypophysectomizedhosts. Conversely, AtT exhibited no TtH or GtHactivity. We assumed, therefore, that productionof AtH is a feature of a specialized cell (At) of thepituitary. This view is negated in part by the observations discussed here and by recent findingsof others suggesting that some human tumors of thethoracic cavity (lung and thymus) have AtH activity (10). It is possible that polypeptides derivedfrom such tumors are functionally related to AtHbut may not be under corticoid control (10).
It is of interest to note that a pituitary cell canhave not only StH and MtH but also AtH activityand that adrenalectomy will enhance both growthand AtH activity. This suggests that the AtH effect is a feature of a large hormone molecule andis not due to a contaminating At cell in the tumoror to an independent "At organelle" in these cells.
Individual differences among MtT strains.—
Three years ago none of the rat MtT assayed gaveevidence of AtH activity (2), nor did morphologicobservation suggest it.
The F4 with AtH activity was induced withestrogen. The rat in which the W5 arose was irradiated. F4 may have lacked AtH potency in thecourse of earlier passages (2) ; W5 is of more recentprovenance. The number of well studied strains isfew, and it remains to be clarified whether AtH activity in MtT is related to genetics, mode of induction, duration of transplantation, or to some otherfactor.
It was noted earlier that a subline of mouseMtT.4 became "atypical" (5). The tumors grew
rapidly, and the hosts became obese. Typical MtTstrains cause weight gain, but only slight or noobesity. Adrenalectomy abolished obesity in fourof six mice of this subline. The tumors grew as wellin males as in females, and the original MtT grewbetter in females. Estrogens appear to be thephysiologic stimulant of Mt (5). The mammo-tropic character of this subline was indicated bythe extensive alveolar stimulation of the breast inall tumor-bearing female mice. The thymus wasatrophie in normal and hypophysectomized mice
7J. Meites, personal communication.
bearing this tumor line. It is possible that thecharacter of hormonal secretions changed in thecourse of transplantations.
Grindeland et al. (9) assayed the AtH activityof a subline of MtT.F4 carried in their laboratoriesby using hypophysectomized rats. They foundthat F4 tumor-bearing rats had at least a tenfoldincrease in corticosterone over that of hypophysectomized controls. The hypophysectomized tumor-bearing animals gained body weight while theircontrols lost weight.
Recent assays of Cohen et oZ.(2) confirmed theseobservations. Thus, some MtT strains possessAtH activity; others do not. It remains to be ascertained whether Mt secretes one or several hormones and whether it is adaptable in secretorycapacity.
The extraordinary increase of thymic weightsin these tumor-bearing adrenalectomized animalsis puzzling. We may recall that the size of thisorgan is greatest during the growth period of lifeduring which MtH activity is minimal. The present observations indicate that an extraordinarythymic enlargement can be caused by StH activity, a moderate enlargement by elimination of corticoid inhibition, and that the two influences cansummate.
Another interesting finding was the decrease inovarian and uterus weights in MtT-bearing animals. Since the literature persists in using luteo-tropin as a synonymous term for prolactin (MtH),it deserves to be emphasized not only that theovaries failed to participate in general somato-tropic stimulation, but also that their weights decreased in tumor hosts (14) and that the uteriweighed less than normal, indicating diminishedlevels of gonadal hormones.
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