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"' ‘ . I J cut.- ‘ n . o .A j- ,1 ‘. On - s y""" .“."“f« .‘ our“ '. . ' -.. -v.,.,._";"':"“j'r-—np ' ‘ .. I ' o . ‘ ‘ rW‘oM rvv - . _ v . fl ' I 0- ....,.,..,.. I: u ‘0". ‘ o¢mp.y A H. c'a W 33? z ‘: D - “'rn'O/b ! "' "’~CFI‘.4~p-'l. ' u 3 < v v '0 ‘ “M‘wo'p'pv . nan-rt, '- ' '~'Ill~_. i! pa 0'- , . 1 pa. . ..4~ - .1? I o a . u o “ "U _ ‘ . ., , vii-nour . ‘. - h...“ ' ' ‘ ' ». . v 0 . . .. _ "as. Jar“, , -- . - o'fl' ’ ' ' "P'l-lu‘t‘o—O' w. . . ’ ‘ ' >':;."vo.-po>~r 9", ' D . l- . . , . Out '0‘" - ‘. ~ , , ....‘ ‘I 'FW .":7 f. - 7—. .'~. D ‘— . may... '3, n-vr- .-.‘ . .l . . .H‘.“ ..-._—.—. .‘ J LIBRARY f- ~.-M‘_‘-—“- _._ Michigan Stats E University “ d In“ J‘s : muons}; a? E? "DAB & SONS' j 300K BINDERY INC. LIBRARY BINDERS Inllnnlt manna“ m ABSTRACT RELATION OF PROLACTIN, ESTROGEN, GH AND PROTEIN DEFICIENCY T0 GROWTH OF DMBA-INDUCED MAMMARY TUMORS IN RATs By Carol Joan Bradley l. The relationship between the growth response of a carcinogen-induced mammary tumor to prolactin and the amount of pro- lactin binding protein in the tumor was studied. Twelve daily injections of l mg ovine prolactin were given to rats bearing 34 DMBA-induced mammary tumors, and tumor response was measured by the increase in the sum of three perpendicular diameters. Dr. Henry Friesen in Montreal, Canada, measured prolactin receptor activity by a radioreceptor assay. Statistical analysis showed a correlation coefficient between tumor growth and amount of receptor protein of r = 0.69, p< .Ol. Tumors with the greatest growth response to pro- lactin exhibited the highest prolactin binding, and vice versa. A negative correlation was noted between the amount of prolactin receptor activity in the liver and the average tumor growth response in indi- vidual rats. These results indicate that the amount of prolactin re- ceptors in a mammary tumor of a rat is a good indication of its ability to show a growth response to prolactin. U 45VE9 Carol Joan Bradley 0) 2. An attempt was made to characterize and quantify estrogen and prolactin dependency of individual rat mammary tumors. The effects of age of tumors on hormone dependency also were studied. Female rats bearing DMBA-induced mammary tumors were subjected to a two-phase treatment regime 2 1/2 or 5 months after DMBA injection. Combinations of ovariectomy with drug or hormone treatments for two weeks caused an estrogen and/or prolactin deficiency in one treatment phase, and corrected the deficiency in the second two-week phase. Tumors were classified as prolactin or estrogen dependent, based upon regression in the absence of estrogen or prolactin and resump- tion of growth upon replacement with estrogen or prolactin. About 29% of the younger tumors and 33% of the older tumors were classified as prolactin dependent. Estrogen dependency was exhibited by 35% of the younger and 43% of the older tumors as determined by ovariectomy and estrogen replacement treatments. However, estrogen dependency as determined by ovariectomy and replacement with estrogen was drastically reduced in both age groups when high prolactin levels were maintained. Younger tumors had a higher regression rate after estrogen or prolactin reduction than older tumors. More tumors regressed independently of hormone levels in the older rats. 3. The effects of growth hormone (GH) and protein deficiency on mammary tumor number and growth rate were studied in female rats bearing DMBA-induced mammary tumors. Animals were fed diets con- taining 6%, 12% or 18% casein for three weeks with or without injections of 1 mg GH. Significant increases in tumor number occured in animals treated with GH above that of saline controls. GH also Carol Joan Bradley increased tumor diameter: the greatest increase occurred in rats fed 6% casein, a lesser increase occurred in rats fed 12% casein, and the smallest increase occurred in rats fed 18% casein. However these increases in tumor diameter were not statistically signi- ficant. Protein deficiency resulted in a significantly smaller increase in tumor diameter but had no significant effect on tumor number. These results suggest that a protein deficiency results in a decrease in mammary tumor growth and that GH administration can partially overcome this reduced growth. RELATION OF PROLACTIN, ESTROGEN, GH AND PROTEIN DEFICIENCY TO GROWTH OF DMBA-INDUCED MAMMARY TUMORS IN RATS By Carol Joan Bradley A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1974 ACKNOWLEDGEMENTS The experiments described in this thesis could not have been carried out without the aid and cooperation of a number of people. I would like to express my thanks to Dr. Joseph Meites for his support and guidance during the course of this work. To the other members of my guidance committee, Dr. c.w. Helsch, Dr. H.A. Tucker and Dr. J. Hook, I express my appreciation for their comments and suggestions for improvements in the manuscript. Thanks and recognition are due to 6.5. Kledzik, Dr. P.A. Ke1ley, R.P.C. Shiu and Dr. H.G. Friesen for their contributions of time and talent in helping to obtain and analyze some of the data in this thesis. ii TABLE OF CONTENTS Page INTRODUCTION ........................ 1 REVIEW OF LITERATURE .................... 3 DMBA-Induced Mammary Tumors in Rats as a Research Model for Human Breast Cancer ............ 3 Estrogen Effects on Mammary Tumor Induction and Development ..................... 4 Estrogen Requirements ............... 4 Mammary Tumor Responses to Estrogen at Different Developmental Stages ......... . 5 Effects of Anti-Estrogens ............. 6 Variability of Tumor Response to Hormones ..... 7 Prolactin Effects on Mammary Tumor Induction and Development ................... 9 Prolactin Requirements .............. 9 Mammary Tumor Responses to Prolactin at Different Developmental Stages .......... 10 Interactions of Estrogen and Prolactin ...... 11 Significance of Receptor Assays and Their Use in Elucidation of Hormone Mechanisms ......... 12 Prolactin Receptor Assays .............. 15 Growth Hormone and Protein Deficiency Effects on Mammary Tumor Induction and Development ....... 17 MATERIALS AND METHODS .................... 21 Research Animals. . . . . .............. 21 Tumor Induction ................... 21 Tumor Measurements .................. 22 Prolactin Receptor Assay ............... 22 EXPERIMENTAL ........................ 25 TABLE OF CONTENTS cont'd Page Relation of Mammary Tumor Growth Response to Prolactin to the Amount of Prolactin Receptor in the Tumor ..................... 25 Objectives ..................... 25 Procedure ..................... 25 Results ...................... 27 Conclusions .................... 30 Individual Mammary Tumor Response to Estrogen and Prolactin at Early and Late Developmental Stages ........................ 31 Objectives ..................... 31 Procedure ..................... 32 Results ...................... 34 Conclusions .................... 42 Effects of Protein Deficiency and Growth Hormone Administration on Growth of DMBA- induced Mammary Tumors in Rats ............ 45 Objectives ..................... 45 Procedure ..................... 46 Results ...................... 49 Conclusions .................... 50 GENERAL DISCUSSION ...................... 52 LIST OF REFERENCES ...................... 66 iv LIST OF TABLES Table Page 1 Tumor Growth and Specific Binding of 125I-Labeied oPRL .......................... 28 2 Tumor Growth and Specific Binding of 125I-oPRL in Liver Membranes of DMBA-Treated Rats ......... 29 3 Treatment Schedule for Rats with Mammary Tumors ..... 35 4 Mammary Tumor Response to Treatments 2 l/2 Months after DMBA Injection .................. 36 5 Mammary Tumor Response to Treatments 5 Months After DMBA Injection .................. 37 6 Classification of Tumors as Prolactin or Estrogen Dependent 2 l/2 Months Post-DMBA ............ 39 7 Classification of Tumors as Prolactin or Estrogen Dependent 5 Months Post-DMBA .............. 4O 8 Composition of Experimental Diets ............ 47 9 Effects of Growth Hormone (GH) and Dietary Protein . Content on Rat Mammary Tumor Size and Number ...... 49 LIST OF FIGURES Figure Page 1 Data Sheet for Recording Mammary Tumor Locations and Measurements .................. 23 vi INTRODUCTION Much of the knowledge of human breast cancer is based on experi- mental work in rats with carcinogen—induced mammary tumors and in mice with spontaneously developed mammary tumors. Studies using these models have yielded information on drug and hormone treatments that already have been used in the treatment of human patients with some degree of success. It has been well established that estrogen and prolactin can promote growth of some mammary tumors and that removal of these hormones can induce tumor regression. However, not all tumors are equally responsive to hormone treatments. Experiments have re- vealed that a correlation exists between the effects of estrogen on growth of mammary tumors and estrogen receptor activity in the tumor. Mammary tumors with high estrogen binding activity are responsive to estrogen treatment or ovariectomy, while those with low estrogen binding activity are not. It has not yet been demonstrated whether the same type of correlation exists for prolactin receptors and mammary tumor dependency on prolactin in rats. Do mammary tumors that show the greatest growth response to prolactin also contain the most prolactin receptor activity? Prolactin and estrogen have been shown to be essential for development and growth of 7,12-dimethylbenz(a)anthracene (DMBA)- induced mammary tumors in rats. Numerous studies have clearly indicated that altering the levels of these two hormones by drug treatment or surgical manipulations can profoundly influence the development and growth rates of populations of tumors, but some individual tumors may be relatively independent of these hormonal influences. The response of individual tumors to these two hormones may also vary with the stage of development and the size of the tumor. Therefore, it was of interest to follow the course of individual tumors in order to establish the percentage of tumors responding to estrogen or prolactin at an early and later stage of development, and thus to clarify the dependency of individual tumors on these two hormones. Previous studies have not yielded definitive results on the effects of protein deficiency on rat mammary tumor growth. Neither is there strong evidence for an effect of GH on rat mammary tumor incidence or growth. However, there are experiments which have shown a relationship between GH and protein in rats. Protein deficiency causes a decrease in pituitary GH (Srebnik g__al,, 1959), while starvation can cause a decrease in both pituitary and plasma GH in rats (Dickerman gt_al,, 1969). Conversely, GH can promote nitrogen retention in normal and protein deficient rats (Gordon gt al., 1947). In view of this interrelationship of GH and dietary protein, it was of interest to examine the effects of these two factors on the growth rate and number of DMBA-induced mammary tumors in rats. REVIEW OF LITERATURE DMBA-induced MammaryATumors in Rats as a Research Model for Human Breast Cancer The study of human breast cancer has been hindered for many years by lack of a suitable animal model with similar characteristics. The model should (1) occur with relative frequency in a reasonable period of time (2) develop in a manner similar to human breast cancer, with similar gross and histological characteristics (3) respond to endocrine, drug, and other treatments in a manner similar to human breast cancers (4) appear in an animal suitable for laboratory study. Most of the earlier work on mammary cancer was performed in inbred mice, particularly the C3H strain. However, these mouse mammary cancers appear to be hormone dependent only during the developmental stage and become autonomous and unresponsive to hormones after they appear (Dux and Mfihlbock, 1969). In this respect they are unlike many human breast cancers. The search for a better model led to the development of carcinogen- induced rat mammary tumors. Investigation of the carcinogenic properties of coal dust resulted in the isolation of several carcinogenic substances, including 3-methylcholanthrene and 7,lZ-dimethylbenz(a)anthracene (DMBA), the latter, one of the most potent carcinogens. These agents are highly carcinogenic upon oral, subcutaneous, or intravenous administration. DMBA is particularly effective for induction of mammary tumors in Sprague-Dawley rats but will produce other cancers as well. A single intravenous injection of a lipid emulsion containing 5 mg DMBA given to female rats at 50 to 60 days of age results in 95 - 100% incidence of mammary adenocarcinomas which appear in 1 to 3 months (Geyer gt_al,, 1953; Huggins gt_al,, 1965). The DMBA- induced cancers do not readily metastasize (Young and Cowan, 1963), in marked contrast to human breast cancer, which is an important exception to the general similarity between the two cancer types. However, DMBA-induced mammary cancers are particularly suited as a model for human mammary cancer research because of their respon- siveness to hormonal, chemical, and immunological factors, which appear to be comparable to many human breast cancers. Estrogen Effects on MammarygTumor Induction and Development Estrogen Requirements The effect of estrogen on DMBA tumor induction and growth is complex. Dao (1962) observed that DMBA fails to induce tumors in ovariectomized rats. This was confirmed by_Ta1walker gt_al,, (1964) who also were able to induce DMBA tumors in ovariectomized rats by estrogen replacement. Ovariectomy also caused regression of established mammary tumors, but when exogenous estrogen was used to replace the missing steroid, tumor growth was maintained (Huggins §t_al,, 1959; Sterenthal gt al,, 1963). Moderate doses of estrogens given to intact rats with established DMBA tumors can increase both tumor growth rate and the rate of appearance of new tumors. Dao and Sunderland (1959) showed that mammary tumor growth in rats was accelerated during pregnancy and pseudopregnancy, presumably due to increased ovarian hormone secretion. Large doses of estrogen have been shown to retard rat mammary tumor growth (Dao, 1964; Huggins, 1965; Meites, 1972). A daily dose of 20 ug estradiol benzoate administered at the critical period for tumor induction in Sprague-Dawley rats (55 - 60 days of age) for 20 days before and 20 days after DMBA administration was shown to inhibit the development of tumors, and to delay the time of appear- ance and the number of tumors appearing (Kledzik, Bradley, and Meites, unpublished data). An attempt to simulate hormonal conditions of pregnancy by administration of various amounts of estrogen and progesterone 15 days after DMBA treatment was reported by McCormick and Moon (1963). This treatment resulted in stimulation of tumor development_with progesterone when a constant amount of estrogen was given. However, tumors were inhibited by graded increases in estrogen, whether given alone or in combination with progesterone. MammarygTumor Responses to Estrogen at Different Developmental Stages Griswald and Green (1970) found a greater regression rate upon ovariectomy of rats with recently developed tumors (4 months post-DMBA) than in ovariectomized rats with older and larger tumors (7 months post-DMBA). They found the size of the tumor was a less important determinant of the regression rate in the younger than in the older tumors, as larger tumors were less likely to regress than smaller tumors following ovariectomy of the older rats. Androgen treatment also caused less regression in large than in small tumors. Thus large mammary tumors are more autonomous and less hormone responsive than small mammary tumors in older rats. Effects of Anti-estrogens Anti-estrogens have also proved to be effective for inhibition of mammary tumor growth. MER-25 (Hm. S. Merrell Co., Cincinnati, Ohio), a potent anti-estrogen, was administered about the time of DMBA treatment by Terenius (1971). This delayed tumor induction by DMBA and decreased the number and size of tumors that appeared. Testosterone and progesterone, while possessing anti-estrogenic properties, failed to exert this inhibitory influence on the time of tumor appearance or number of tumors appearing. Terenius (1971) attributed this discrepancy to a different mechanism of estrogen antagonism for steroids than for MER-ZS. However, many other investigators have reported that early treatment with androgens or progestins can inhibit mammary cancer development in rats. Huggins gt al. (1959) found that 1 mg dihydro-testosterone injected intramuscularly for 84 days delayed the mean time of appearance of DMBA-induced mammary tumors from 78.9 days in controls to 177.2 days in androgen-treated rats. HER-25 is believed to act by occupy- ing estrogen receptor sites and reducing the ability of the estrogen to interact with the target tissue. In contrast, Keightley and Okey (1973) have shown, by a charcoal dextran technique, that di- hydrotestosterone doesn't show competition for estradiol binding sites in mammary tissues. Thus, the early effect of the MER-25 to inhibit tumor development appears to depend upon the occupation of the estrogen receptor sites, while the later inhibition by testos- terone apparently works through some other mechanism. In rats bearing DMBA-induced tumors, ovariectomized and immediately treated with testosterone proprionate, the tumor re- gression rate was less than in untreated ovariectomized rats (Griswald and Green, 1970). This could have resulted from metabolic conversion of the androgen to partially replace the estrogen loss, or from some direct effect of the androgen itself. Recent work by Quadri, Kledzik and Meites (1974) showed the ability of dromostanolone (a potent androgen) to inhibit growth of DMBA-induced mammary carcinomas. Prolactin injections were able to overcome the androgen-induced regression. The authors attributed these results to peripheral blocking of the effect of prolactin on mammary tumors. Variability of Mammary Tumor Response to Hormones Mammary tumors are not as predictable in their responses to hormones as might be inferred from the above discussion. There are some tumors which do not follow the generally observed pattern of regression following ovariectomy. Others may fail to show increased rates of growth with elevated estrogen or prolactin levels. Esta- blished tumors are sometimes observed to undergo spontaneous re- gression, regardless of the prevailing hormonal state (Young and Cowan, 1963). These spontaneous regressions can't be attributed to any particular factor, but can lead to erroneous conclusions when treatment groups are small. Young §t_al,, (1963) reported that isolated areas within a single DMBA-induced rat mammary tumor could continue growing while the tumor as a whole was regressing. This confirmed the results t al. (1959) who studied the response to ovariectomy of of Huggins different cell populations within one 3-methylcholanthrene-induced tumor. Thus, even in tumors which had been classified as hormone- dependent or independent, there were groups of cells which did not fall into this classification. Prior to hormonal treatment, there was no histological indi- cation which allowed the prediction of the response to an endocrine change (Young gt al,, 1963). Some tumors which failed to respond to ovariectomy were found to regress under massive estrogen doses (Teller gt_gl,, 1969). However, this latter was probably due to an interference with prolactin action (Meites §t_al,, 1971). Daniel and Pritchard (1963) concluded that the tissue may be truely inde- pendent of estrogen in cases in which tumors fail to regress in response to ovariectomy, or there may be a very small degree of estrogen influence, and other factors which influence tumor growth take precedence. Since the results of most tumor experiments are presented in terms of average tumor response versus the average response of con- trols, the response of the individual tumor is often lost in the averaging process. This masks the presence of autonomous and variant tumors which may be relatively frequent because of the heterogeneous nature of the mammary carcinoma. These are important considerations in evaluating the response of individual tumors to hormone treat- ments in both animals and humans. Prolactin Effects on Mammary Tumor Induction and Development Prolactin Requirements Prolactin has been shown to play an important role in the induction and growth of mammary tumors. Pearson gt_al, (1969) and Meites (1972) concluded that the presence of prolactin in the serum was necessary for the development and growth of DMBA-induced mammary tumors. Nagasawa gt_al, (1973) observed normal serum levels of prolactin in rats with growing DMBA-induced mammary tumors, indicat- ing that tumor growth can occur in the presence of normal prolactin levels. Work by Boyns gt al, (1973) indicated that strains of rats with higher prolactin serum levels have a higher tumor incidence following DMBA administration. When prolactin levels were raised above normal, the rate of growth of existing DMBA-induced mammary cancers increased dramatically (Meites, 1972). Welsch gt_al, (1968) found that increasing serum prolactin levels by grafting 4 pitui- taries inside the kidney capsule accelerated the growth of esta- blished DMBA-induced mammary tumors. Pituitary stalk section or median eminence lesions, which remove the pituitary from hypothalamic inhibition of prolactin release, also cause mammary tumors to grow more rapidly (Clemens §t_al,, 1968). Drugs that increase prolactin secretion, such as reserpine, methyldopa, and haloperidol, stimulate mammary tumor growth in rats (Welsch and Meites, 1970; Quadri gt a1,, 1973; Lu and Meites, 1971). 10 A reduction in prolactin secretion inhibits development of DMBA-induced mammary cancers and reduces growth of established can- cers. Drugs that lower serum prolactin levels also have been shown to cause tumor regression. L-DOPA, which acts by increasing hypo- thalamic catecholamines, thus increasing prolactin inhibiting factor (PIF), decreased prolactin secretion and caused regression of DMBA- induced mammary tumors (Quadri §t_al,, 1973). Ergot drugs are believed to increase hypothalamic PIF release (Wuttke gt_al,, 1971) and to inhibit pituitary prolactin release directly (Zeilmacher and Carlson, 1962), thereby reducing prolactin secretion. Ergot drugs have been used to reduce DMBA—induced tumor growth (Nagasawa and Meites, 1970; Heuson gt_al,, 1970). Butler and Pearson (1971) administered rat prolactin antibodies to rats with DMBA-induced tumors and reported reduced tumor growth and tumor regression. Mammary Tumor Responses to Prolactin at Different Developmental Stages Clemens gt 31. (1968) demonstrated that median eminence lesions placed before administration of DMBA raised serum prolactin levels and inhibited the mammary tumor induction process. When prolactin levels were increased by haloperidol injection, administered daily for 20 days before and 20 days after DMBA treatment, a delay of tumor appearance and a reduction in the size and number of tumors resulted (Kledzik, Bradley, and Meites, unpublished data). Stimulation of the normal mammary tissue by increased prolactin levels is believed to render the mammary gland refractory to DMBA. Evidence for the necessity of a critical level of prolactin at the time of DMBA administration comes from the above study in which 11 L-DOPA was injected for 20 days before and 20 days after DMBA treat- ment. This treatment lowered serum prolactin and reduced tumor inci- dence (Kledzik, Bradley, and Meites, unpublished data). A normal level of prolactin, therefore, seems to be necessary for DMBA to promote mammary tumor development, and either an excess or defi- ciency of prolactin can inhibit development of DMBA-induced mammary tumors. With existing mammary tumors, an excess of prolactin stimu- lates growth, whereas a deficiency results in tumor regression. Interactions of Estrogen and Prolactin Pearson and Ray (1959) hypothesized that estrogen stimulation of mammary tumors in humans was mediated through the pituitary, rather than only by a direct effect on the tumor. They found estro- gen was ineffective in reactivating mammary cancer growth follow- ing hypophysectomy in women, although it was successful following oophorectomy. This has been confirmed in DMBA-induced mammary tumors in rats. Estrogen permitted continued growth in ovariectomized- adrenalectomized rats, while it was ineffective after hypophysectomy (Sterental gt_al,, 1963). Talwalker _t_al, (1964) found that either estrogen or prolactin was able to permit mammary tumor induction by DMBA in ovariectomized rats. This would seem to support a secondary role for estrogen as compared to prolactin. Furth and Clifton (1957) were also strong proponents of prolactin as the primary hormonal stimulator of mammary tumors, with estrogen as a secondary hormone. They postulated that estrogen stimulates prolactin secretion and perhaps synergises or sensitizes the tissue to prolactin. Further 12 evidence for pituitary mediation of the estrogen effect on tumors is the finding of Nicoll and Meites (1962, 1964) and Meites and Nicoll (1966) that estrogen can promote synthesis and release of prolactin by exerting its actions on both the hypothalamus and pituitary. However, it must be noted that evidence exists that estrogen as well as prolactin is essential for DMBA-induced mammary tumor growth. A recent report by Sinha gt_al, (1973) showed that intact rats with growing DMBA tumors produced the expected acceleration of tumor growth rate when median eminence lesions were placed in the hypothalamus, whereas ovariectomized rats with regressing DMBA tumors failed to respond to median eminence lesions with accelerated growth. Reimplantation of the ovaries resulted in the re-establish- ment of the accelerated growth rate without an increase in serum prolactin levels. This is in agreement with similar earlier work by Clemens gt_gl, (1968). These studies do not completely contra- dict Pearson and Ray's (1959) belief that estrogen is secondary to prolactin in promoting tumor growth, since the ovarian reimplanta- tion was done in animals with an already elevated prolactin serum level produced by the median eminence lesion. However, both estrogen and prolactin have been shown to be essential for maintenance of established mammary tumor growth. Significance of Receptor Assays and Their Use' in Elucidation of Hormone Mechanisms Recent development of methods to measure hormone receptors in target tissues has opened new avenues of investigation for determination 13 of mechanisms of hormone action. Specific tissues can be profoundly affected by a hormone carried in the general circulation because of selective uptake by tissue receptors. Assays reported thus far have made use of the principle of specific displaceable uptake of radio- labeled hormone as an indication of the quantity of receptor protein present for that hormone. These assays were designed for jn_yjtrg_ quantification of receptors using tissue slices or selected fractions of tissue protein. Early workers in the field of estrogen receptors had varying degrees of success with the particular assay technique used. Sup— port for the role of the target organ in selective estrogen uptake was provided by jg_yjyg studies which showed that the uterus and vagina, organs exhibiting known responses to estrogen, could con- centrate radio-labeled estradiol (Jensen and Jacobson, 1962). Concentration of estrogen also was shown by DMBA-induced mammary tumors in rats (King gt_al,, 1965). Once the ability to concen- trate estrogen in tisSues was established, researchers worked to establish quantitative assays. The sucrose density gradient method of separating fractions of tissue cytosol provided a precise method for measuring receptor protein. Jensen §t_al, (1971) used this technique to develop a two- stage binding mechanism theory for estrogen action. He observed that the labeled fraction of the sucrose density band was initially near the 85 region, but with time, an increasing percentage of the label was localized in the 4s band. The sucrose density gradient assay was more precise than other methods (including Sephadex filtra- tion) used to purify the radio-labeled bound estrogen. 14 More precise assays made possible quantitative detection of receptors in tissues, whereas previous studies had been limited to determination of the presence or absence of receptor protein. Terenius (1973) assayed 23 human breast tumor samples for estrogen receptors by both the tissue slice and cytosol binding techniques. There was good correlation between the two methods for detection of receptors with only one exception, however, the ranking of the tumors by amount of estrogen binding differed by the two techniques. Researchers working with DMBA tumors in rats or with human mammary cancer biopsies have repeatedly found differing amounts of receptor in the samples. McGuire and Chamness (1973) presented evidence which showed that the range of estrogen receptor protein content in cytosol of 40 human breast cancers was from 612 femtomoles/ mg cytosol protein to non—detectable levels. Varying amounts of estrogen receptor in human breast cancers were also measured by Korenman and Dukes (1970), while measurement of estrogen receptor in rat mammary carcinomas was demonstrated by Wittliff gt_al, (1972). Attempts to correlate presence of receptor protein with sub- sequent response to endocrine therapy have proved highly successful. Jensen (1971) found only one of 29 patients whose breast cancer biopsies indicated no estrogen receptors present experienced breast cancer remission after adrenalectomy. However, in human mammary tumors exhibiting estrogen binding, 10 of 13 showed some degree of regression following adrenalectomy. Clinical use of this finding could be of immense value in indicating which patients are likely to benefit from endocrine ablative surgery. It could spare the trauma of surgery to patients unlikely to benefit from adrenalectomy or ovariectomy. 15 In addition to the findings correlating receptor protein and tumor response to estrogen, Feherty gt 31, (1971) found that a higher percentage of carcinomas than benign biopsies possessed estrogen receptors. In biopsies from benign breast tumors, only 3 of 41 possessed detectable estrogen receptors. Receptors were present in 37 or 53 mammary carcinomas, indicating that malignant cancers of the breast are more likely to be hormone responsive than the benign tumors. Prolactin Receptor Assays Prolactin receptor assays have been developed more recently than the estrogen receptor assays. A particulate tissue prolactin receptor assay was described by Turkington (1974). This method used lactoperoxidase-125I-labeled prolactin to measure the dis- placeable binding of the hormone. This system was highly specific for prolactin although growth hormone and human placental lactogen cross-reacted to some degree. The receptor was determined to be a protein since pretreatment with trypsin reduced the prolactin bind- ing, while pretreatment with DNase and RNase was without effect on binding. Specific binding was also localized in the plasma membranes with no specific binding found in either the nuclear or ribosomal fractions (Turkington gt al., 1973a). Shiu gt_gl, (1973) reported a radioreceptor assay which is based on tissue membrane uptake of prolactin. This is described as an assay for prolactin using rabbit mammary tissue membranes, but has also been modified to quantify the amount of binding which can be exhibited by membranes prepared from other tissue types (personal 16 communication). The membrane receptors described (Shiu gt al,, 1973) have been shown to be specific for lactotropic hormones. Using a modification of the technique developed by Shiu and coworkers, Costlow gt_gl, (1974) measured the prolactin receptors in tissue slices from the transplantable R3230AC rat mammary carci- noma, a tumor which is not dependent on estrogen or prolactin for growth. High affinity prolactin binding sites were present in the tumor. A calculated dissociation constant for the prolactin binding sites in the tumor was similar to the dissociation constant calcu- lated for receptor sites in normal lactating mammary tissue. Extra- polation of a Scatchard plot of the binding data to the absissa yielded the number of binding sites. This was found to be 0.99 t 0.39 femtomoles receptor/ug DNA for the lactating gland, and 0.61;: 0.28 femtomoles/ug DNA for the R3230AC carcinoma. This tissue slice determination has the drawback of making the receptor sites less readily available to the labeled hormone in the incubation medium than is true for the purified membrane preparation. It does have the advantage, however, of being closer to the normal physiological condition of the tissue. Other work on prolactin receptors in tumor cells appeared about the same time in a study comparing mouse C3H tumors with DMBA- induced and R3230AC carcinomas in the rat (Turkington, 1974). He fbund similar amounts of receptors in lactating mammary tissue and DMBA-induced tumors (15.5 x 10’13 and 14.5 x 10’13 moles/mg protein, respectively). The dissociation constants were the same for these two tissues (7.1 x 10’9M). The R3230AC carcinoma receptor sites, however, had less affinity (Kd = 6.0 x 10‘9M) and were fewer in number (2.0 x 10'13 moles/mg protein). This agrees with the known 17 prolactin dependency of the DMBA tumor and the lack of prolactin ‘ dependency of the R3230AC carcinoma. Turkington (1974) found that different DMBA carcinomas from the same animal could vary considerably in the amount of receptor present, possessing 30 - 80% of the amount of receptor found in the lactating gland. The R3230AC tumor con- tained only 15% of the receptors found in the lactating rat mammary gland, while the prolactin independent mouse C3H tumor contained no detectable receptor. It is difficult to compare the quantitative results of this and the previous study (Costlow _t__1,, 1974), even though the R3230AC carcinoma is described in both, because of the difference in technique, as well as the difference in mode of ex- pressing the concentration of the receptor. The trends in both studies are definitely in agreement. Growth Hormone and Protein Deficiency Effects on Mammary Tumor Induction and Development Growth hormone (GH) is secreted by the anterior pituitary and has been shown to have numerous metabolic effects. Greenbaum and McClean (1953) followed the time course of the effects of GH on lipid mobilization and showed an increase in lipid in rat liver and plasma 3 to 6 hours after GH injection. These concentrations re- turned to control values by 24 hours after treatment. GH can induce hypoglycemia and has anti-insulin effects (Young, 1953; Di Bodo and Altszuler, 1957). GH can synergize with steroids and other anterior pituitary hormones in a variety of physiological functions including development of the mammary gland (Li, 1956; Moon, 1961; Lyons gt_al,, 18 1958). GH also promotes nitrogen retention. Rats treated with GH while on a protein deficient diet were found to excrete less nitrogen in the urine than rats not given GH injection (Gordon gt al,, 1947). The effects of GH on nitrogen excretion were also observed in rats fed a normal diet containing adequate protein. This relation of OH to nitrogen retention can be directly correlated with conservation of protein in the animal (Bennett gt_al., 1948). Contrary to findings in mice and humans, GH levels in the pituitary are reduced in the protein deficient and starved rats (Srebnik gt 11., 1959; Dickerman gt 31., 1969). While experiments in mice and rats have generally shown that establishment of tumors is inhibited by protein deficiency, there is disagreement as to the effects on growth of established tumors (Tannenbaum, 1953). Severe protein deficiency caused transplanted mouse adenocarcinomas to grow at a rate only 74% of that observed in controls after three weeks of observation (White and Belkin, 1945). On the other hand, Green gt a1. (1950) found no difference in the rate of growth of transplanted Walker 259 (granulosa cell) tumors in rats on low pro- tein diets once the tumors had become established. In a series of experiments on spontaneous mammary tumors and induced skin tumors and sarcomas in mice (Tannenbaum and Silverstone, 1949), no difference in tumor growth rate was found with diets of 9% to 45% protein. None of the studies in rats have attempted to relate the observed effects on tumors to a reduction in OH or the ability of GH to promote nitrogen retention. While a complete listing of all the functions of GH under different physiological conditions in beyond the scope of this review, it is important to consider the metabolic effects of l7 prolactin dependency of the DMBA tumor and the lack of prolactin dependency of the R3230AC carcinoma. Turkington (1974) found that different DMBA carcinomas from the same animal could vary considerably in the amount of receptor present, possessing 30 - 80% of the amount of receptor found in the lactating gland. The R3230AC tumor con- tained only 15% of the receptors found in the lactating rat mammary gland, while the prolactin independent mouse 03H tumor contained no detectable receptor. It is difficult to compare the quantitative results of this and the previous study (Costlow et al,, 1974), even though the R3230AC carcinoma is described in both, because of the difference in technique, as well as the difference in mode of ex- pressing the concentration of the receptor. The trends in both studies are definitely in agreement. Growth Hormone and Protein Deficiency Effects on Mammary Tumor Induction and Development Growth hormone (GH) is secreted by the anterior pituitary and has been shown to have numerous metabolic effects. Greenbaum and McClean (1953) followed the time course of the effects of OH on lipid mobilization and showed an increase in lipid in rat liver and plasma 3 to 6 hours after GH injection. These concentrations re- turned to control values by 24 hours after treatment. GH can induce hypoglycemia and has anti-insulin effects (Young, 1953; Di Bodo and Altszuler, 1957). OH can synergize with steroids and other anterior pituitary hormones in a variety of physiological functions including development of the mammary gland (Li, 1956; Moon, 1961; Lyons gt_gl,. 18 1958). GH also promotes nitrogen retention. Rats treated with GH while on a protein deficient diet were found to excrete less nitrogen in the urine than rats not given GH injection (Gordon gt_al,, 1947). The effects of GH on nitrogen excretion were also observed in rats fed a normal diet containing adequate protein. This relation of GH to nitrogen retention can be directly correlated with conservation of protein in the animal (Bennett gt filo: 1948). Contrary to findings in mice and humans, GH levels in the pituitary are reduced in the protein deficient and starved rats (Srebnik et_al,, 1959; Dickerman gt g1., 1969). While experiments in mice and rats have generally shown that establishment of tumors is inhibited by protein deficiency, there is disagreement as to the effects on growth of established tumors (Tannenbaum, 1953). Severe protein deficiency caused transplanted mouse adenocarcinomas to grow at a rate only 74% of that observed in controls after three weeks of observation (White and Belkin, 1945). On the other hand, Green §t_gl, (1950) found no difference in the rate of growth of transplanted Walker 259 (granulosa cell) tumors in rats on low pro- tein diets once the tumors had become established. In a series of experiments on spontaneous mammary tumors and induced skin tumors and sarcomas in mice (Tannenbaum and Silverstone, 1949), no difference in tumor growth rate was found with diets of 9% to 45% protein. None of the studies in rats have attempted to relate the observed effects on tumors to a reduction in GH or the ability of GH to promote nitrogen retention. While a complete listing of all the functions of GH under different physiological conditions in beyond the scope of this review, it is important to consider the metabolic effects of 19 GH and the effects of GH on protein retention when assessing the results of experiments involving the relationship of GH to mammary tumor development. Long-term injections of GH result in increased incidence of neoplasms in rat mammary glands (Evans and Simpson, 1931). This was confirmed by Moon gt_al, (1950) who also reported increased incidence of lung and lymph tissue neoplasms in rats. However, later Moon gt_al, (1951) were unable to observe any increase in incidence of tumors in hypophysectomized rats given GH injections for a prolonged period. GH injections were unable to reactivate growth of mammary tumors in rats bearing tumors which had been classified as stable (Young and Cowan, 1963). Nandi gt_al, (1960) reported that GH in combination with estrogen and progesterone could promote mammary tumor growth in hypophysectomized C3H/Crgl mice to the development seen in control intact mice. Sinha gt_al, (1974) found higher serum levels of GH in C3H/St mice, a strain which has a high incidence of spontaneous mammary tumors, than in CS7Bl/St mice, which have a lower mammary tumor incidence. This same correlation did not hold for prolactin levels in these two strains of mice. Pearson and Ray (1959) found an increase in urinary calcium excretion in 2 of 5 breast cancer patients who had been hypophy- sectomized and later were treated with human GH. The increased calcium excretion was considered a sign of progression of the osteoly- tic metastasized breast cancer. This increase in calcium excretion with GH administration was found only in women who had experienced no regression of breast cancer in response to hypophysectomy. These findings were confirmed by Lipsett and Bergenstal (1960), although 20 they considered their results paradoxical. They reasoned that if GH promoted breast cancer growth, hypophysectomy should cause a GH dependent tumor to regress, and subsequent GH injections should reactivate growth of the regressing mammary tumor. However, they found GH effective in stimulating calcium excretion only in patients who experienced no mammary tumor regression upon hypophysectomy. Thus, they believed that their results did not support a role for GH in promotion of breast cancer growth. Inconclusive experiments leave the role of GH in mammary can— cer development in doubt. This area of research lacks the substan- tial body of evidence that exists for the influence of estrogen and prolactin on mammary cancer development. Further studies are necessary to provide a clear understanding of any actions that GH may have on the induction or growth of mammary cancers. MATERIALS AND METHODS Research Animals All animals in these experiments were female Sprague-Dawley rats, 50-55 days of age when obtained from Spartan Research Animals, Haslett, Michigan. They were housed, 4 animals to a cage, in plastic cages in a temperature-controlled (25 :_1°C) room, with 14 hours of light daily (5:00 AM - 7:00 PM). Animals were fed ag_1ibitum on a diet of tap water and Wayne Lab Blox pellets (Allied Mills, Chicago, Ill.). Tumor Induction The rats were given a single injection of 5 mg 7,12-dimethyl- benz(a)anthracene (DMBA) in 1 ml of a lipid emulsion via the tail vein at 55 - 60 days of age, according to the procedure of Huggins (1965). Injections were given under light ether anesthesia. Rats were checked for tumor development by palpation once each week from one month after DMBA injection until commencement of treatment. Tumors appeared in l to 3 months in all animals injected, with less than a 5% mortality rate occurring the first 60 days after DMBA injection. 21 22 Tumor Measurements Animals were carefully palpated at weekly or more frequent intervals to locate all tumors, and shaved in those areas where tumors were detected. Tumors were measured using calipers while the animal was under light ether anesthesia. Each tumor was pulled up from beneath the skin and held between the thumb and forefinger as measurements of length, width, and depth were taken. These were re- corded on a data sheet prepared for each animal which showed the location of the tumor on a diagram (See Fig. l). Diameters were re- corded to the nearest millimeter for each of the 3 dimensions of a tumor, and the sum of these 3 measurements (length + width + depth) was used for data analysis. Prolactin Receptor Assay The prolactin receptor assay was performed in Dr. Henry Friesen's laboratory (Royal Victoria Hospital, McGill University, Montreal, Canada) by his personnel according to the procedure des— cribed by Shiu et_al, (1973). This technique requires a lactating mammary membrane preparation obtained from rabbits injected intra- muscularly for 4 days with 10 mg human placental lactogen and 5 mg hydrocortisone to induce lactation. The mammary tissue was removed, homogenized, and filtered through cheese cloth. The filtrate was centrifuged at 15,000 g and the resulting supernatant was centrifuged at 15,000 g and then at 100,000 g. The final pellet containing the microsomal membranes was resuspended in 0.025 M tris-HCl buffer at 23 Figure 1 DATA SHEET FOR RECORDING MAMMARY TUMOR LOCATIONS AND MEASUREMENTS _—————_—————~ —_——————_——— r . j—‘T ‘ --*~ —-~ “'"““"11'""'”""“ "m r-W" Wk 3 Wk 2 1Wk 1 ' Pre-‘ 1 Pre- Wk‘lIWk .21 Wk 3 2 fr tx I tx I 1 um. --” «.n. - .. 1 1 1 e 1 1 . . .1 e ,_._..__i 1 I - E. Total No. of Tumor 24 pH 7.6 containing 10 mm CaClz and diluted to a final concentration of 100 - 300 ug of protein per 0.1 ml. This constituted the stan- dard prolactin receptor membrane preparation. Purified prolactin was labeled with 1251 by a lactoperoxidase and hydrogen peroxide method which permitted the prolactin to retain its biological potency. This method has been described by Thorell and Johansson (1971). One-tenth ml of membrane preparation was incubated with labeled prolactin and brought to a final volume of 0.5 ml with tris buffer. This was incubated at 25°C for 90 minutes with graded amounts of un- labeled hormone in 0.1 ml to provide a standard. At the end of the incubation period, 3 m1 ice cold buffer was added and the sample filtered through a millipore filter under suction. The sample was then washed twice with 5 ml cold buffer before counting the filter membrane in a plastic tube in a gamma spectrometer. Replacing the rabbit mammary tissue preparation with a simi- larly prepared rat mammary carcinoma preparation allowed assay of the receptor content of this tissue. The carcinoma tissue was incu- bated with a known amount of labeled and unlabeled prolactin and the amount of displaceable binding per 300 mg of tumor protein was determined. RELATION 0F MAMMARY TUMOR GROWTH RESPONSE TO PROLACTIN TO THE AMOUNT OF PROLACTIN RECEPTOR PROTEIN IN THE TUMOR Objectives While estrogen receptor assays are of some value in predict- ing the response to endocrine ablative therapy (Jensen, 1971), studies seeking to establish a relationship between prolactin re- ceptors and tumor dependency have not been reported. The develop- ment of relatively sensitive prolactin receptor assays (Turkington, 1973; Shiu gt_gl,, 1973) has provided an opportunity to study the mechanism of prolactin action in promoting mammary tumor growth by measuring the prolactin receptors present in tumor membranes. Some tumors respond to prolactin excess or lack with a greatly acceler— ated or decreased growth rate, respectively, while others may be indifferent to altered prolactin levels. It was of interest, therefore, to determine whether this growth response to prolactin could be correlated with the amount of prolactin that was specifi- cally bound by the tumor membrane. Procedure Female Sprague-Dawley rats, 55 - 60 days of age were given a single intravenous injection of an emulsion containing 5 mg 25 26 7,12-dimethylbenz(a)anthracene (DMBA). Two and one half months later when tumors had developed to approximately 2 cm in diameter, 10 rats were given daily subcutaneous injections of 1 mg NIH ovine prolactin (oPRL, 26 IU/mg) dissolved in 0.85% saline made slightly basic with 0.1 N NaOH. Tumors were measured with calipers for length, width, and depth, initially and every 4 days throughout the 12-day treatment period. Following a six-day non-treatment period the animals were sacrificed and a total of 34 tumors were excised, weighed, and frozen on Dry Ice. One to 8 cancers were removed from each animal as well as liver tissue to be included in the prolactin receptor assay. The difference in the sum of the three diameters of each tumor (length + width + depth) at the beginning and end of the treat- ment period was calculated and defined as the growth index for each tumor. This was considered to indicate the degree to which the individual tumor responded to prolactin treatment. The 34 tumors present were ranked from 1 to 34 according to their growth response (growth index). The excised tumors and livers were homogenized in 0.3 M sucrose and the membrane fraction was prepared for assay of specific 1251- labeled prolactin binding for the radio receptor assay. The results of the assays were compiled before the growth index ranking was made known to Dr. Friesen's laboratory in order to rule out possible bias in the assays. 27 Results The tumors were arranged in 4 groups according to their rank as shown in Table 1 together with the growth index and the specific binding of 125I-labeled prolactin exhibited by the membrane prepara- tion of each tumor. Those tumors showing the largest growth re- sponse to prolactin also showed the greatest amount of specific prolactin binding to membrane preparations (13.4 - 34.1%). Conversely, those tumors showing little or no growth response to prolactin, were found to bind the smallest amount of labeled prolactin (2.0 - 18%). Analysis by linear regression gave a correlation coefficient of r = 0.69 (p <0.01) for 125I-prolactin binding and tumor responsive- ness to prolactin. The livers from the 10 animals were also ranked according to the combined growth indices of all tumors from each rat, and separated into 3 groups based on this ranking. An inverse relation- ship was found, with the rats showing the largest tumor growth re- sponses to prolactin possessing livers with the lowest prolactin binding, and vice versa. These liver groupings, average growth indices, and prolactin binding figures are shown in Table 2. The calculated correlation coefficient from analysis by linear regression was r = -0.69 (p <0.05) for 125I-prolactin and average tumor growth index. This is precisely the negative of the tumor prolactin bind- ing correlation with the growth response. .mpaEmm on“ op umuu< mpcaoo _mpoh mo pcmugma m we commogaxm .Acwuumpoga uwpmnmpcz mmwuxm yo mucmmmga cw mpcsouv mcmgnsmz op ucaom mpcsoo owewumqmicoz m::_z muzcwz\mpcaou quokww com: on» mo goggm ugmu:mum.fi mmmgw>< « 28 ogmmg osmoé Ndmmm %-m~ emuodp oémma manna £-m_ wmmkmp wane; named :-m e.~.H _.NN ~.o.u ~.~ .P._.H _.e m - P ARV Aggy Asov «wazfiosz “NHW ¥z<¢ Ammo-um~p UHLHumam xuozn :hzomw «asap s< mew: mgosah mcp Fp< seem mmuwucn gpzogu on» .Los:» mco cash ago: vmmmmmmod mama =mg3« 29 mamugp _5m¢d o_-m m.m.H o.o_ F.o.fl m.o A - m 04mm; flout, ¢-_ ARV Asa. quasz xuozfi xezomu xz<¢ Ammo-Hm~. UHLHomam «mo<¢u>< mh~4 zH Jamaiummp no wzHosz magnummm oz< :hzomw mozah .N mpnmh 30 Conclusions These data indicate that there is a high correlation between the tumor growth response to prolactin and the amount of tumor membrane prolactin binding. There is also a strong negative corre- lation between the tumor growth rate in response to prolactin and the amount of prolactin binding to rat liver membrane fractions. Thus mammary tumor responsiveness to prolactin appears to depend on the number of prolactin binding sites present in the tumor tissue, and bears a negative correlation to the number of prolactin binding sites found in the liver. INDIVIDUAL MAMMARY TUMOR RESPONSE TO ESTROGEN AND PROLACTIN AT EARLY AND LATE STAGES 0F.TUMOR DEVELOPMENT Opjectives The role of estrogen and prolactin in promoting mammary cancer growth is well established in both rats and mice, but further evi- dence is necessary to establish the role of prolactin in human breast cancer. Estrogen in small doses and prolactin in any dose can accelerate mammary tumor growth in rats, and a deficiency of either of these hormones results in a general decrease in tumor growth. These observations have been based on the general trend or average response of entire tumor sample populations. However, within a large sample of tumors, varying degrees of response can be observed, with some tumors showing marked growth, others with moderate or slight growth, some with no response, and some that actually regress under estrogen and prolactin treatments. These variations in response to estrogen and prolactin may indicate that some tumors are relatively indifferent to these hormones, or that other factors exist which are exerting a stronger influence on tumor growth at a particular stage of development. This experiment was designed to examine the response of indi- vidual rat mammary tumors to estrogen and prolactin deprivation and replacement, when tested singly or in combination. The effects of 31 32 these two hormones at both early and later stages of tumor develop- ment also were studied to determine whether age of the tumors at initiation of testing could influence the response to estrogen and prolactin. Procedure Virgin female Sprague-Dawley rats, 55 - 60 days of age were given a single intravenous injection of a lipid emulsion containing 5 mg of 7,12-dimethylbenz(a)anthracene (DMBA). The DMBA was ob- tained from the Upjohn Company, Kalamazoo, Michigan. Two and one half months later, 100 animals were randomly separated into groups of 20 rats each and subjected to 4 weeks of treatment, divided into two phases of two weeks each. The pattern of treatment is shown in Table 3. Group A received injections of 0.85% NaCl throughout both phases of treatment and served as a control group with normal pro- lactin and estrogen levels. Group B was ovariectomized (OVX) for the first phase of treatment to remove the primary source of estro- gen, and 3.75 ug of estradiol benzoate (E8) was given during the second phase to correct the estrogen deficiency. Group C was given injections of 0.5 mg ergocornine methanesulfonate (EC) obtained from the Sandoz Company, Basel, Switzerland, to reduce prolactin levels during the first phase of treatment, followed by ovariectomy and in- jections of 120 ug of haloperidol (HAL) obtained from McNeil Laboratories Inc., Fort Washington, Pa., to reduce estrogen and raise prolactin during the second phase. Animals in group D were first 33 ovariectomized and given injections of 0.5 mg EC to reduce both estrogen and prolactin levels, then 3.75 ug EB and 120 ug HAL were injected to increase the estrogen and prolactin levels during the second phase of treatment. Group E was ovariectomized and given 120 ug HAL during the first phase of treatment to reduce estrogen while maintaining high prolactin levels, while the second treatment of 2.75 ug EB and 0.5 mg EC raised estrogen levels and reduced prolactin. All injections were given subcutaneously daily between 10 and 11:00 AM in a volume of 0.2 ml.. EB and HAL were suspended in corn oil. EC was dissolved in 70% ethanol and diluted with 0.85% NaCl to a final concentration of 14% ethanol. A pretreatment measurement of tumor size and number and animal body weights were recorded. During the treatment the tumors were measured weekly for length, width, and depth to the nearest mm in each dimension using calipers. The sum of the length, width, and depth of each tumor at the beginning of treatment was compared with the sum of the three diameters at the end of treatment. Each tumor was classified as growing, regressing, or stable at the end of both the first and second treatments. A tumor which had increased by 3 mm or more in the sum of its measurements was classified as 9595: ing, while those which had decreased by 3 mm or more were classified as regressing. A tumor which had changed by less than 3 mm in the sum of its diameters was considered staple, The second phase of treatment was started immediately after the first two weeks of treat- ment in order to determine the effects of changing estrogen and pro- lactin levels in opposite directions from the first treatment phase. Thus the same tumor could be subjected to both hormone deprival and replacement. 34 A second experiment with 100 rats was started 5 months after DMBA injection and followed the same treatment schedule as those begun at 2 1/2 months after DMBA injection (see Table 3). Results The classification of tumors is presented in Tables 4 and 5 for rats at 2 1/2 and 5 months after DMBA injection. Table 4 shows that the control rats at 2 1/2 months (group A) had 79 tumors at the beginning of treatment and 6.3% were regressing, 17.7% were stable, and 76% were growing. During the second 2 week period there were 87 tumors of which 23% were regressing, 19.6% were stable, and 57.4% were growing. Ovariectomy (group 8) caused 89.2% of 65 tumors to regress, while only 6.2% continued to grow. EB injec- tions during the second phase of treatment brought the number of tumors growing up to 41.8% while 27.3% regressed under this treat- ment. EC injections into intact rats (group C) bearing 56 tumors resulted in 64.3% of the tumors regressing while 14.3% grew. Ovariec- tomy and HAL injections to these same rats caused 32.1% of the tumors to regress while 41.5% grew. Ovariectomized rats receiving EC in- jections (group D) had regression of 96.2% of the 52 tumors and none grew. When HAL and EB were given during the second phase of treat- ment only 31.7% regressed while 61% grew. Ovariectomized and HAL treated rats (group E) had 58.1% of 43 tumors regress and 37.2% grow. EC and EB treatments raised the percentage of regressing tumors to 79.1% during the second 2-week period and only 11.6% of the tumors grew. 35 mpmo~=ma _o_cmgumm u mu Fou_gmaopwz u 4o u x>o um me m.o .mm a: mu.m .x>o so m s<= an own .mu m: m~.m .x>o am as m.o .x>o o so um 92 m.o .puach 8 mm a: m~.m .x>o Fonz Rmm.o .x>o m Fonz Rmm.o .poaa=H .uaz Rmm. .uaapcm < szzpmo u x>o Axo.vam Aam.m We Aa_.mkv4m me mm + um + x>o MN A&~.~mvmp Awe.e N Aap.mmvm~ me a s<= + x>o _ m Aao._mvm~ Ann.“ vm ANN.Fmva ,4 m_ mm + s<= + x>o AN Ago vo Aam.m VN Aa~.omvom mm um + x>o A_ o Aam.F¢V~N Age.m~v¢. m_.NmVA_ mm 44: + x>o AN “Rm.epvm AN4.FNVNP Rm.eovmm om e_ um A. u Mam.F¢ mm am.omwsp Axm.umwmp mm op mm + x>o MN am.o e No.¢ m AN~.mm mm mm x>o F m Axe.~mVOm Aaa.m_vep “no.mmvom Am mp _uaz amm.o AN Amo.mevom AR~.~PV¢F Axm.o vm a“ Puaz Rmm.o A. < mxozah memo u x>o ANN.ON.¢. ANo.N.Vm AN..eevoe mm .. mm + UN + x>o AN AN¢.N¢.NN ANe.m.... ANN.4N.NN No so A. N AN..mm.oe ANN.eNvN. AN4.N.VN An N. mm + Sex + x>o AN ANN.¢ .N ANN.. .m ANm.NNV.o so am + x>o A. o Aao.om.NN ANN.NN.0. Anm.NN.¢. Ne N. Sex + x>o AN AN¢.o..o. ANe....N ANN.NA.¢¢ .e am A. u NO.N¢..N NO. ON o. ANo.N. m on N. N. + x>o MN NN.N.vo NN. N.o Anm.mN .N me x>o . N ANN.om.eN ANN..N.N. AN¢.mNV¢N on .. .oaz amm.o MN AN¢.vamN ANo.m..N. ANQ.NN.¢. .o .oaz Nmm.o . < mmozs. m.<¢ No some N.N<.m oummmmumm s<.o. mmmz=z .zmz.me. cwuee.ege use :eeegpme peace: we eecemege use cw :ewmmegmeg meemceeceem Ne empeewe=.. .eewgeee.ez u Ao u x>o Azmgm .N . emmmegmeg .PV Am¢\.v NN.N Azegm .N . eemmegmeg ..V AmeANN. N¢.mN mAeemmeLaew .N . eemmogmeg ..V NANNAOV No mm + om Anemmegmeg .N i 3egm ..V + x>o aN Ao A. N mm + 4o UN + x>o 4o MN AeNAe.. NN.NN em mm + x>o N x>o . m AA 0"“. qu4e. .eELe: we eecemege one c. eewmmegmeg meeeceuceem an eeueewe=.. .eewgeee.e= u Ao u x>o mm + um Azegm .N . eemmegmeg ..V Aeemmegmeg .N . 3ega ..v + x>o ANeAe. NN.N ANe\.N. Ne.em .«x + x>e mm + 4o um + x>o Azegm .N . eemmegmeg ..v 4o A.e\eNv Nm.NN em Azegm .N . eemmegmeg ..v mm + x>o Am¢\.N. NN.N¢ x>e mAeemmegeeg .N . eemmegeeg ..V NzAAe.u .zceeaeu .eewseseewm .ecewpwgpaz. ucmwez “ewe mx\msegm ecumegeeg mgeeE=c ..<« a eee. e eee. e eee. .aee. ..e .N@ .NN amoaesm AN AN AN .AmN. oz e2. meaex.s e.am NN NN NN . .egepxwe cwsepw> om. em. om. ..o caoe a.o~az om. eN. wee .=.amae .N.o we.o .m.o z.emmm11 gm) or small (4< 500 mg) tumors. However, the time at which the tumors were tested was not stated, and the sample size was small. Samples at different times after exposure to DMBA might reveal differences with time. Tumor prolactin dependency was tested in rats bearing mammary tumors by administration of prolactin antiserum for 36 days (Butler and Pearson, 1971). Under these conditions, 50% (19/20 tumors) regressed and 35% (7/20 tumors) grew, versus 13% (3/23 tumors) which regressed and 57% (13/23 tumors) which grew in controls with- out prolactin antiserum. Thus, after adjusting for controls, the percentage of prolactin dependent tumors appears to be about 37% which is only slightly higher than the 29% to 34% prolactin dependency 62 determined in this thesis. Butler and Pearson (1971) failed to state the elapsed time after DMBA administration, which may be a factor contributing to the differences in prolactin dependency in the two studies. Spontaneous mammary tumor regressions were reported in 13% of 23 untreated rats in the above study (Butler and Pearson, 1971). The age of the tumors at the time of the study wasn't given, but the percentage falls within the range of 0% at 2 1/2 months and 16% at 5 months post-DMBA determined in this thesis. Young and Cowan (1963) were impressed by the large number of DMBA-induced rat mam- mary tumors that regressed spontaneously or reached a plateau in growth. They found 27% (49/181 tumors) regressing, 52% (95/181 tumors) stable, and 21% (37/181 tumors) growing when tested at 26 weeks post-DMBA.’ These figures compare with the 36% regressing, 27% stable, and 36% growing observed at 23-25 weeks post-DMBA in the present study. Because the age of the tumors in these two studies is very close, some inherent difference in the strain of SpragueeDawley rat may account for the differences in the growth patterns of the tumors in the two studies. Although earlier studies are generally in agreement that chronic low protein diets can delay the appearance of mammary tumors (Tannenbaum, 1953), the experiment reported in this thesis showed no significant effect of protein deficiency on the number of tumors appearing during the 3 weeks of this study. However, in the present study some tumors aleardy were present at the initiation of the protein deficiency whereas this was not always true of the earlier studies. This study showed a definite decrease in growth of esta- blished tumors with a low protein diet. Previous studies in mice 63 were unable to show differences in growth rates of spontaneous mammary tumors when the animals had been full-fed or placed on a restricted diet months before the appearance of the tumors (Tannen- baum, 1940). However, in agreement with this study, mammary tumor growth was restricted in tumors which arose in full-fed animals which were subsequently placed on limited diets. Thus, there may be some adaptive processes which occur with chronic underfeeding that result in delayed tumor appearance, but have less effect on tumor growth rate once the tumors appear. Following this view further, an animal with established tumors that was suddenly placed on a low protein diet would have no chance to adapt physiologically to the deficiency. This is one possible explana- tion for the lower tumor growth rate which resulted from protein deficiency in the present experiment. 1 GH levels are reduced in pituitaries and serum of rats on restricted diets (Srebnik gt_gl,, 1959; Dickerman pt 11:9 1969). Dickerman §t_al, (1969) concluded that a reduction of growth hor- mone releasing factor (GH-RF) during starvation resulted from a lack of amino acids and energy available for GH-RF synthesis. However, Yamomoto (1974) presented results that suggested that in- creased release of GH-RF caused the lower hypothalamic GH-RF levels. He found decreased synthesis of GH in the pituitary of starved rats, but an increase in the percentage of GH release. However, the exact relationship of GH-RF synthesis and release during starvation has not yet been firmly established. The observation that GH injections to rats on 6% and 12% protein diets resulted in increases in tumor number and diameter 64 over control rats on diets of the same protein content, suggests that part of the effect of protein deficiency may be a result of GH reduction. However, the increases in tumor diameter with GH injections were not significant, even though a progressive increase with more severe protein deficiency was strongly suggested. Further studies with larger sample sizes are required to determine whether the suggested graded response to OH is real. GH did have a significant effect on the number of tumors appearing, while the primary effect of protein deficiency was on mammary tumor growth rate. This suggests that some of the effects of protein deficiency on tumor growth are of a different nature than might be caused by GH deficiency alone. The decrease in mammary tumor growth during protein de- ficiency may result from a lack of nitrogen necessary for growth of the tumor tissue. However, White and Belkin (1945) reported that transplanted mammary tumors in mice fed low protein diets were able to concentrate nitrogen and grow despite a negative nitrogen balance in the host. They concluded that the tumor was able to draw on the animal's protein stores at the expense of the host. However the mammary tumors in mice often are highly autonomous and may be less affected by dietary insufficiencies than hormone-responsive rat mammary tumors. GH may partially compensate for protein deficiency indirectly, through its ability to promote nitrogen retention (Gordon gt_gl,, 1947). 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