Serum estrogens and estrogen responsiveness in 7,12-dimethylbenz[a]anthracene-induced mammary tumors as influenced by dietary fat.

The effect of dietary fat on mammary tumor incidence, estrogen-binding capacity as related to the hormone dependency of the tumors, and circulating estrogen levels in Sprague-Dawley rats given an oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) was investigated. Rats were fed diets consisting of 0.5, 5, or 20% corn oil starting at weaning and were administered 5 mg DMBA at 50 days of age. Tumor incidences were 13, 46, and 75% for the groups given 0.5, 5, and 20% fat, respectively, when the experiment was terminated 20-22 weeks later. Serum estradiol, measured at proestrus at 50 days of age and at the end of the experiment, was slightly depressed at both time points in rats fed the 0.5% fat diet but was similar in the other 2 groups. Serum estrone levels were not significantly different at either time point. Estrogen receptor levels in the tumor were the same in the groups given 5 and 20% fat but were lower in the group given 0.5% fat. No difference was detected in the progesterone receptor concentrations. Furthermore, most (approximately 70%) of the tumors in all 3 dietary groups regressed in response to ovariectomy, which suggested that dietary fat has very little influence on the estrogen dependence of the tumor. This observation suggested that fat intake does not result in any intrinsic difference in the biochemical action of estrogen.

Combined therapy with 5-azacytidine and hydrocortisone in glucocorticoid-sensitive and -resistant mouse P1798 lymphosarcoma.

Experiments were undertaken to test the hypothesis that 5-azacytidine (AZA) treatment might make the glucocorticoid-resistant line of the mouse P1798 lymphosarcoma sensitive to cortisol. A modest (30%), though significant, effect in making cells cortisol sensitive was observed. Moreover, AZA enhanced tumor growth inhibition by cortisol in the corresponding sensitive line. The AZA alone was cytostatic, but it had no effect on DNA synthesis. The drug did not alter cytoplasmic glucocorticoid receptor levels or affinity, but nuclear levels were increased in the sensitive tumor. Results suggest that glucocorticoid resistance may be partially reversible and that the effect of AZA may be at the gene-expression level.

Androgen control of cytosol progesterone receptor levels in the MT-W9B transplantable mammary tumor in the rat.

The effect of androgen on the levels of the cytosol progesterone receptor was examined in the transplantable rat mammary tumor MT-W9B and in the normal uteri of inbred WF rats. Progesterone receptor levels were barely detectable in tumors grown in male WF rats but were increased after castration or administration of 17 beta-estradiol. Both effects were blocked by testosterone. In tumors grown in intact female rats, both testosterone and dihydrotestosterone decreased progesterone receptor levels in a dose-dependent manner, and testosterone completely blocked the estradiol-induced increase in progesterone receptor levels in tumors from ovariectomized rats. The inhibitory effect of testosterone in female rats was blocked by the antiandrogen flutamide, suggesting an androgen receptor-dependent mechanism. Neither dihydrotestosterone nor testosterone had any effect on basal levels of progesterone receptor in tumors from ovariectomized rats. In uterus, up to 5 mg dihydrotestosterone/kg did not affect progesterone receptor levels, and a dose of 5 mg/kg was also uterotropic. This fact plus the finding that testosterone only partially blocked the estradiol-induced increase in uterine progesterone receptor levels suggested stimulation of different cell types by testosterone and estradiol. This did not appear to be the case in the tumor, however. Androgen is suggested to act as a negative modulator of progesterone receptor levels, which might have clinical relevance in terms of hormone therapy of breast cancer.

Effect of a high-protein-low-carbohydrate diet on toxicity and antitumor activity of 5-fluorouracil in mice.

The effect of different levels of dietary protein on 5-fluorouracil (FUra) toxicity and antitumor activity was assessed in female (BALB/c X DBA/2Ha)F1 (CDF1) and BALB/c mice bearing the P1798 lymphosarcoma. In the toxicity experiments, CDF1 mice were fed diets containing 5% [low protein (LP)], 20% [normal protein (NP)], or 60% [high protein (HP)] casein for 22 days and were then given a single ip injection of 275 mg FUra/kg. As determined by effects on the white blood cell count and by changes in body weight, FUra was least toxic in mice fed the HP diet. Moreover, mortality was 77.0, 42.9, and 11.4% for mice fed the LP, NP, and HP diets, respectively. In the antitumor studies, BALB/c mice bearing the P1798 lymphosarcoma were fed one of the 3 diets starting 5 days after transplant. Mice were given injections of 50 mg FUra/kg or the vehicle control on days 14 and 15 after transplant. As measured by tumor weights on day 16, FUra was least effective in mice fed the LP diet and most effective in mice fed the NP and HP diets. Survival time of mice given injections of FUra on days 14 and 15 was increased when compared to the survival time of control mice, but no difference in the percent increase in survival time was noted among the 3 dietary groups. If mice were given two cycles of FUra therapy (days 14 and 15 and days 20 and 21), mice fed the HP diet survived significantly longer than did mice fed the LP or NP diet. These data demonstrate that the feeding of an HP diet markedly decreases the toxicity of FUra while preserving or actually enhancing the antitumor effect.

Characterization of estrogen-binding proteins in variants of a rat mammary tumor.

Estrogen-binding proteins have been characterized in variants of the MTW-9B rat mammary tumor in an attempt to determine the functional significance of the low affinity cytosolic estrogen binder. In vivo selection of tumor variants was carried out by transplant of the tumor into intact and castrated male and female Wistar-Furth rats for four or five successive transplant generations. Tumors that developed in each of the four lines were taken for retransplant into intact and castrated male and female rats and all recipient tumor groups were compared for high and low affinity estrogen-binding proteins using isoelectric focusing analysis. No alterations in the isoelectric focusing profiles were observed in tumors that developed after repeated passage in ovariectomized female or castrated male rats when compared to the profile of the estrogen-binding proteins of the parent tumor carried routinely in intact female rats. However, a tumor variant containing only the low affinity, more basic estrogen-binding protein resulted from repeated passage of the MTW-9B mammary tumor in male rats. The high affinity estrogen receptor was absent in this variant and could not be induced by retransplant of the tumor into intact female hosts. Growth of the parent tumor and each of the variants was shown to be ovarian independent, suggesting that the presence of the low affinity estrogen-binding protein is not predictive of estrogen responsiveness. This suggestion is further supported by the observation that estrogen-stimulated progesterone receptor synthesis could be demonstrated in the parent tumor which demonstrated both binders, but not in the tumor variant developed in male rats which contained only the low affinity estrogen-binding protein. Progesterone receptor synthesis in this latter tumor appeared to be constitutive. Studies are continuing in an attempt to identify a role for the cytosolic low affinity estrogen-binding protein.

Effects of high dietary fat on the growth and development of ovarian-independent carcinogen-induced mammary tumors in rats.

This study examined the influence of high dietary fat intake on the development of ovarian-independent mammary tumors in both vehicle-treated controls and rats made deficient in estrogen and prolactin during tumor induction. The majority of 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors in rats are dependent on estrogen and prolactin for growth, and suppression of prolactin and estrogen at the time of tumor initiation causes a reduction in tumor incidence and increase in tumor latency. However, the majority of mammary tumors which do develop in these animals exhibit ovarian-independent growth. Sprague-Dawley rats were given 7.5 mg DMBA p.o. at 57 days of age. Starting 1 day prior to and continuing for 7 days after DMBA administration, rats were given daily injection of vehicle or the combination of tamoxifen (20 micrograms/rat) plus bromocryptine (5 mg/kg). At the end of drug treatment, rats in each treatment group were equally divided and placed on normal fat (5% corn oil) or high fat (20% corn oil) diets for the duration of the experiment. Vehicle-treated rats were ovariectomized 27 wk and drug-treated rats 47 wk after DMBA administration to determine tumor ovarian dependency. Vehicle-treated rats fed high fat diets showed significant increases in mammary tumor incidence and number as compared to similarly treated rats fed a normal fat diet, with approximately 80% of the tumors in each group being ovarian dependent. Likewise, tamoxifen-bromocryptine-treated rats fed a high fat diet showed a significant enhancement in mammary tumor number, although not incidence, as compared to similarly treated rats fed a normal diet. Tumors in these drug-treated groups displayed essentially the same incidence of ovarian dependence (23%). Tamoxifen-bromocryptine-treated groups displayed a 2-fold increase in latency of tumor appearance as compared to vehicle-treated controls; however, this long latency was not reduced when these rats were fed a high fat diet. These results demonstrate that high dietary fat stimulates ovarian-dependent and -independent mammary tumorigenesis in rats but does not influence the hormonal responsiveness of these tumors.

Requirement of essential fatty acid for mammary tumorigenesis in the rat.

In an attempt to determine the requirement of essential fatty acid for dimethylbenz(a)anthracene-induced mammary tumorigenesis, rats were fed diets containing different levels of linoleate: 0.5, 1.1, 1.7, 2.2, 3.5, 4.4, 8.5, or 11.5%. Each diet contained 20% of fat by weight, with varying amounts of coconut oil and corn oil added to achieve the desired levels of linoleate. Mammary tumorigenesis was very sensitive to linoleate intake and increased proportionately in the range of 0.5 to 4.4% of dietary linoleate. Regression analysis indicated that a breakpoint occurred at 4.4%, beyond which there was a very poor linear relationship, suggesting the possibility of a plateau. From the intersection of the regression lines in both the upper and lower ranges, the level of linoleate required to elicit the maximal tumorigenic response was estimated to be around 4%. The differences in tumor yield could not be correlated with changes in prostaglandin E concentration in the mammary fat pads of normal animals maintained on similar diets, suggesting that linoleate may act by some other mechanism to stimulate mammary tumorigenesis.

Similarity between trans fat and saturated fat in the modification of rat mammary carcinogenesis.

Commercial hydrogenation of vegetable oils results in the introduction of trans fatty acids. In the present study, we have investigated the effect of feeding a fat which contained approximately 38% trans isomers (designated trans fat) on the induction of mammary tumors by dimethylbenz(a)anthracene in rats. The corresponding control fat (designated cis fat), which had a similar fatty acid composition, consisted of only cis isomers. Since both the trans and cis fats were rather saturated, a comparison was also made between these 2 types of fat and corn oil, which contains about 60% linoleic acid (C18:2). Each fat was present in the diet at 2 levels, 5 and 20% by weight. Although rats fed the 20% trans fat or cis fat diets had a slightly higher tumor incidence and yield than did those on the corresponding 5% fat control diets, the difference was not statistically significant. In contrast, rats fed the 20% corn oil diet developed a much greater number of tumors than did rats fed a diet containing only 5% corn oil. Further analysis of the data showed that diets containing either trans fat or cis fat were much less effective than were the corn oil diets in promoting the development of mammary neoplasia at either the 5 or 20% level. Our results thus suggest that trans fat behaves very much like a saturated fat in the modification of mammary tumorigenesis. A determination of the fatty acid content of the mammary fat pad indicated that its composition generally reflected the dietary fatty acid intake, with the incorporation of trans isomers into the mammary tissue found to be dependent on the quantity of trans fat in the diet.

Antitumor efficacy in rats of CGP 19984, a thiazolidinedione derivative that inhibits luteinizing hormone secretion.

The antitumor efficacy and the hormonal effects of the thiazolidine-dione derivative (sodium methyl((-3-methyl-2- ([5-methyl-3-(2-methylallyl)-4-oxo-2 thiazolidinyliden]hydrazono)-4-oxo-5-thiazolidinyl)) phosphate, CGP 19984, have been studied in in vivo rat prostatic and mammary cancer models. CGP 19984 significantly inhibited growth of the androgen-dependent Dunning R3327 rat prostate adenocarcinoma. Concomitant with tumor inhibition, a significant decrease in circulating luteinizing hormone and testosterone levels was observed, suggesting that the antitumor effects of drug treatment resulted primarily from inhibition of luteinizing hormone release and subsequently decreased testosterone synthesis. Drug treatment had little effect on serum prolactin or corticosterone levels. Animals showed no adverse effects from CGP 19984 except for a modest loss of body weight. In female rats, growth of the estrogen-independent MTW-9B rat mammary tumor was also inhibited by CGP 19984 and uterine weight and tumor progesterone receptor levels were reduced. The latter suggests that CGP 19984 treatment decreases circulating estrogen in female rats. However, the inhibitory effect of CGP 19984 on the growth of the MTW-9B tumor does not appear to be mediated by the action of the drug to lower estrogen levels, since this tumor is not dependent on estrogen for growth, and lower doses of CGP 19984 were found to be equally effective in reducing uterine weight, but had no antitumor activity. The ability of CGP 19984 to suppress gonadal function and to inhibit tumor growth suggests that this drug may have potential clinical application in the treatment of both hormone-dependent and -independent prostate and breast cancers.

Functionality of estrogen receptor and tamoxifen treatment of R3327 Dunning rat prostate adenocarcinoma.

The functionality of the estrogen receptor as determined by the effect of estrogen on progesterone receptor levels and the effect of tamoxifen on tumor growth has been examined in the R3327 Dunning rat prostate adenocarcinoma. The progesterone receptor was absent in 78% of prostate tumors grown in male Copenhagen X Fischer F1 rats but was induced in tumors taken from rats given injections of 25 microgram estradiol benzoate per kg for 10 days. This result suggested that the tumor might be sensitive to the antiestrogen tamoxifen, and it was subsequently shown that treatment of rats with tamoxifen (0.5 mg/kg) 5 times/week for 2 to 7 months resulted in marked suppression of tumor growth in 91% of the tumors examined. In vitro binding analysis demonstrated that tamoxifen competed for the estrogen receptor in the tumor but not for the androgen receptor. These data suggest that tamoxifen might be acting directly on the tumor, although an indirect effect cannot be ruled out, since plasma testosterone levels were reduced as a result of tamoxifen treatment. Regulation of androgen and estrogen receptors was also observed in this tumor system. Thus, administration of estradiol benzoate for 10 days resulted in increased levels of androgen receptor with no change in estrogen receptor. Long-term tamoxifen treatment had a similar effect. Short-term castration, which appeared to induce the progesterone receptor, also resulted in increased levels of both androgen and estrogen receptors. This latter observation suggests that tamoxifen might be even more effective in castrated rats.

The truncated glucocorticoid receptor in the P1798 mouse lymphosarcoma is associated with resistance to glucocorticoid lysis but not to other glucocorticoid-induced functions.

Glucocorticoid receptors in lines of the P1798 mouse lymphosarcoma either sensitive or resistant to glucocorticoid-induced lysis have been characterized and their functional significance determined. The glucocorticoid receptor from the cortisol-sensitive tumor is an Mr approximately 98,000 protein with a Stokes radius of 7.4 nm in the oligomeric, non-DNA-binding state and 5.6 nm in the transformed, DNA-binding state. This receptor binds glucocorticoid and reacts with the BUGR-2 monoclonal antibody. In contrast, two abnormal receptor species were identified in the cortisol-resistant tumor. One is an Mr approximately 98,000 non-steroid-binding but immunologically reactive protein. The other is an Mr approximately 45,000 species which contains both steroid- and DNA-binding sites but exhibits little or no reactivity with BUGR-2, suggesting that its NH2 terminus is truncated in a region within or adjacent to the BUGR epitope. This species had Stokes radii of 5.8 and 3.5 nm in nontransformed and transformed states, respectively. In both tumor lines, glucocorticoids stimulated the activities of glutamine synthetase and 5'-nucleotidase and the synthesis of glucocortin. However, glucocorticoid-induced tumor regression occurred only in the cortisol-sensitive tumor. Additionally, the glucocorticoid inducibility of a specific protein in the sensitive, but not in the resistant, tumor was demonstrated, as well as the presence of a protein specific to the resistant line. Taken together, these results suggest that the truncated glucocorticoid receptor in the P1798 lymphosarcoma is functional, although possibly in a more restricted gene-specific manner, and that the lysis defect, while possibly resulting from a truncated receptor, may also result from the inability of glucocorticoids to induce a critical protein in the pathway of programmed cell death and/or from the presence of a protein which inhibits the lytic response.

Comparative effects of different animal and vegetable fats fed before and during carcinogen administration on mammary tumorigenesis, sexual maturation, and endocrine function in rats.

The purpose of this investigation was to determine whether diets high in animal or vegetable fat affected mammary tumorigenesis when fed to rats only prior to and during the initiation phase of carcinogenesis. Weanling 21-day-old female Sprague-Dawley rats were divided into different dietary treatment groups and were allowed to feed and libitum on one of the following diets: 5% (normal fat) corn oil; 20% (high fat) corn oil; 20% palm oil; 20% beef tallow; or 20% lard. At 52 days of age, all rats were given p.o. 7.5 mg 7,12-dimethylbenz(a)anthracene (DMBA). One week following DMBA administration, all rats were switched to the 5% corn oil control diet and were maintained on this diet for the duration of the experiment. Rats fed a 20% lard diet during the treatment period showed a significant increase in mammary tumor incidence and number 19 weeks after DMBA administration, when compared to all other dietary treatment groups. Rats fed a 20% beef tallow diet during this same time period also demonstrated enhanced mammary tumor development, during the 10- to 19-week time period after DMBA. Mammary tumor development in rats fed 20% corn oil or palm oil diets during this treatment period was similar to that of normal fat controls. Estrogens are potent stimulators of mammary tumor growth and development in rats. Because mammary tumorigenesis was enhanced in rats fed high animal, but not vegetable fat diets, it was possible that estrogens present in animal fat might be responsible for this stimulation. Further studies demonstrated however, that increased mammary tumorigenesis in rats fed diets high in animal fat could not be explained on the basis of endocrine stimulation. Average day of vaginal opening for all groups fed 20% fat diets was similar and occurred earlier than in normal fat controls. In addition, 50- to 65-day-old rats in the different dietary treatment groups showed no differences in basal or surge levels of serum prolactin, luteinizing hormone, or estradiol. Rat diestrus uterine weight also showed no significant differences among dietary treatment groups. Thus diets containing high levels of animal fat caused little if any increased estrogenic activity in rats. In conclusion, high dietary intake of lard and beef tallow, but not vegetable fat, fed from weaning until only 1 week after DMBA administration, significantly enhances mammary tumorigenesis in rats. The mechanism(s) by which animal fat induces this stimulation is not clear, but it does not appear to result from endogenous or exogenous endocrine stimulation.

Effect of the prostaglandin synthetase inhibitor indomethacin on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats fed different levels of fat.

The effect of the prostaglandin synthetase inhibitor indomethacin on the dietary fat enhancement of 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis has been examined in female Sprague-Dawley rats. Rats were fed either a normal-fat or high-fat diet (5 or 18% corn oil, respectively) with or without 0.004% indomethacin, starting 3 days after a single intragastric intubation of 5 mg 7,12-dimethylbenz(a)anthracene. Results of this experiment demonstrated that indomethacin completely blocked the stimulatory effect of fat on tumorigenesis, as measured by a decreased tumor incidence, a decreased number of tumors per group, a decreased tumor size, and an increased latency. No effect of indomethacin was observed in rats fed the normal-fat diet. These data suggest that at least part of the stimulatory effect of polyunsaturated fat on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis may be mediated through an increased synthesis of prostaglandins.

Dichotomous effects of tamoxifen on a transplantable rat mammary tumor.

The effect of the antiestrogen tamoxifen on growth of the transplantable autonomous mammary tumor MTW-9B was compared with its effects on progesterone and estrogen receptor levels. Growth of the tumor was similar in intact rats, ovariectomized rats, or ovariectomized rats given estradiol, in both the presence and the absence of tamoxifen. Progesterone receptor levels, however, were reduced by ovariectomy and restored by estrogen administration. Tamoxifen antagonized this effect of estrogen, although when administered by itself to ovariectomized rats it acted as an agonist, since progesterone receptor levels were induced. Cytosol estrogen receptor was depleted by tamoxifen in both intact and ovariectomized rats. In uterus from the same animals, tamoxifen also showed both antagonist and agonist properties, although here too there was an apparent dissociation between effects on growth and progesterone receptor levels. We conclude from these experiments that: (a) the estrogen receptor complex may be acting at more than one site on the genome; and (b) regulation of progesterone receptor levels by estrogen (or tamoxifen) does not necessarily predict sensitivity of tumor growth to antiestrogens.

Interaction of dietary fat and the thymus in the induction of mammary tumors by 7,12-dimethylbenz(a)anthracene.

The interaction of dietary fat and the thymus in the induction of mammary tumors by dimethylbenz(a)anthracene has been examined in female Sprague-Dawley rats. In these experiments, rats fed diets of 0.5% (low fat), 5% (normal fat), or 20% (high fat) corn oil from weaning (21 days of age) were thymectomized or sham thymectomized at 35 days of age and were given 5 mg of dimethylbenz(a)anthracene at 55 days of age. Thymectomy exerted a protective effect in rats fed low and normal fat diets, and this was not reversed by Thymosin Fraction V. In high fat-fed rats, tumorigenesis was increased compared to the low fat groups, and in addition, the protective effect of thymectomy was absent. This differential effect of thymectomy could not be explained on the basis of changes in prolactin concentration, since prolactin levels were decreased in all dietary groups. Neither diet nor thymectomy affected corticosterone levels or the estrus cycle of mature rats. Peripheral blood lymphocytes were, however, decreased by both thymectomy and increasing the fat content of the diet. It is hypothesized that the promoting effect of dietary fat on dimethylbenz(a)anthracene-induced mammary tumorigenesis is mediated via the immune system, although a role for the endocrine system still cannot be ruled out.

Expression, tissue distribution, and cellular localization of the antiapoptotic TIP-B1 protein.

TIP-B1 is a novel 27-kDa protein isolated from the cytosol of tumor necrosis factor (TNF)-stimulated cells. Cells preincubated with TIP-B1 are protected from TNF-induced apoptosis. This study showed that, as with normal fibroblasts and U937 histiocytic lymphoma, human MCF7 mammary adenocarcinoma cells were protected from TNF in a concentration-dependent manner by pretreatment with either TNF or purified TIP-B1. Immunoblot and immunohistochemical analyses indicated expression of both TIP-B1 mRNA and protein in MCF7 cells and heart, kidney, brain, liver, ovary, uterus, thymus, spleen, lymph node, and mammary gland cells throughout their development. Expression of TIP-B1 was heterogeneous, with staining of specific cell types within tissues. Based on the ability of TIP-B1 to protect both normal and tumor cells from TNF-induced apoptosis and its broad tissue distribution, with expression only in select cells within those tissues, a role for TIP-B1 in the regulation of TNF-induced effects is strongly indicated.

Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation.

Protein kinase C (PKC) is involved in signaling that modulates the proliferation and differentiation of many cell types, including mammary epithelial cells. In addition, changes in PKC expression or activity have been observed during mammary carcinogenesis. In order to examine the involvement of specific PKC isoforms during normal mammary gland development, the expression and localization of PKCs alpha, delta, epsilon and zeta were examined during puberty, pregnancy, lactation, and involution. By immunoblot analysis, expression of PKC alpha, delta, epsilon and zeta proteins was increased in mammary epithelial organoids during the transition from puberty to pregnancy. In mammary gland frozen sections, PKCs alpha, delta, epsilon and zeta were stained in the luminal epithelium and myoepithelium, in varying isoform-and developmental stage-specific locations. PKC alpha was found in a punctate apical localization in the luminal epithelium during pregnancy. During lactation, PKC epsilon was present in the nucleus, and PKC zeta was concentrated in the subapical region of the luminal epithelium. Additionally, marked staining for PKCs alpha, delta, epsilon, and zeta was observed in the myoepithelial cells at the base of ducts and alveoli. This basal ductal and alveolar staining differed in intensity in a developmentally-specific fashion. During most time points (virgin, pregnant, lactating, and early involution), myoepithelial cells of the duct were more intensely stained than those lining the alveoli for PKCs alpha, delta, epsilon and zeta. During late involution (days 9-12), the preferential staining of ducts was lost or reversed, and the myoepithelial cells lining the regressing alveolar structures stained equally (PKCs epsilon and zeta) or more intensely (PKCs alpha and delta), coincident with the thickening of the myoepithelial cells surrounding the regressing alveoli. The increased PKC isoform staining at the base of alveoli during involution suggests that alveolar regression may be influenced by alterations in signaling in the alveolar myoepithelium.

Protein kinase C eta upregulation and secretion during postnatal rat mammary gland differentiation.

The mammary gland has the ability to undergo repeated cycles of tightly regulated postnatal proliferation, differentiation, and apoptosis-mediated regression, providing a model to investigate potential regulators of mammary epithelial growth and differentiation. Protein kinase C eta is a candidate regulator of mammary epithelial differentiation, as increased expression of PKC eta is often observed during the terminal differentiation of many epithelial tissues. In this study, PKC eta expression and localization were characterized during puberty, pregnancy, lactation and involution in isolated rat mammary epithelial cells (MEC), as well as in paraffin-embedded and frozen rat mammary gland sections. By Western blot analysis of whole cell lysates from purified MEC, PKC eta protein expression increased during the shift from resting to a pregnant state. This increased PKC eta protein expression during pregnancy was associated with alveolar rather than ductal development, as immunohistochemical staining for PKC eta was increased in differentiating secretory alveoli, but not ducts. By immunofluorescent staining, PKC eta was stained intensely in an intracellular reticular meshwork throughout the cytosol of alveolar epithelial cells from pregnant mammary gland. During lactation, PKC eta was abundant in apocrine bodies budding from the alveolar epithelium, in the lumen of alveoli, and was present in milk, in association with casein, while being decreased in the cytoplasm of the luminal alveolar epithelium. Staining intensity of alveoli for PKC eta decreased further during involution. Western blotting of subcellular fractions from isolated mammary epithelial cells demonstrated that PKC eta remained associated with the membrane and particulate fractions throughout development. The upregulation of PKC eta in alveolar but not ductal epithelium during pregnancy suggests an association with functional secretory differentiation.

Selective changes in EGF receptor expression and function during the proliferation, differentiation and apoptosis of mammary epithelial cells.

Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.

Characterization and hormonal regulation of estrogen binding proteins in the MTW-9B transplantable rat mammary tumor.

A two-component estrogen (E2)-binding system has been characterized in the E2-independent MTW-9B rat mammary tumor by Scatchard analysis, sucrose gradient analysis, and isoelectric focusing. One cytosol receptor protein (type I) conforms to the classical estrogen receptor with a high affinity (Kd = 0.45 nM) for E2 and limited binding capacity [maximum binding (Bmax) = 53 fmol/mg protein]. The second component (type II) demonstrates a high number of sites (Bmax = 164 fmol/mg protein) and low E2-binding affinity (Kd = 22.3 nM). The type I and type II E2-binding components were shown to sediment on sucrose gradients at 9.6S and 4.5S, respectively, and to focus at isoelectric points of 6.6 and 8.0, respectively. The addition of 0.4 M KCl to the homogenization buffer converted the high affinity receptor species to a form that cosedimented and cofocused with the low affinity E2-binding protein. When tumors were grown in intact male rats, the ratio of the type II to the type I protein, as assessed by sucrose gradient analysis, increased 4.5-fold relative to that in tumors from intact females. Concomitantly, the Kd values of the type I and type II proteins were increased 9- and 2-fold, respectively, and the Bmax of the type I protein was decreased. No changes in the ratios of the E2-binding proteins were observed in tumors grown in ovariectomized female or castrated male rats; however, the Kd for both proteins was increased in tumors from the latter group. Relative to that in intact females, tumor growth was retarded in intact male rats, but was unaffected by ovariectomy or castration. These studies demonstrate that the presence of the low affinity E2-binding protein does not necessarily predict E2 responsiveness. While the role of the type II cytosolic protein has not yet been established, it is possible that it could act as a reservoir for E2, which is required to activate specific biochemical functions such as progesterone receptor synthesis. Alternatively, it could be a nonactivated, perhaps norphosphorylated, form of the E2 receptor which binds E2 with only a very low affinity.