Milk-like fluid in a mammary adenocarcinoma: biochemical characterization.

The milk-like accumulation in the R3230AC mammary adenocarcinoma that follows treatment with estrogen contains lactose, fatty acids, and proteins with electrophoretic properties similar to those of casein and whey of rat milk. This mammary tumor retains the biochemical capacity of the mammary gland in its lactational response to administration of hormone.

Hormonal dependence of DNA synthesis in mammary carcinoma cells in vitro.

Explants of C3H-mouse and rat R3230AC mammary carcinomas were cultured on chemically defined medium for study of the effects of the hormonal environment on synthesis of DNA. Synthesis in the more slowly proliferating C3H carcinoma cells is stimulated by insulin and inhibited by estrogenic hormones as in normal mammary epithelial cells. Rat R3230AC mammary carcinoma cells can initiate synthesis of DNA independently of insulin or estrogenic hormones. Autonomous growth with respect to these hormonal controls correlates with rapid proliferation, but it is not an essential characteristic of the neoplastic mammary cell.

Relationship of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate to growth of dimethylbenz(a)anthracene-induced mammary tumors in rats.

Alteration of growth of dimethylbenz[a]anthracene-induced mammary tumors was caused by removal of estrogen (ovariectomy), or insulin (diabetes), or by inhibition of prolactin secretin (treatment with an ergoline derivative). The levels of cyclic AMP (cAMP) and cGMP were measured in carcinomas classified as growing, static, and regressing. The amount of cAMP, expressed as pmoles/mg tumor weight or pmoles/mg protein, was lowest in growing tumors, intermediate in static tumors, and highest in those regressing. No correlation was seen between tumor growth and cGMP levels. Cyclophosphamide-induced tumor stasis did not elevate cAMP levels. The data suggest a role of cAMP in arrest of hormone-induced tumor growth.

Failure of indomethacin to inhibit growth of the R3230AC mammary tumor in rats.

The relationship between the dietary lipid-induced growth of the R3230AC mammary tumor and prostaglandin E2 (PGE2) levels as well as the effect of the prostaglandin synthetase inhibitor indomethacin (Ind) on these parameters was examined. F344 rats fed a high-fat (HF) diet containing 20% corn oil demonstrated more rapid tumor growth and higher tumor and plasma PGE2 levels than rats fed a 20% hydrogenated cottonseed oil (HCTO) diet. Addition of 0.004% Ind to the HF diet markedly reduced tumor and plasma PGE2 levels. However, Ind had no effect on tumor growth. Neither the fatty acid composition nor the insulin-binding capacity of the tumor plasma membranes was affected by Ind. Membranes from animals fed HF diets with or without Ind bound more 125I-labeled insulin than membranes from HCTO-fed rats. The results suggest that, for the R3230AC mammary tumor, reduction in both tumor and plasma PGE2 levels by Ind did not result in reduced tumor growth in animals fed diets high in polyunsaturated fatty acids.

Membrane-associated phosphatidylinositol kinase of R3230AC mammary tumors and normal mammary glands and effects of insulin on tumor enzyme activity.

Phosphatidylinositol (PI) kinase was characterized, its activity was measured in plasma membrane-enriched fractions of R3230AC rat mammary tumors, and these results were compared to enzyme activity in normal mammary glands at various stages of differentiation. PI kinase activity was found to be highest in mammary adenocarcinomas, whereas the mammary gland displayed the following order of decreasing activity: late lactation greater than early lactation greater than late pregnancy. Although diabetes only slightly increased tumor membrane PI kinase activity, insulin treatment of tumor-bearing diabetic rats, which reduced R3230AC tumor growth, caused a significant reduction (30 to 40%) in PI kinase activity. These results imply that PI kinase activity may be correlatable with normal mammary gland differentiation and with mammary tumor growth behavior. Formation of phosphatidylinositol-4-phosphate in tumor membranes was inhibited by low concentrations of calcium (microM range), suggesting the presence of calcium-sensitive polyphosphoinositide metabolism in the R3230AC carcinoma.

Effects of estrogen to alter amino acid transport in R3230AC mammary carcinomas and its relationship to insulin action.

The effects of estrogens on transport and incorporation of amino acids into the R3230AC mammary adenocarcinoma were studied in vivo and in vitro. Dissociated tumor cells from ovariectomized rats, like those from diabetic rats, displayed elevated transport of proline, representing entry by the A system; transport of phenylalanine (L system) was unaltered, as was glucose transport and its utilization. Administration of estradiol valerate decreased the entry of proline into tumor cells from intact, diabetic, or ovariectomized animals; the response to the steroid hormone was greater in ovariectomized or diabetic rats compared to intact animals. The time course of the effects of estrogen treatment was examined in diabetic rats. By 72 hr, transport of both proline and leucine was significantly decreased; incorporation of leucine into proteins and uridine into RNA was significantly reduced by 24 hr after injection of estradiol valerate. The effects of estrogen in vivo to reduce transport of amino acids and their incorporation into proteins appeared to correlate with the reduced tumor growth observed. Experiments were performed to examine the effects of 17 beta-estradiol in vitro on amino acid transport into dissociated cells from ovariectomized or diabetic rats. Under these experimental conditions, 17 beta-estradiol (10(-6)M) inhibited proline transport with little or no effect on leucine transport in cells from ovariectomized rats; in cells from diabetic rats, proline transport and leucine incorporation were significantly reduced by estradiol, whereas phenylalanine transport was slightly inhibited (approximately 20%). The effect of estradiol in vitro was also manifest in tumor cells obtained from diabetic rats treated in vivo with estradiol valerate; estradiol in vitro caused a further reduction in proline transport but not in leucine transport, results that imply some specificity to the action of estrogen on the A system. Since we had earlier shown that insulin action on transport in these tumor cells were directed towards the A system, we examined the effects of insulin, estradiol, and their combination in vitro on proline and leucine transport. Insulin (10(-8) M) stimulated proline transport; 17 beta-estradiol, at a selected lower level of 10(-8) M, inhibited proline transport. When both were added in vitro, estradiol (10(-8 M) was capable of significantly reducing the insulin (10(-8) M)-induced increase in proline transport. Leucine transport was not altered in any of these experiments. Together, these data suggest that estrogens are capable of inhibiting amino acid transport into the R3230AC mammary carcinoma, an effect that is compatible with reduced tumor growth. The possible relationship of estrogen and insulin at the level of amino acid transport remains to be elucidated.

Regulation of estrogen-binding capacity by insulin in 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats.

The role of insulin as a regulator of estrogen receptors (ER's) was investigated in 7,12-dimethylbenz(a)anthracene-induced mammary tumors in diabetic and insulin-treated rats. Induction of diabetes with streptozotocin produced regression of lesions classified as insulin dependent; lesions that continued to grow in diabetic rats were classified as insulin independent. Compared to tumors in intact hosts [ER, 39 +/- 4 (S.E.) fmol/mg cytosol protein], regressing insulin-dependent lesions had ER levels of 8.5 +/- 0.7 fmol, and insulin-independent tumors had ER levels of 24 +/- 3 fmol. Treatment of diabetic rats with insulin 8 IU/day, caused insulin-dependent regressing tumors to resume growth; these lesions had ER levels of 53 +/- 10 fmol. Insulin-independent lesions in diabetic rats demonstrated two patterns after treatment with insulin; continued growth resulted in tumors having ER levels of 28 +/- 11 fmol, whereas insulin-induced tumor regression resulted in tumors that demonstrated ER levels of 40 +/- 6 fmol/mg cytosol protein, a value equal to the level of ER in tumors growing in intact rats. Scatchard analysis of the saturation-binding data gave linear representations, and the estimated Kd for the ER was comparable for all groups, ranging from 0.44 to 1.90 x 10(-9) M. Several additional tumors were classified as demonstrating static growth. When this behavior represented a response due to insulin treatment, ER levels were elevated. Static tumors remaining static after insulin treatment demonstrated low ER levels. We conclude that (a) cessation of tumor growth after induction of diabetes resulted in reduction of ER levels, (b) treatment with insulin that resulted in an altered tumor growth was accompanied by elevated ER levels, and (c) insulin may play a role in regulation of ER independent of tumor growth.

Relationship between insulin and estrogen binding to growth response in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

Insulin and estrogen binding have been determined in 7,12-dimethylbenz(a)anthracene-induced mammary tumors of rats in various endocrine states. Hormonal therapy, such as diabetes and ovariectomy, resulted in differential effects on growth patterns and hormone binding of tumors coexisting in the same host or in different hosts. It was observed that tumors that continued to grow after the host was made diabetic (insulin independent) or started to regress after ovariectomy (ovarian dependent) demonstrated decreased insulin binding. Tumors that regressed in diabetic hosts (insulin dependent) or continued to grow in ovariectomized animals (ovarian independent) showed an increased insulin-binding capacity. No significant change in insulin binding was observed in tumors that remained static after ovariectomy or induction of diabetes. Estrogen binding in tumor cells from diabetic rats paralleled the pattern of tumor growth response to diabetes; insulin-independent tumors demonstrated a significant increase in binding compared to tumors from intact hosts, and insulin-dependent tumors showed decreased estrogen receptor levels. From these results, we conclude that (a) insulin plays a positive role in regulating estrogen-binding capacity, (b) ovarian hormones may play a role in regulating insulin-binding capacity, and (c) a relationship between insulin and ovarian hormones and the growth of 7,12-dimethylbenz(a)anthracene-induced tumors is strongly suggested and may have therapeutic implications.

Influence of insulin on estrogen-induced responses in the r3230ac mammary carcinoma.

The R3230AC mammary adenocarcinoma was not dependent on insulin; tumor growth was equal to or greater in diabetic rats than in intact animals. However, tumor growth was reduced when daily doses of insulin were administered. Treatment with estrogen inhibited growth of the R3230AC carcinoma, either in diabetic rats or in intact animals simultaneously treated with insulin. The effects of insulin plus estrogen treatment appeared to be additive in causing inhibition of tumor growth. Tumors from diabetic rats showed few metabolic alterations as reflected by little or no changes in the activities of selected glycolytic enzymes, pyruvate kinase, phosphofructokinase, and hexokinase, nor any striking changes in the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, representing the pentose phosphate pathway. A modest reduction in the ratio of utilization of (1-14C)glucose: (6-14C)glucose was seen in vitro by tumors from diabetic rats. It was concluded that insulin, along with estrogen and prolactin, should be considered as a hormonal factor that influences growth of this automonous, hormone-responsive adenocarcinoma.

Photosensitization of mitochondrial cytochrome c oxidase by hematoporphyrin derivative and related porphyrins in vitro and in vivo.

Mitochondrial cytochrome c oxidase activity was inhibited by exposure to hematoporphyrin derivative followed by photoirradiation. Inhibition of enzyme activity in vitro was a dose- and time-related event, the log of percentage inhibition being linear with time. Exposure of mitochondria to hematoporphyrin or hydroxyethylvinyldeuteroporphyrin sensitized mitochondria to photoirradiation, whereas protoporphyrin IX was only weakly active as a photosensitizer for inhibition of cytochrome c oxidase. Mitochondria from mammary glands of pregnant rats showed hematoporphyrin-induced photosensitivity similar to those from R3230AC mammary adenocarcinomas. Mitochondria prepared from tumors of animals given injections of hematoporphyrin derivative in vivo and then photoirradiated in vitro demonstrated considerable sensitivity to light as reflected by significant inhibition of cytochrome c oxidase activity. A side by side comparison of hematoporphyrin derivative with hematoporphyrin, using this in vivo-in vitro experimental protocol, indicated that photosensitivity was retained for a longer time after treatment with hematoporphyrin derivative. Taken together, these data provide a potential mechanism of action, i.e., inhibition of respiration, for porphyrin-induced photosensitivity, and they offer a useful assay to investigate this family of therapeutic agents.

A role of estrogens and insulin binding in the dietary lipid alteration of R3230AC mammary carcinoma growth in rats.

The importance of estrogens in the dietary lipid alteration of R3230AC mammary carcinoma growth and insulin binding was studied. Animals were divided into three groups [intact, ovariectomized, and ovariectomized treated with estradiol valerate (EV)] and were fed diets containing either 0% fat (fat free), 0.5% corn oil (low fat), or 20% corn oil (high fat). An alteration of tumor burden between animals fed high-fat versus either low-fat or fat-free diets was observed and appeared to be influenced by the estrogen status of the animal. The difference in tumor burden attributed to dietary lipid seen in intact rats was less in ovariectomized rats and greater in ovariectomized rats treated with EV, despite the fact that absolute tumor burden was reduced by this treatment. A similar relationship was observed for dietary lipid-induced differences in insulin binding to plasma membranes from these tumors. Reduction of tumor growth resulting from estrogen treatment was greater in low-fat- and fat-free-fed animals than in high-fat-fed rats. Again, tumor growth behavior appeared to be related to the reduction of insulin binding induced by estrogen treatment; insulin binding to plasma membranes from animals fed a low unsaturated lipid diet was decreased to a greater extent by EV treatment than in membranes from high-fat-fed rats. Altered tumor growth and membrane insulin binding, resulting from dietary perturbations and/or EV treatment, were not invariably related to serum insulin levels, nor to differences in membrane preparation, as reflected by 5'-nucleotidase activity, nor to membrane fatty acid composition or uptake of proline. Taken together, these results suggest a potential role of estrogens and insulin receptors as mediators of the dietary lipid alterations of growth of the R3230AC mammary carcinoma.

Influence of hormonal alteration of host on estrogen-binding capacity in 7,12-dimethylbenz(a)anthracene-induced mammary tumors.

The estrogen-binding capacity of mammary tumors induced by 7,12-dimethylbenz(a)anthracene was measured in lesions from animals after the ovariectomy, deprival of insulin (diabetes), or treatment with lergotrile mesylate to inhibit prolactin secretion. The average estrogen-binding capacity was 30 fmoles/mg cytosol protein in growing or static carcinomas from intact (control) animals. A significant reduction in estrogen-binding capacity was observed in regressing but not static mammary tumors from ovariectomized animals. In regressing and static tumors from diabetic rats, estrogen-binding capacity was significantly lower than in lesions from intact animals; this effect was not seen in growing tumors from diabetic rats. Tumors that were growing or static in lergotrile-treated animals showed reduced capacity to bind labeled estradiol. The effects of duration of hormone treatment or time of tissue storage on estrogen-binding capacity were examined and did not appear to be correlated with the decreased binding in tumors from treated animals. The results suggest that hormones capable of producing altered neoplastic growth may influence the level of estrogen receptors.

Identification and characterization of the insulin receptor in the R3230AC mammary adenocarcinoma of the rat.

The binding of labeled insulin to dissociated R3230AC mammary adenocarcinoma cells from diabetic and intact rats was investigated in vitro. At 20 degrees, specific binding (total binding minus binding in the presence of 1000-fold excess or 10(-6) M unlabeled insulin) reached a plateau at 45 to 60 min and was directly related to the number of cells used. Degradation of labeled insulin, as measured by trichloroacetic acid precipitation, was related to the number of cells used, was not prevented by trasylol or phenylmethylsulfonyl fluoride (general proteolytic enzyme inhibitors), but was prevented by addition of 1 to 2% bovine serum albumin to the incubation medium. Specificity of insulisulin, and desoctapeptide insulin were capable of competing for insulin binding in an order of potency related to their relative biological activity; prolactin and glucagon were unable to compete for insulin binding. Scatchard analysis of the binding data demonstrated a curvilinear-plot; specific binding (over the concentration range of 10(-11) to 10(-10) M insulin) showed a high affinity (Kd approximately 1 to 3 X 10(-10) M), and the estimated number of sites was greater in tumors from diabetic animals than in tumors from intact animals or intact animals given insulin prior to sacrifice. Reversibility of insulin binding was studied by dissociation experiments; dissociation was enhanced in the presence of added unlabeled insulin compared to dissociation examined under conditions of "infinite" dilutions only. Maximum dissociation of bound insulin was observed in the presence of 10(-7) M unlabeled insulin, with less of an effect at lower or higher concentrations of added insulin (no effect seen at 10(-10) M insulin). Two techniques were investigated for separating cells from unbound labeled insulin; the procedure using centrifugation was found to be more efficient. Thus, in the R3230AC mammary adenocarcinoma, data obtained on saturability, reversibility, and specificity of insulin binding indicate the existence of an insulin receptor with properties similar to those found in normal cells.

Effects of diabetes and sex steroid hormones on insulin receptor tyrosine kinase activity in R3230AC mammary adenocarcinomas.

Insulin binding and receptor tyrosine kinase activity were investigated in the insulin-responsive R3230AC mammary adenocarcinoma. Insulin receptors, partially purified by wheat germ agglutinin-agarose chromatography, displayed electrophoretic properties similar to those of normal tissues and demonstrated autophosphorylation of the beta subunit. Tyrosine kinase activity of tumor preparations was measured by incorporation of 32P from ATP into the synthetic polypeptide substrate glutamic acid80:tyrosine20. The Km (app) for ATP, 15 to 30 microM in tumors from ovariectomized or intact rats, appeared to be increased by 10(-7) M insulin in vitro, with the calculated Vmax increased by 3- to 5-fold; the Km (app) for glutamic acid80:tyrosine20 was 2 to 3 microM and insulin increased the Vmax by 25 to 50%. The effects of diabetes and insulin treatment and of various doses of estradiol, progesterone, estradiol plus progesterone, or tamoxifen on insulin binding, basal tyrosine kinase activity, and insulin-inducible tyrosine kinase activity in vitro were studied in tumors from treated animals. Preparations from diabetic rats had elevated insulin binding and basal tyrosine kinase activity and displayed a striking dose-related increase in the ability for insulin induction of tyrosine kinase activity in vitro compared to intact animals; these effects of diabetes were prevented by administration of insulin. Over comparable doses, insulin growth factor 1 added in vitro induced tyrosine kinase activity minimally versus that seen for insulin. Treatment of rats with pharmacological doses of sex steroid hormones produced changes in insulin binding capacity and/or basal tyrosine kinase activity and, depending on dose, usually resulted in increased basal kinase activity relative to insulin binding. The insulin-inducible increase in tyrosine kinase activity in vitro was not altered by treatment with estradiol or estradiol plus progesterone in vivo, whereas high doses of progesterone attenuated the response. A consistent finding with increasing doses of sex steroids was an increase in the half-maximum dose or 50% maximum induction dose for insulin, implying reduced responsiveness. Tamoxifen administered to intact rats increased insulin binding and blunted the insulin-induced increase in tyrosine kinase in vitro; these effects were not seen in ovariectomized rats...

Photosensitizing effects of hematoporphyrin derivative and photofrin II on the plasma membrane enzymes 5'-nucleotidase, Na+K+-ATPase, and Mg2+-ATPase in R3230AC mammary adenocarcinomas.

The sensitivity to photodynamic treatment of three plasma membrane enzymes in R3230AC mammary adenocarcinomas was assessed. The activities of Na+K+-ATPase, Mg2+-ATPase and 5'-nucleotidase in isolated membranes were measured after exposure of membranes to either hematoporphyrin derivative or Photofrin II plus light in vitro or in tumor membranes prepared from animals previously injected with 25 mg/kg Photofrin II and sacrificed at various times prior to exposure to light (in vivo-in vitro protocol). The activities of both Na+K+-ATPase and Mg2+-ATPase were inhibited at equivalent rates by Photofrin II in vitro; inhibition was drug dose and light dose related. For 5'-nucleotidase in vitro, a 10-fold higher porphyrin concentration was required to achieve a similar rate of enzyme inhibition as that for the ion-activated ATPases. Injection of Photofrin II in vivo followed by preparation of tumor plasma membranes, which were subsequently exposed to light in vitro, produced no photosensitization of 5'-nucleotidase activity at any time studied (up to 72 h after Photofrin II administration). Under the same conditions Na+K+-ATPase activity was reduced by 40-60% from 2 to 72 h after drug injection, whereas Mg2+-ATPase activity was inhibited by 10-25% over the same time course. The differential sensitivity of these three enzymes observed in this in vivo-in vitro protocol suggests that each enzyme may possess different characteristics, such as three-dimensional configuration or membrane location, that afford varying susceptibility to porphyrin photosensitization. The data also suggest that photosensitivity-induced damage to these ion-activated plasma membrane ATPases could have deleterious effects on tumor cell survival.

Effects of estradiol and tamoxifen on creatine kinase in rodent mammary carcinomas.

The effects of altered estrogenic environments on creatine kinase (CK) and adenylate kinase (AK) were studied in two rodent mammary tumor systems, R3230AC and primary 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinomas, to determine whether response of these enzymes could be related to their hormone dependence. The hormonal perturbations studied were ovariectomy and administration of various doses of estradiol valerate or the antiestrogen tamoxifen. Total CK activity and AK activity were assessed by a spectrophotometric assay followed by electrophoretic separation of the CK isozymes to determine their relative activities. In the ovarian-independent R3230AC tumors, estrogen treatment produced a dose-related decrease in CK activity, whereas CK was not responsive in ovarian-dependent DMBA-induced tumors. Adenylate kinase activity remained unchanged regardless of the hormonal perturbation. Glucose-6-phosphate dehydrogenase and lactate dehydrogenase, which were studied for comparative purposes, were both estrogen responsive. While both estrogenic and antiestrogenic effects on enzyme activities were observed in the DMBA-induced tumors, the effect of tamoxifen in the R3230AC tumors was generally estrogenic. We conclude that the effect of estrogen on CK-BB in DMBA-induced tumors is not sufficient to be used as a biochemical marker of hormone dependence.

Photosensitizing effects of Photofrin II on the site-selected mitochondrial enzymes adenylate kinase and monoamine oxidase.

The response to Photofrin II-induced photosensitization on the activities of mitochondrial monoamine oxidase (MAO) and adenylate kinase (AK) were studied in order to gain further insight into site specific effects. Utilizing intact mitochondria in vitro, both MAO, located on the cytoplasmic side of the outer mitochondrial membrane, and AK, located in the intermembrane space, were inhibited by exposure to Photofrin II plus light; inhibition was drug-dose and light-dose dependent. However, MAO activity was inhibited to a greater extent than AK; at 35 micrograms/ml of Photofrin II and 160 J/cm2, MAO activity was decreased by 80% whereas AK activity was inhibited by 30%. Higher doses of Photofrin II had no further effect on AK activity. Studies of photosensitization of AK in different mitochondrial preparations demonstrated that inhibition of activity was evident only when mitochondrial membranes containing sequestered porphyrins were present in the reaction mixture. Using an in vivo-in vitro protocol and sampling at 2 to 72 h after administration of 25 mg/kg of Photofrin II, photosensitization of MAO (30% inhibition) was seen at 2 h after drug treatment but inhibition of activity was not observed at later times. AK activity was unchanged over the entire time course. Compared to cytochrome c oxidase, located in the inner mitochondrial membrane and which displayed a sustained inhibition of activity, we suggest that inhibition of MAO or AK activities probably does not contribute to the tumor cytotoxicity under the usual conditions used for photodynamic therapy.

A statistical model for predicting response of breast cancer patients to cytotoxic chemotherapy.

A binary logistic model is used for predicting response to cytotoxic chemotherapy for a breast cancer patient on the basis of her tumor enzyme activity profile. The enzymes used in the model are lactate dehydrogenase, nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase, and phosphoglucomutase, all of which were measured on primary tumor specimens from each patient. The statistical model provides an estimate of the probability that an individual will respond to treatment. Chemotherapeutic treatment consisting of combination cytotoxic drugs and subsequent evaluation of patient response followed cooperative group protocol guidelines, including outside review to confirm the patient evaluation. The model based on this study, which represents 5 years of patient follow-up, correctly predicts clinical outcome in 32 of the 37 cases available.

Prolactin binding to mammary gland, 7,12-dimethylbenz(a)-anthracene-induced mammary tumors, and liver in rats.

Specific binding of radioactively labeled prolactin was determined in membrane preparations from mammary glands and livers of rats during pregnancy and lactation. Prolactin binding to mammary gland increased throughout late pregnancy and early lactation, reached a maximum on Day 11 of lactation, and then declined. Maximum prolactin binding to liver membrane preparations was observed during late pregnancy and declined throughout lactation. Estradiol benzoate (20 mug/day), administered on Days 5 to 10 of lactation, reduced prolactin binding to mammary gland by 55%, increased binding to liver 2-fold, and reduced litter weight gain by 25%. Prolactin binding to 7,12-dimethylbenz(a)anthracene-induced mammary tumors was 3 times higher than that observed in lactating mammary gland. Administration of prolactin enhanced tumor growth but decreased specific prolactin binding to tumors. Lergotrile mesylate inhibited and estradiol benzoate (2 mug/day) enhanced tumor growth, but neither treatment affected prolactin binding to tumor membrane preparations. In contrast, higher doses of estradiol benzoate (20 mug/day) inhibited tumor growth and reduced prolactin binding. Prolactin binding varied widely within all groups of mammary tumors and was not clearly related to growth response or to altered circulating estrogen and/or prolactin levels. Hormone dependence in this animal tumor model is complex and may not be predicted on the basis of prolactin-binding capacity alone.

PIP: Experiments designed to study the changes in prolactin (PRL) binding to rat mammary tissue and liver during pregnancy and lactation, to study the relationship between PRL binding and growth in 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and to measure PRL binding in DMBA tumors, lactating mammary gland and liver following administration of pharmacological doses of estrogen are described. Specific binding of radioactive PRL binding to mammary gland increased throughout late pregnancy and early lactation, reached a maximum on Day 11 of lactation and then declined. Maximum binding to liver membrane preparations was observed during late pregnancy and declined throughout lactation. The administration of 20 mcg estradiol benzoate (EB/day on Days 5-10 of lactation, reduced PRL binding to mammary gland by 55%, increased binding to liver 2-fold and reduced litter weight gain by 25%. PRL binding to DMBA-induced mammary tumor was 3 times higher than that observed in lactating mammary gland. PRL administration enhanced tumor growth but decresed specific PRL binding to tumors. Lergotrile mesylate inhibited and 2 mcg EB enhanced tumor growth, but neither treatment affected PRL binding to tumor membrane preparations. However, 20 mcg EB inhibited tumor growth and reduced PRL binding. PRL binding varied widely within all groups of mammary tumors and was not clearly related to growth response or to altered circulating estrogen and/or PRL levels. It is concluded that hormone dependence in the rat tumor model is complex and may not be predicted on the basis of PRL-binding capacity alone.

A comparative study of inner membrane enzymes and transport systems in mitochondria from R3230AC mammary tumor and normal rat mammary gland.

Mitochondria from a rat mammary tumor (R3230AC) have been compared with mitochondria from pregnant and lactating rat mammary glands, with particular attention paid to inner membrane enzymes and Transport proteins. In the tumor the mitochondrial adenosine triphosphatase was not activated by 2,4-dinitrophenol, in contrast to the mammary mitochondria from lactating or pregnant rats. Translocation of adenosine diphosphate across the inner membrane was found to be more rapid in the tumor by virtue of lovered Km adenosine diphosphate and raised Vmax. Transport of phosphate and dicarboxylic acids occurred at similar rates in all three types of mitochondria. The inner membrane proteins were also examined directly by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and some differences are noted. These results, although they indicate subtle differences between the inner mitochondrial membranes of tumor as compared with those of pregnant or lactating rat mammary glands, cannot form the basis of an explanation for enhanced glucose utilization and aerobic lactic acid production in this tumor.