The conversion of an ovariectomy-nonresponsive to an ovariectomy-responsive mammary tumor strain.

MTW9, a transplantable mammary tumor in Wistar Furth rats, shows little growth unless host serum prolactin is increased. This study compares the response to ovariectomy of MTW9-MtT, a tumor developed in rats bearing the mammosomatotropic tumor MtTW10 (serum prolactin 500 to 7000 ng/ml) with MTW9-P developed in rats given chronic perphenazine treatment (4 mg/kg/day). Serum prolactin concentrations were 150 to 600 ng/ml in MTW9-P bearing rats. MTW9-MtT does not regress after ovariectomy but does regress after surgical removal (resection) of MtT. Ovariectomy plus MtT resection leads to greater tumor regression than MtT resection alone. MTW9-P does not regress when perphenazine administration is stopped but does regress after ovariectomy, whether or not rats are given perphenazine. Administration of estradiol (10 mug/day) to rats with complete ovariectomy-induced regression of MTW9-P results in regrowth of tumor. These data suggest that MTW9-P may represent a clone of MTW9 with a lower requirement for prolactin.     

Prolactin and estrogen binding in transplantable hormone-dependent and autonomous rat mammary carcinoma.

A hormone-dependent subline of the transplantable rat mammary tumor MTW9 contains binding sites for both prolactin and estrogen. Prolactin binding is saturable (K-d similar to 2 times 10-9 M), hormone specific, and destroyed by proteases. By contrast, an autonomous subline derived from the same parent tumor has lost more than 75% of both prolactin- and estrogen-binding sites, although binding affinities for both hormones are unchanged. This reduction in binding sites for both prolactin and estrogen in the autonomous line may result in an incomplete recognition of the tumor cells as a target for the circulating hormones with a subsequent loss of hormone-dependent growth characteristics.     

Prolactin binding and localization in rat mammary tumor mast cells

We found that prolactin is taken up by mast cells residing in prolactin-dependent, 7,12-dimethylbenzanthracene-induced rat mammary tumors. Light and electron microscopic immunocytochemistry showed that mast cells concentrate prolactin in their cytoplasmic granules. No prolactin was found on mast cell surface membranes or in their nuclei. In primary cultures of tumor cells, mast cells were found mainly in the periphery of dome structures and these cells concentrated prolactin. When purified rat peritoneal mast cells were incubated with 125I-labeled prolactin, uptake was time, energy, and temperature dependent. Seventy % of accumulated prolactin was released intact from cytoplasmic granules by C48/80-induced degranulation. A mouse mastocytoma cell line also took up and released prolactin. These cells contained prolactin receptors (Kd = 4.5 nM) as determined in whole cells (approximately 3150 sites/cell) and in crude membranes (approximately 180 fmol/mg protein). We conclude that mast cells might significantly influence mammary tumor growth by accumulating and releasing prolactin within tumor tissue.     

Prolactin receptor in human mammary carcinoma.

The specific binding of labelled human prolactin was determined in 83 human breast carcinomas. Twenty-seven tumors (32.5%) contained specific binding for prolactin of at least 1% and were considered prolactin receptor positive. The binding was found linearly related to membrane protein concentration and specific only for lactogenic hormones. By Scatchard analysis the dissociation constant appeared similar to that observed in other target tissues, with a low binding capacity.     

Prolactin binding to R3230AC mammary carcinoma and liver in hormone-treated and diabetic rats.

Specific 125I-labeled prolactin binding was measured in membrane particles prepared from R3230AC mammary carcinoma and liver of tumor-bearing Fischer rats after either prolactin, estrogen, or lergotrile mesylate treatment, or after the induction of diabetes by streptozotocin. Hormone binding to tumors was decreased by treatment with prolactin (0.5 or 1 mg/day) or estradiol valerate (7.5 mg/kg/week). In contrast, prolactin treatment did not affect prolactin binding to liver membrane particles, but estradiol valerate treatment resulted in a four-fold increase in prolactin binding to this tissue. Lergotrile mesylate, which lowers plasma prolactin levels, did not affect tumor growth or prolactin binding to either tumor or liver. Prolactin binding to both tumor and liver was significantly reduced in diabetic rats, suggesting that insulin may play an important role in controlling tissue sensitivity to prolactin. Specific binding of 125I-labeled prolactin to enzymatically dissociated cells from R3230AC tumors was demonstrated in vitro. The binding capacity of the cells was found to be of the same order of magnitude as the binding capacity in membrane preparations when appropriate corrections were applied for yields of cells and membranes. R3230AC tumor, which is responsive to prolactin appears therefore to be a useful model system for further study aimed at elucidation of growth and metabolic response to the hormone prolactin in breast cancer.     

Prolactin binding in benign and malignant mammary tissue of female dogs

Prolactin receptor (PRL-R) concentrations were determined in membrane preparations of canine mammary tumours and of non-affected mammary tissues by a radioreceptor-assay using ovine prolactin (oPRL) both for 125I-labelling and for displacement. Receptor levels greater than or equal to 3 fmol/mg membrane protein were considered positive. Histologically non-affected samples of mammary tissue from 6 dogs were PRL-R positive (12-195 fmol/mg protein). These levels were positively correlated with epithelium content (based on surface area in microscopic sections; r = 0.943, P less than 0.02). In tumour samples where pre-existing mammary epithelium (PME) was present (3 non-malignant and 6 malignant tumour samples; PME content 5-10%), the cut-off limit for PRL-R positivity was increased to 50 fmol/mg protein to forestall false positives due to non-affected tissue. If no PME was present the general limit of 3 fmol/mg protein was maintained. All 18 non-malignant tumours showed PRL-R (18-162 fmol/mg protein). The PRL-R levels were positively correlated with levels of oestrogen-(ER; r = 0.735, P less than 0.002) and progesterone receptors (PgR; r = 0.556, P less than 0.02) as measured by a multi-concentration dextran-coated charcoal method. ER and PgR levels were also proportional (r = 0.660, P less than 0.01). In 6 dogs bearing primary cancers with 5-10% PME, 1 out of a total of 6 tumours was PRL-R positive. In 9 dogs bearing primary or locally recurrent cancers without PME, significant PRL-R levels were measured in 2 out of a total of 10 tumours. Three metastatic sites in 2 other dogs were PRL-R positive. In 2 dogs (1 with a PRL-R negative local recurrence) the metastatic lesions were PRL-R negative. Thus 5 dogs of a total of 18, had PRL-R positive mammary cancers (3-377 fmol/mg protein). Unlike in non-malignant lesions the ER, PgR, and PRL-R levels were not related. In mammary cancer the presence of PRL-R was less common (P less than 0.001), and the ultimate levels less high (P less than 0.001) than in non-malignant tumours. In comparative studies using pooled membrane preparations from benign mammary tissues, oPRL was far more effective than canine prolactin (cPRL) in displacing 125I-oPRL; canine growth hormone (cGH) in this respect was ineffective. It is concluded that non-malignant mammary tissue in the dog generally is PRL-R positive; only some mammary cancers retain the PRL receptors.     

Prolactin binding by human mammary carcinoma: Relationship to estrogen receptor protein concentration and patient age

Summary Biopsy specimens of 55 human mammary carcinomas (38 primary and 17 metastatic) were assayed for prolactin receptors (PrlR). Prolactin bound specifically to 32 (58%) of the tumor biopsy specimens. The apparent K _d for PrlR in individual tumors ranged from 15 pM to 2. 3 nM (mean 600 pM,n = 5) and the concentration of PrlR ranged from 0 to 44.5 fmoles/mg protein. Estrogen receptors (ERP) were also detected in 28 of the 32 tumors which had PrlR. Overall, there was no correlation between PrlR and ERP. However, the mean concentration of PrlR was significantly higher (p <0.01) in tumors with 6–100 fmoles/mg protein ERP ( 13 fmoles PrlR) than in tumors with either <6 or >250 fmoles ERP (4.0 ± 0.4 and 6.5 ± 1.8 respectively fmoles PrlR). Analysis of PrlR concentration as a function of patient age also showed no overall correlation, but the mean PrlR in tumors from women aged 60–70 was significantly higher (p <0.01) than in those from either younger or older women. A higher concentration of PrlR was observed in tumors which were classified histologically as medium or well differentiated (6.1 ± 1.2 and 11.1 ± 2.1, respectively) than in those classified as poorly differentiated (3.3 ± 1.2) (p <0.03). There was a negative correlation between PrlR concentration and membrane yield from the tumors (r = 0.43,p <0.002). The membrane yield correlated with the ratio of tumor cells to stroma (histologically) (r = 0.63,p <0.001). In tumors from 12 patients with metastatic disease on whom follow up after endocrine-related therapy was available, the mean PrlR concentration was significantly higher in the non-responding group (8.2 ± 3.0) than in the responding group (3.4 ± 4.2,p = 0.05). Address for reprints: Dr. Barbara Rae-Venter, Department of Surgery, University of Texas Medical Branch, Galveston, TX 77550.     

Prolactin and aging: X-irradiated and estrogen-induced rat mammary tumorigenesis

Both sexes of inbred WF rats at either 8 or 28-60 weeks of age were exposed to 200 rad whole-body radiation, 2.5 or 5.0 mg 17 beta-estradiol (E2), or both agents The female rats treated with E2 alone or with both X-rays and E2 at 8 weeks of age showed a high incidence of mammary carcinomas (MCA), a large increase in pituitary weight, and a rise in serum prolactin (PRL) levels. However, the same treatments to males did not induce MCA despite a moderate increase in both pituitary weight and serum PRL. Ovariectomy prior to E2 treatment failed to modify the occurrence of MCA or pituitary tumors. When X-rays and E2 were given to female rats at 28-60 weeks of age, pituitary weight, serum PRL levels, and the incidence of MCA were unaffected. When the E2 pellet was kept for the first 24 weeks and withdrawn during the last 12 weeks, the incidence of MCA, pituitary weight, and serum PRL was low. It was concluded that: 1) the pituitary glands of young female rats were susceptible to E2 treatment but were insensitive in older females, and 2) the occurrence of MCA in female rats appeared to be promoted by elevated PRL levels secreted by E2-induced pituitary tumors. Mammary tissue of male rats was less sensitive to PRL levels in the development of MCA.     

Prolactin receptors in explant cultures of carcinogen-induced rat mammary tumors

The turnover, down-regulation and role of intracellular organelles in the down-regulation of prolactin (PRL) receptors have been investigated in N-nitrosomethylurea (NMU)-induced rat mammary tumors cultured in short-term explants. Tumor explants are capable of maintaining PRL receptors for 24–48 hr. This maintenance reflects a dynamic phenomenon involving receptor synthesis, since addition of cycloheximide (1 μg/ml) in the culture medium results within 12 hr in a marked decline of PRL receptor levels. A down-regulation of total PRL receptors (measured after exposure of membranes to 3M MgCl₂) is observed in cultures containing concentrations of 20 μg/ml or greater of ovine PRL (oPRL). Lysosomotropic agents, such as chloroquine (100 μM) are ineffective in either increasing basal PRL receptor levels or in preventing the PRL-induced down-regulation in NMU-induced mammary tumor explants. Cytochalasin B (20 μM), without effect on basal PRL binding, prevents the down-regulation of PRL receptors, whereas colchicine (10 μM) results in a decline of PRL receptor levels both in the absence and in the presence of oPRL. The present data suggest a different pattern of PRL receptor regulation in vitro for tumors compared to normal rabbit mammary explants.     

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

Growth rates of 7,12-dimethylbenz(a)anthracene-induced mammary tumors and the specific 125I-labeled prolactin binding to membrane fractions prepared from livers and tumors were studied in rats made diabetic by streptozotocin injection. Growth was inhibited in a majority of tumors and prolactin binding was reduced in both tumors and livers from diabetic animals. Prolactin binding to individual tumors varied over a wide range in both intact and diabetic animals. Scatchard analysis of binding data revealed that the apparent affinity of prolactin binding to liver and tumor membranes was similar (Ka approximately 3.0 X 10(9) M-1) and was not affected by diabetes. We suggest that the reduction in prolactin binding to tumors may render these tissues less responsive to prolactin and thereby explain, at least in part, the observed inhibition of tumor growth in diabetic rats. However, some tumors in diabetic animals regressed despite relatively high levels of prolactin binding activity. Therefore, additional factors most certainly play important roles in the mechanism(s) by which the growth of 7,12-dimethylbenz(a)anthracene-induced tumors is impaired in the diabetic rat.     

Prolactin and murine mammary tumorigenesis: a review.

It is unequivocal that prolactin is an influential hormone in murine mammary tumorigenesis. The Berenblum hypothesis (7), a well-known theoretical model of tumorigenesis that depicts this oncogenic process as a two-step mechanism, i.e., initiation and promotion, is a conceptual scheme in which the action of prolactin in mammary tumorigenesis may be understood. According to this conceptual model, prolactin would participate in both the initiation and promotion steps of mammary tumorigenesis, In the initiation phase, variations in prolactin secretion appear to influence the metabolism of the mammary epithelium, so that the epithelium would be either more receptive to or refractory to an initiating agent (e.g., chemical carcinogen, physical carcinogens, oncogenic viruses, ets.) i.e., a permissive action. In the promotion phase, prolactin may act as either a promoter or an antipromoter of the "transformed" mammary epithelium. In promotion, the hormone may either directly or indirectly (via the ovary) stimulate mitotic activity of the "transformed" epithelium. In antipromotion the hormone, in the presence of requisite hormones (e.g., glucocorticoids), may synergistically induce differentiation (e.g., lactation) in the "transformed" epithelium. A tumor would result in the former (promotion) but not in the latter (antipromotion) case. Whether or not prolactin is significantly influential in human breast tumorigenesis remains to be determined. This is an extremely important area of research which is justifiably receiving increased attention. For if prolactin can be shown to influence human breast epithelium in a manner similar to its effect on rodent mammary tissue, then prophylactic and/of chemotherapeutic control of human breast tumorigenesis may be feasible by appropriate drug-mediated prolactin suppression.     

Prolactin receptors in human breast carcinoma.

A preliminary investigation into prolactin receptors in human breast carcinomas provided strong evidence that specific binding of prolactin was occurring in at least three of the nine specimens examined (eight human breast carcinomas and one scalp metastasis). These "prolactin receptor positive" tumors were all from premenopausal patients. Three of the tumors of postmenopausal women also suggested the occurrence of specific prolactin binding but, as saturation of the receptors had not been achieved in these assays, the results require confirmation. Some general trends in the binding characteristics of the tumors of premenopausal and postmenopausal patients were also observed.     

Prolactin and mammary gland carcinogenesis. The problem of human prolactin

The ultimate objective of experimental cancer research must be to apply the findings obtained to the prevention or treatment of the disease in humans. In this review it is shown that prolactin is suspected of being one of the hormones mainly responsible for the development of mammary carcinoma in mice. Investigations into the question of whether this might also be true in man are hampered by the fact that the existence of prolactin in this species is still a matter of debate. Because of the intrinsic prolactin-like activity of purified human growth hormone, the need for the presence of prolactin as a separate hormone might be questioned. It is shown, however, on the basis of a number of biological arguments and clinical observations, that it is extremely unlikely that all prolactin-like effects in man are due to one of the manifold activities of growth hormone alone; consequently the urgent need to analyse the role of prolactin as a separate hormone in man becomes evident, especially in the field of breast cancer. Tentatively, ways are indicated by which this objective might be reached.     

Prolactin levels and molecular heterogeneity in rat strains with high and low incidence of DMBA-induced mammary tumors

Summary We compared the following parameters in Long-Evans (LE) and Sprague-Dawley (SD) female rats: 1) mammary tumor incidence after DMBA, 2) plasma prolactin (PRL) during the estrous cycle before and after DMBA, 3) plasma PRL in immature females from 0900 hr on day 29 to 0900 hr on day 30, 4) plasma PRL from 1200 to 1700 hr and before and 10 min after i.p. TRH administration in ovariectomized (OVX) rats treated with 200 µg polyestradiol phosphate (PEP), 5) anterior pituitary (AP) PRL concentration in OVX rats treated with 200µg PEP, and 6) levels and Sephadex G-100 gel filtration patterns of plasma PRL 10 min after i.p. TRH administration in OVX rats treated with 200µg PEP. We observed marked mammary tumor incidence in SD rats from one supplier (S-SD, Spartan) (96%) compared to SD from another supplier (CRSD, Charles River) (40%) or LE rats (10%). Plasma PRL was significantly decreased on metestrus-diestrus and increased on proestrus-estrus in SD (both suppliers) but not in LE rats 90 days after DMBA compared to rats not given DMBA and sacrificed at same stages of the estrous cycle on day 55 of age. Immature LE and SD-CR females exhibited significant late afternoon and early morning prolactin surges on days 29–30 whereas SD-H rats had either no surges or poorly synchronized surges at the same times. Ovariectomized mature females of the tumor-resistant strains had significantly more AP PRL than the tumor-sensitive strain when given PEP, however there were no differences between the strains in estrogen-induced afternoon PRL surges or in TRH-induced PRL release in the mature OVX, PEP-treated rats. On the other hand, Sephadex G-100 gel filtration patterns of plasma PRL in OVX LE and the tumor-resistant SD group treated with PEP and sacrificed 10 min after TRH administration were markedly different when compared with tumorsensitive SD rats. These studies indicate that there are differences in PRL secretion between strains of rats with high and low mammary tumor incidence but not all of these differences are directly related to the variation in tumorigenesis. The most promising parameters appear to be PRL secretion in immature rats and molecular heterogeneity of plasma PRL. These factors are currently under further investigation.A visiting scientist from the Department of Animal Science, Nihon University, Tokyo, Japan.     

Prolactin induces ERalpha-positive and ERalpha-negative mammary cancer in transgenic mice

The role of prolactin in human breast cancer has been controversial. However, it is now apparent that human mammary epithelial cells can synthesize prolactin endogenously, permitting autocrine/paracrine actions within the mammary gland that are independent of pituitary prolactin. To model this local mammary production of prolactin (PRL), we have generated mice that overexpress prolactin within mammary epithelial cells under the control of a hormonally nonresponsive promoter, neu-related lipocalin (NRL). In each of the two examined NRL-PRL transgenic mouse lineages, female virgin mice display mammary developmental abnormalities, mammary intraepithelial neoplasias, and invasive neoplasms. Prolactin increases proliferation in morphologically normal alveoli and ducts, as well as in lesions. The tumors are of varied histotype, but papillary adenocarcinomas and adenosquamous neoplasms predominate. Neoplasms can be separated into two populations: one is estrogen receptor alpha (ERalpha) positive (greater than 15% of the cells stain for ERalpha), and the other is ERalpha- (<3%). ERalpha expression does not correlate with tumor histotype, or proliferative or apoptotic indices. These studies provide a mouse model of hormonally dependent breast cancer, and, perhaps most strikingly, a model in which some neoplasms retain ERalpha, as occurs in the human disease.     

Multiple growth factor independence in rat mammary carcinoma cells

In previous studies we demonstrated that rat mammary tumor (RMT) cells that are serially transplantable consist of cells that are independent of growth factors strictly required by normal rat mammary epithelial (RME) cells for growth in serum-free culture. The present studies were designed to determine the extent of the growth factor independence of several cell lines derived from these tumors and to determine if the cells that expressed growth factor independencein vitro are also tumorigenicin vivo. Cells from a transplantable mammary carcinoma (8–12 RMT) were seeded into culture in serum-free medium in the absence of either insulin (IN), epidermal growth factor (EGF), or cholera toxin (CT), and cell populations independent of the individual factors were developed. Next, the three growth factor independent populations were tested for their ability to grow in the absence of multiple growth factors. 8–12 RMT cells did not lose proliferative potential when multiple growth factors were deleted from the medium. Indeed, 8–12 RMT cells could be serially propagated in serum-free medium supplemented solely with bovine serum albumin (BSA) and ethanolamine. Cell lines independent of single growth factors were also developed from two other transplantable tumors (1–9 RMT and 7–15 RMT). In contrast to the 8–12 RMT-derived cell lines, deletion of additional growth factors from the media of the 1–9 RMT and 7–15 RMT-derived cells resulted in dramatic losses in growth potential. These results suggest that independence of individual growth factors is mediated by different mechanisms, since cells from different tumors can stably express independence of one, two, or three or more factors. Examination of conditioned media of four different RMT cell lines indicates that independence of EGF is mediated by autocrine factors. By contrast, there is no evidence for an autocrine factor that mediates independence of insulin-like growth factors. Thus, cell lines derived from serially transplantable RMTs are independent of either single or multiple growth factors, and independence of individual growth factors appears to be mediated by separate mechanisms.