[CANCER RESEARCH 32, 48—56,January 1972J

secreted by different cells. If so, these pituitary tumors shouldbe composed exclusively or predominantly of mammotropes,somatotropes, and adrenotropes. On the contrary, in ourexperience all of the many pituitary tumors arising inacidophilic cells secrete MtH and StH and some also AtH invarying ratios.

It was reported independently from 3 laboratories thatMtT.W5 that originated in a female W/Fu rat which had beenirradiated over the head with 800 R (22) and was transplantedin series in normal W/Fu female rats lost the ability to produceMtH but not 5tH. MacLeod et aL (1 1) discovered this by thefailure of the rat to support the growth of a MtH.dependentmammary tumor and by the character of the hormone profilein polyacrylamide gel electrophoresis of the pituitary extracts.Tashjian et a!. (19, 20) isolated a similar variant by cloning cellcultures of this tumor, and Hollander and Hollander (7) did sofrom an ovarian metastasis.

Because of the theoretical and practical importance ofhaving monomorphous, transplantable, monohormonalneoplasms, we have undertaken a characterization of theHollander variant with conventional and more recentlyintroduced sensitive techniques.

MATERIALS AND METHODS

OriWn and Variants of MtT.W5. MtT.W5 was isolated in1961 from a rat 563 days after the rat had received 800 R overthe head and neck (22). When the tumor became fast growingand metastasizing, Hollander and Hollander (7) isolated froman ovarian metastasis a variant named MtT.W5/OM. Earlier,the similar variantsisolated from this tumor in the 2 otherlaboratories were named StW5 (1 1) and GH3 (20),respectively. Following the conventional terminology ofgeneticists, we propose to name the 3 somatotropic variants ofMtT.W5 as follows: W5/St.M, W5/St.T, and W5/St.H.Characters following the shilling indicate the character of thevariant (St = somatotrope) followed by the initial letter of thesenior investigator.

In our laboratory, W5/St.H was transplanted s.c. in 6consecutive passages of males and in 8 consecutive passages offemales, in a total of 47 normal and 4 ovariectomized femalesand 49 normal and 4 castrated males. The grafted tumors werepalpable at about 30 days and grew very fast in both femalesand males, reaching approximately 3 cm across (weighingabout 21 g) in about 50 days. They grew equally well incastrated female and male rats.

At autopsy, all endocrine glands and their target organswere carefully examined, sectioned, and stained byhematoxylin and eosin routinely. IHS was performed in some

48 CANCER RESEARCH VOL. 32

Growth Hormone-secreting Variants of a Mammotropic Tumor'

Akihiro Ito,2 JacobFurth,andPeggyMoy

Institute ofCancer Researchand Department ofPathology, Columbia University, New York 10032

SUMMARY

Three investigators described independently somatotropicvariants (mutants) of the originally typical mammotropicMtT.W5, which was isolated from a rat that had been X-rayed(800 R) over the head and neck. Studies of one of thesevariants (W5/St.H) led us to conclude that the three variantsare basically identical; all possess high somatotropic hormone(growthhormone,5tH)andverylowmammotropichormone(prolactin, MtH) activity. However, persistence of secretion ofsome MtH is indicated by our tests with the highly sensitiveradioimmunoassay and immunohistochemical staining.

The mammary gland stimulation of the variant studied byus is characterized by slight to moderate alveolar hyperplasiawith some milk secretion, periglandular and perivascularfibrosis, some ability to support the growth of ahormone-responsive mammary tumor, and serum levels of MtHelevated 8 to 23 times normal. The variant is stable and fullyautonomous, growing equally well in rats of both sexes.

5tH secretion was about as high as that of the originaltumor as judged by similar criteria. StH serum levels wereincreased to about 220 times normal in rats of both sexes.

Immunohistochemical staining of the tumors was distinctfor StH but not for MtH. Staining of the pituitaries of tumorhosts for MtH was reduced as compared with normalpituitaries. These findings suggest that the quantity of thishormone secreted per cell in the tumors was very small.

This study supports our earlier findings of the intimaterelation of MtH and 5tH production and is consistent with thehypothesis that the same neoplastic acidophils can produceboth hormones.

INTRODUCTION

Primary pituitary tumors arising in acidophils arepredominantly mammotropic. When transplanted in isologoushosts, they gain in body and organ weight and exhibit markedsomatotropic and some adrenotropic potency (6). Uponsuccessive transplantation, the ability to secrete thesehormones greatly decreases but is rarely lost completely.

It is widely held that in rodents MtH3 , StH, and AtH are

I This work was supported by Grant CA-06215 from the National

Cancer Institute.2 Research Fellow of the Damon Runyon Memorial Fund for Cancer

Research, Inc.3The abbreviations used are: MtH, mammotropic hormone; 5tH,

somatotropic hormone; AtH, adrenotropic hormone; W/Fu,Wistar-Furth; MtT, mammotropic tumor; IHS, immunohistochemicalstaining; RIA, radioimmunoassay; MT, mammary tumor.

Received June 30, 197!; accepted August 25, 1971.

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No MtT in hostW5/St.H in hostW10inhostNo.orrat443Carcass

(g)202 ±7.3327 ±JO256 ±10.5MT(g)0.21 ±0.060.69 ±0.345.7 ±1.7MtT(g)15.0 ±2.036.0 ±5.2Thymus

(mg)188 ±10(90 ±4.9)381 ±41(116 ±12.5)150 ±15(59 ±6.0)Adrenal(mg)36±6.7(17±3.3)54±7.0(17±2.1)90±5.8(35±2.3)Ovary

(mg)55 ±7.6(26 ±3.8)112 ±11.2(35 ±3.4)79 ±10.0(31 ±3.9)Uterus(mg)204 ±4.6(95 ±2.3)329 ±98(102 ±30)209 ±10(84 ±4.6)Spleen(g)0.44 ±0.08(0.21 ±0.04)1. 06 ±0. 18(0.32 ±0.06)1. 17 ±0.27(0.46 ±0.10)Kidney(g)0.75 ±0.03(0.36 ±0.13)1.08 ±0.06(0.33 ±0.02)1.42 ±0.02(0.56 ±0.08)Liver(g)7.0±0.3(3.4±0.12)17.2±2.1(5.2±0.6)14.8±1.1(5.8±0.4)

Growth Hormone-secreting Tumors

tumors and pituitaries of the tumor-bearing hosts. Allmammary pads were examined at autopsy; but for precisecomparative histological studies, only the right inguinal pad,including the lymph node, was sectioned.

RIA. Serum StH and MtH values were measured by meansof the double-antibody technique (17). The biologicalpotencies of rat MtH (NIAMD-RP-I-l) and of 5tH(NIAMD-RGH-I-l) were 30 and 1.5 units/mg, respectively.The corresponding antthormones were designatedNIAMD-Anti-RP-S-l and Anti-RGH-S-I . The basic technicaldetails used have been described (8). Because of its simplicity,the charcoal method (1 5) was used to duplicate theconventional double-antibody assays for 5tH in S tests.

In each assay, sera with known high and low values weretested with the unknowns. All serum samples were tested induplicate, some in 2 or 3 dilutions; the values stated wereaverages. Some sera were tested repeatedly. The standard curvewas made with hormone concentrations from 0 to 25 ng/0.lml. Five tests were made for MtH and 5 were made for 5tH.The most sensitive areas of the curve on which the calculationswere based were between 0.5 to 10 ng/0.1 ml.

IHS. This was done by peroxidase-labeled antibodiesfollowing the technique of Nakane and Pierce (14) withtumors, pituitaries of the tumor hosts, and normal pituitariesas controls. The tissues were fixed in Bouin solution andembedded in paraffin as recommended by Baker et a!. (1).Rabbit antthormones to rat 5tH, rat MtH (18), and humanStH/YA were prepared by us. The goat anti-rabbit v-globulinwas prepared and conjugated with horseradish peroxidase, andthe substrate used was 3,3'-diaminobenzidine.

Test for MtH for Stimulation of MtH-responsive MT.MtT.W5/St.H was tested for stimulation of MtH-responsiveMT. A hormone-responsive mammary tumor (MT.W9A) wasderived from a combined treatment of subcarcinogenic dosesof 3-methylcholanthrene and MtH (9). It is transplantable innormal female rats but grows much faster in MtT-bearing(MtH-stimulated) hosts.

Four experiments were performed. The MtT mince wasinjected s.c. in the back of the neck, and MT was injected inthe right groin. Each experiment contained 2 additional groupsof rats. In one, MT was cografted with the high-MtH-secretingMtT.Wl0, and in the other, normal females received nopituitary tumor graft. MT and MtT were grafted at the sametime but MtT grew faster than MT. In Experiments 1 and 2,the rats were sacrificed in extremis. In Experiments 3 and 4,the rats were sacrificed in matched “trios―when the MtT wasvery large.

RESULTS

Transplantation and Anatomical Evidence of MtH and 5tHSecretion. Transplantation of W5/St.H was successful in all104 rats, and none of the tumors regressed. The rate of growthwas equal in males and females and was not affected bycastration.

In the original tumor (MtT.W5), the tumor cells were fairlyuniform in size and shape (Fig. 1). The only distinctmorphological change in W5/St.H was a moderate anaplasia.This is indicated by much greater variability in the size andshape of the tumor cells, especially their nuclei and increasednumber of mitoses (Fig. 3). In some areas the tumor cells weredetached, superficially resembling reticulum cell sarcomas.

The marked increase in body weights and a proportionalincrease in organ weights indicated 5tH secretion in W5/St.H(Table 1). The increases in weights are based on those of ratsnot grafted with MtT's. These figures indicate a markedsomatotropic activity of the variant W5/St.H. The changesnoted were in direct relation to tumor size and tumor age(Table 2). When the normal control females weighed about328 g, the W5/St.H.bearing female rats weighed 455 g and themales weighed 5 18 g. The thymus weight in this experimentrose from a range of 180 to 200 mg to 540 mg in rats withtumors weighing 4 to 6 g and measuring 1.5 to 2.0 cm in

Table 1Tumor, organ,and carcassweightsofrats in Experiment 1 in Table3

The figures not in parentheses give the actual weights.Those in parentheses refer to weights/100g of carcass.The italicizedfigures in MtT-bearing rats, based on those of normal rats, are significant at p < 0.01 leveL

The data suggest that the adrenal enlargement in W10 hosts is due mainly to AtH, while that ofW5/St.H is probably due togeneral 5tH effect of this hormone. Enlargements of the liver can be caused by both 5tH and AtH. The kidneys are onlyslightly (not significantly) enlarged by W5/St.H while in W10 rats they are markedly (significantly) enlarged, predominantlydue to AtH activity. The marked increaseof the thymus weight in W5/St.Hrats is clearly due to 5tH, sinceAtH is thymolytic,as is well known.

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RatTumorfeaturesHormones

inseraBiological5tHMtHNo.SexTimea

(days)Size― (cm)Body weightMammaryhyperplasiaNo.of

ratsLevel (@g/ml)No.of

ratsLevel(@sg/ml)14F0198

±193bo140.04 ±0.02k'0.057 ±001b1F511.0260±10.1410.05SF66(44—78)2.0—3.2320±24.2±to+±64.91±

1.5460.112±0.0175F71(62—81)4.0—6.0455 ±8.5++ to ++±69.24 ±3.9660.435 ±0.1316M0328±3.380160.03±0.01160.014±0.0014M1

16 (79—190)‘@-5.0518 ±40.0+ to ++46.55 ±0.7240.33 ±0.2

Akihiro Ito, Jacob Furth, and Peggy Moy

Table 2Hormone levels,body weights,and mammarygland hyperplasiain rats bearingMiT. W5/St.H

The mammary gland hyperplasia was graded on the basis of microscopic appearance of the gland around the right inguinal node as ifiustrated inFigs.5 to 7.

a Mean and range.

b Mean ±S.E.

average diameter. With further growth of the tumor, thethymus weight decreased to about 40 mg. This may be due to“stress―-inducedAtH secretion. At the same time, adrenalweights increased disproportionately. However, microscopicexamination failed to indicate the type of stimulation by AtHas seen in AtH-secreting MtT's. In Table 1, the weight changesin W5/St.H rats are contrasted with those of the usual type ofMtH-, 5tH-, and AtH-secreting, MtT.Wl0-bearing rats.

Mammary Gland Hyperplasia (Figs. S to 7). This was oftenmissed at autopsy in rats with small tumors because afibromuscular fascia obscured the underlying mammary gland.It became, however, conspicuous at autopsy only when thetumor-bearing period was long and the tumors were large. Milksecretion, which is characteristic of the usual MtT strain (Fig.2), was slight or absent. Diffuse periductal and perivascularfibrosis is characteristic of these tumors (Figs. 6 and 7), incontrast to usual MtT's (Fig. 2), in which it is rare. Thisfibrosis was more marked in males than in females. Mast cellswere abundant in the stimulated gland in several rats (Fig. 6)and absent in others.

mA'S in Relation to Biological Features. The serum level of

both StH and MtH was progressively increased with the tumorsize in rats of both sexes (Table 2).

In 1 female, a tumor 1 cm in average diameter causeddistinct elevation of 5tH but not of MtH. With tumors withdiameters of 2 to 3.2 cm, the serum 5tH increased to about120 times normal and MtH increased to about twice normal.With tumors with diameters of about 4 to 6 cm, 5tH levelsreached a peak of 230 times normal and MtH levels were 8times normal.

In males, 5tH levels with tumors with a mean diameter ofabout 5 cm were 220 times normal and MtH levels were 23times normal.

These figures indicate an increase of the serum levels ofboth hormones in both sexes directly related to tumor age andtumor size. Whereas in the usual type of MtT.Wl 5 the ratios ofserum levels between 5tH and MtH were 1:6.2 in females, theywere I :4.4 in males; in W5/St.H rats, they were about 20: 1 inboth sexes.

In this present study, MtT.WlO was used to test the ability

of MtH to support the growth of a hormone-responsive MT,grafting both on the same rat. The RIA's of MtH of the sera ofrats bearing this MtT averaged 5 .4 jig/ml with a range of 3.6 to6.6 pg/mi in 4 female rats that were assayed.

IHS. Sections from tumors and pituitaries ofW5/St.H-bearing rats were stained with peroxidase-labeledantibodies for 5tH and MtH. The tumors stained well for 5tH(Figs. 8 and 14), but the intensities of the staining in differentcells varied greatly. 5tH in both normal and tumor cellsencircles the spherical nucleus.

Pituitaries for 5tH in tumor-bearing hosts are shown in Figs.9 and 15. In general, the staining per cell was more intense inthe pituitary than in the tumor. In the pituitary, the stainingfor 5tH was less intense in the tumor-bearing hosts than innormal controls (Fig. 12).

Staining of the tumor with anti-MtH was weakly positive(Figs. 10 and 16), often barely detectable; many cells wereunstained. The number of mammotropic cells in the pituitariesof tumor hosts (Figs. 11 and 17) were, however, fewer, andthe intensity of staining was lighter than that of mammotropiccells in normal pituitaries (Fig. 13). Localization of thehormone in the cytoplasms of mammotropic cells ischaracteristic. Unlike 5tH, it is concentrated in one “pole,―forming a cap over the unstained nucleus (Fig. 16). Anti-AtHfailed to stain the tumor cells (Fig. I 8).

Test for Ability of W5/St.H to Support the Growth of aHormone-responsive Mt. Failure to stimulate the growth of thehormone-dependent MT9 led MacLeod et al. (1 1) to thediscovery of his 5tH. Hollander and Hollander (7) observed thesame behavior of their variant, derived from an ovarianmetastasis.

The ability of MtT.W5/St.H to stimulate growth of thehormone-responsive MT9A was tested by us in 4 experiments,u@ng for comparsion the MtH-secreting MtT.WlO and normalfemales (with no MtT cografts). Two of these experiments aredetailed in Table 3. All grafted MtT's grew progressively. Thegrowth promotion of the hormone-responsive MT by thehigh-MtH-secreting MtT.Wl0 is indicated by shortened latencyperiod of MT in Experiments 1 and 2 and by marked increaseof the size of MT in Experiment 1. In Experiments 3 and 4

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Experiment 1Experiment2No.MTNo.MTCograftsMT/no.LatencysizeMT/no.Latencysizeof

MtTinjected(days)(mm)injected(days)(mm)None3/570

±5.3 “4±l.Oa2/32705W5/St.H4/567±6.06.5 ±1.63/4103 ±8.1@13 ±3.3―W105/5<4014

±2.83/3<6012 ±4.4

Growth Hormone-secreting Tumors

Table 3Testfor ability ofMtT. W5/St.H to support the growth ofMtH-responsive MT. W9A

a Mean ±S.E.

(not shown in Table 1), all rats were sacrificed at 43 and 54days, respectively, because of the rapid growth of MtT.WlO. Inthese experiments, 8 of 11 rats, cografted with MtT.WlO, hadgrafted MT; but no MT was detected in the other 2 groups.

These experiments suggest that both the high-MtH-secretingMtT.WlO and the low-MtH-secreting W5/St.H can promote thegrowth of the hormone-responsive MT. However, MtT.WlOdoes it more efficiently than the predominantly 5tH-secretingW5/St.H.

INCIDENTAL OBSERVATIONS WITH COMMENTS

Staining Human Pituitaries with Anti-Human GrowthHormone. Postmortem Bouin-fixed human somatotropic cellsin pituitaries can be well visualized, not only by anti-humangrowth hormone (Fig. 19) but also by anti-rat and anti-bovinegrowth hormone by means of IHS. Since the human growthhormone has inherent prolactational activity, we also tried tostain the human pituitary with anti-rat MtH. In simultaneoustests, rat pituitary stained intensely while the human pituitaryfailed to do so.

Virus in Mammosomatotropic Tumors. Chopra and Taylor(4) found C-type viral particles in both the original W5 strainand its W5/St.H variant. There is no evidence at present thatthese viral particles play any role in tumorigenesis in the rat orwere in any way influential in the findings here described.From MtT.W10 culture fluids, collected in our laboratory, gs3viral antigen was identified. No disease occurring in the W/Fustrain could be connected with this virus.

Extensive experiments aimed at acceptance by W/Fu rats ofadministered Bittner's mammary tumor virus have beenunsuccessful (G. Ueda and J. Furth, unpublished data).

DISCUSSION

The Problem. Since the discovery that all estrogen-inducedpituitary tumors, originally named mammotropic, alsopossessed somatotropic properties (6), large numbers ofspontaneous, variously induced and transplanted pituitarytumors were studied in our laboratory, but none was foundthat did not secrete some of both hormones.

The constant association of MtH with 5tH puzzled usbecause of the currently accepted view that these hormonesare secreted by 2 different types of acidophils, “orangophils,―which secrete 5tH, and “carminophils,―which secrete MtHELacour(10)]. Lacour'sfindingshavebeenconfirmedby

electron microscopists, who noted ultrastructural differencesbetween somatotropic and mammotropic cells (1 6). However,immunofluorescent labeling of rat pituitaries with anti-MtH byEmmart et a!. (5) showed bright fluorescence of all normalacidophils. This tends to support the view that one cell mayhave overlapping MtH and 5tH activity. In contrast, by the useof IHS some differences were noted between mammotropicand somatotropic cells (1 , 14). Conclusive experiments tosettle this problem are wanting.

The problem was further complicated by the findings ofhigh levels of AtH in some MtT's. Studies of MtT.F4 with veryhigh AtH activity (2, 12) led us to conclude that this tumorderived from acidophils is composed of cells which can secrete3 hormones: MtH, 5tH, and AtH (J. Furth and P. Moy,unpublished data). With the W5/St.H variant, IHS failed todisclose the secretion of AtH. The adrenals are only slightlyenlarged, and the thymus becomes involuted only when thetumors are large. This is a common event in rats with largetransplated tumors of diverse types. This atrophy is attributedto adrenotrope-mediated, stress-induced thymus involution bythe glucocorticoids of the host (21).

Our observations suggest the hypothesis that neoplastic“acidophils―can secrete both MtH and 5tH and that some canalso secrete AtH. Neoplastic cells are transformed normal cells;in the course of transplantation some cells can undergo furthermodifIcation.

On the basis of extensive experience, the natural history ofMtT's appears to be as follows. With primary tumors thedominant anatomic feature is hyperplasia of the mammarygland. However, RIA's (8) reveal elevated levels of both MtHand 5tH in the sera of rats with primary tumors, although invarying ratios. In the course of transplantation, 5tH activityusually becomes manifest as early as the 1st transplantgeneration. In the course of further transplantations, the trendis increase in the rate of reproduction, and increase inanaplasia of tumor cells, with diminution of secretion of bothMtH and 5tH, predominantly the former. Some tumors loseability to secrete hormones altogether; others become stable ata fixed secretory rate which may include AtH. The latterseems to be associated with MtH. Why, when, and how AtH isacquired are unknown.

Somatotropic Pituitary Tumor Variants. All 3 somatotropictumors reported (7, 11, 20) were derived from the sameMtT.W5 . The original tumor is being preserved in liquidnitrogen in the Hormone Related Tumor Bank of the NationalCancer Institute. It was resuscitated upon our request by Dr.

JANUARY 1972 51

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Akihiro Ito, Jacob Furth, and Peggy Moy

A. E. Bogden, who found that it still possessed high MtHactivity.

The 3 reports on 5tH are not overlapping and supplementeach other remarkably. The 3 variants reported have 2 basicfeatures in common: secretion of large quantities of StH anddiminution or possibly complete loss of ability to secrete MtH.Two basic issues remain to be clarified. Did W5/St.H losecompletely the ability to secrete MtH or was it composed of 2cell types (mammotropic and somatotropic) of which one (themammotrope) was lost in the course of transplantations? Thetechniques used by others were not sensitive enough to provecomplete loss of MtH secretion. Reasons for the possiblefailure of others to detect mammary gland stimulationmorphologically have already been stated. Disc electrophoresisin acrylamide gel detects only about 10 to 20 j.zgprotein pergel of MtH and 5tH, whereas RIA detects 1 to 10 ng (m@ig).

It is well known that circulating hormones depress theproduction of the corresponding hormone in the pituitary.This has been well documented for 5tH by MacLeod et al.( 1 1). In our study with IHS of the pituitaries of rats bearing

larger tumors, the depression was only moderate. This remainsto be explained.

The Biological Potentialities of 5tH. We found no reportpertaining to the question of whether 5tH alone can stimulatethe mammary gland but suppose that it may do so onprolonged administration. If it does, in what way does it differmorphologically from stimulation by MtH?

Bates et al. (3) found that bovine 5tH administered to ratsfor 10 days failed to increase the weight of the mammarygland; while ovine MtH, similarly administered, increased it 4times normal without visible milk production. Thecombination of MtH, 5tH, and AtH increased the weight ofthe gland 10-fold, and the alveoli were filled with milk.

Tests for MtH by promotion of the growth ofhormone-responsive MT is supported by the findings ofNagasawa and Yanai (1 3), who found that bovine MtH but notbovine 5tH strongly stimulated hormone-responsive MT.

MtT.W5/St.H secretes homologous, nonantigenic 5tH inmuch higher quantities and over longer periods of time thanhitherto administered 5tH. Therefore, it is possible that StHalone accounted for some mammary gland stimulation in ourstudies of W5/St.H. However, the elevated RIA levels for MtHand the secretion of milk in the mammary alveoli suggest someMtH activity. The marked periductal and perivascular fibrosisin the mammary gland of MtT.W5/St.H may be due to 5tH.The effects of StH on the mammry gland with and withoutglucocorticoids remain to be studied.

ACKNOWLEDGMENTS

We thank Dr. K. C. Hsu (Columbia University, New York, N. Y.) forpreparation of the goat anti-rabbit 7-globulin conjugated withhorseradish peroxidase; Dr. P. K. Nakane (University of Colarado,Denver, Cob.) for supplying anti-AtH; Miss G. Geering, forimmunotyping the virus particles in MtT.W10; Mr. Harold McQuilla forskillful technical assistance;Mr. Edward Hajjarand Mr.Martin Rothgarfor the photomicrographs; and Miss Rona Esterman for the histologicalpreparation. NIAMD-Anti-RP-S-1 and Anti-RGH-S-1 were kindlysupplied by the National Institute of Arthritis and Metabolic Diseases.

CANCER RESEARCH VOL. 3252

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Tumor Variants in the Rat. Cancer Res., 28: 1963—1975, 1968.22. Yokoro, K., Furth, J., and Haran-Ghera, H. Induction of

Mammotropic Pituitary Tumors by X-rays in Rats and Mice. TheRole of Mammotropes in Development of Mammary Tumors.CancerRes.,21: 178—186,1961.

Mammary Tumors. VI. Immunoassays of Rat Prolactin and GrowthHormone. Proc. Soc. Exptl. BioL Med., 127: 70—74, 1968.

19. Tashjian, A. H., Bancroft, F. C., and Levine, L. Production of BothProlactin and Growth Hormone by Clonal Strains of Rat PituitaryTumorsCells. J. CellBiol.,47: 61—70,1970.

20. Tashjian, A. H., Yasumura, Y., Levine,L., Sato, G. H., and Parker,M. L. Establi@ment of Clonal Strains of Rat Pituitary Tumor CellsThat Secrete Growth Hormone. Endocrinology, 82: 342—352,1968.

Figs. 1 to 7 are stained with H & E. All sections are from female rats.Fig. 1. The original MtT.W5. [Reproduction of Fig. 4 of Yokoro et a!. (22).l x 1000.Fig. 2. Mammary gland of a rat bearing the original MtT.W5. Note the marked hyperplasia with moderate secretion. [Reproduction of Fig. 9 of

Yokoro et al. (22).l X 50.Fig. 3. Variant MtT.W5/St.H. Note the increased anaplasia with mitotic figures and a giant cell. X 1000.Fig. 4. The mammary gland of a young adult normal female in proestrus. x 50.Fig. 5. Mammary gland of a rat bearing MtT.W5/St.H, showing minimal hyperplasia, rated ±.x 50.Fig. 6. Mammary gland of a rat bearing the same type of tumor, showing moderate hyperplasia, rated +±, showing marked periglandular fibrosis

and numerous mast cells. X 50.Fig. 7. Mammary gland of a rat bearing the same type of tumor, showing marked hyperplasia, rated ÷+±with slight fibrosis and moderate milk

secretion. X 50.Figs. 8 to 11. IHS of tumors and pituitaries of MtT.W5/St.H-bearing female rats. X 390.Fig. 8. Tumor stained for 5tH.Fig. 9. Pituitary stained for 5tH.Fig. 10. Tumor stained for MtH.Fig. 11. Pituitary stained for MtH.Figs. 12 and 13. IHS ofpituitaries ofa normal female rat. x 390.Fig. 12. Pituitary stained for 5tH.Fig. 13. Pituitary stained for MtH.

Figs. 14 to 17. IHS of tumors and pituitaries of MtT.W5/St.H-bearing male rats. X 390.Fig. 14. Tumor stained for 5tH.Fig. 15. Pituitary stained for 5tH.Fig. 16. Tumor stained for MtH.Fig. 17. Pituitary stained for MtH.Fig. 18. IHS for AtH ofa MtT.W5/St.H tumor. X 390.Fig. 19. Human female pituitary stained for 5tH. x 390.

53JANUARY 1972

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1972;32:48-56. Cancer Res   Akihiro Ito, Jacob Furth and Peggy Moy  Growth Hormone-secreting Variants of a Mammotropic Tumor

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