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Pancreatic ductal adenocarcinoma (PDAC) is associated with significant fibrosis. Recent findings have highlighted the profibrotic activity of tissue-resident macrophages in the pancreatic cancer microenvironment. Here, we show that neoplastic pancreatic epithelium, as well as a subset of tissue-resident macrophages, expresses the prolactin-receptor (PRLR). High mobility group box 1-induced prolactin expression in the pancreas maintained FAK1 and STAT3 phosphorylation within the epithelium and stroma. Gain-of-function and loss-of-function experiments demonstrated the essential role of prolactin in promoting collagen deposition and fibrosis. Finally, the signaling cascade downstream of prolactin/PRLR activated STAT3 rather than STAT5 in PDAC. These findings suggest that targeting prolactin together with IL6, a known major activator of STAT3, could represent a novel therapeutic strategy for treating pancreatic cancer. SIGNIFICANCE: Prolactin is a key factor in the cross-talk between the stroma and neoplastic epithelium, functioning to promote fibrosis and PDAC progression., (©2019 American Association for Cancer Research.)

BCL6 is a transcriptional repressor that recognizes DNA target sequences similar to those recognized by signal transducer and activator of transcriptions 5 (Stat5). BCL6 disrupts differentiation of breast epithelia, is downregulated during lactation, and is upregulated in poorly differentiated breast cancer. In contrast, Stat5a mediates prolactin-induced differentiation of mammary epithelia, and loss of Stat5 signaling in human breast cancer is associated with undifferentiated histology and poor prognosis. Here, we identify the mammary cell growth factor prolactin as a potent suppressor of BCL6 protein expression in human breast cancer through a mechanism that requires Stat5a, but not prolactin-activated Stat5b, MEK-ERK, or PI3K-AKT pathways. Prolactin rapidly suppressed BCL6 mRNA in T47D, MCF7, ZR75.1, and SKBr3 breast cancer cell lines, followed by prolonged reduction of BCL6 protein levels within 3 hours. Prolactin suppression of BCL6 was enhanced by overexpression of Stat5a but not Stat5b, was mimicked by constitutively active Stat5a, but did not require the transactivation domain of Stat5a. Stat5 chromatin immunoprecipitation demonstrated physical interaction with a BCL6 gene regulatory region, and BCL6 transcript repression required histone deacetylase activity based on sensitivity to trichostatin A. Functionally, BCL6 overexpression disrupted prolactin induction of Stat5 reporter genes. Prolactin suppression of BCL6 was extended to xenotransplant tumors in nude mice in vivo and to freshly isolated human breast cancer explants ex vivo. Quantitative immunohistochemistry revealed elevated BCL6 in high-grade and metastatic breast cancer compared with ductal carcinoma in situ and nonmalignant breast, and cellular BCL6 protein levels correlated negatively with nuclear Stat5a (r = -0.52; P < 0.001) but not with Stat5b. Loss of prolactin-Stat5a signaling and concomitant upregulation of BCL6 may represent a regulatory switch facilitating undifferentiated histology and poor prognosis of breast cancer.

The activation of the Janus-activated kinase 2 (Jak2) tyrosine kinase following ligand binding has remained incompletely characterized at the mechanistic level. We report that the peptidyl-prolyl isomerase (PPI) cyclophilin A (CypA), which is implicated in the regulation of protein conformation, is necessary for the prolactin (PRL)-induced activation of Jak2 and the progression of human breast cancer. A direct correlation was observed between the levels or activity of CypA and the extent of PRL-induced signaling and gene expression. Loss of PRLr-CypA binding, following treatment with the PPI inhibitor cyclosporine A (CsA), or overexpression of a dominant-negative PRLr mutant (P334A) resulted in a loss of PRLr/Jak2-mediated signaling. In vitro, CsA treatment of breast cancer cells inhibited their growth, motility, invasion, and soft agar colony formation. In vivo, CsA treatment of nude mice xenografted with breast cancer cells induced tumor necrosis and completely inhibited metastasis. These studies reveal that a CypA-mediated conformational change within the PRLr/Jak2 complex is required for PRL-induced transduction and function and indicate that the inhibition of prolyl isomerases may be a novel therapeutic strategy in the treatment of human breast cancer.

Angiogenesis plays a key role in promoting tumorigenesis and metastasis. Several antiangiogenic factors have been shown to inhibit tumor growth in animal models. Understanding their mechanism of action would allow for better therapeutic application. 16-kDa prolactin (PRL), a NH2-terminal natural breakdown fragment of the intact 23-kDa PRL, exerts potent antiangiogenic and antitumor activities. The signaling mechanism involved in 16-kDa PRL action in endothelial cells remains unclear. One of the actions of 16-kDa PRL is to attenuate the production of nitric oxide (NO) through the inhibition of inducible NO synthase (iNOS) expression in endothelial cells. To delineate the signaling mechanism from 16-kDa PRL, we examined the effect of 16-kDa PRL on interleukin IL-1beta-inducible iNOS expression, which is regulated by two parallel pathways, one involving IFN regulatory factor 1 (IRF-1) and the other nuclear factor-kappaB (NF-kappaB). Our studies showed that 16-kDa PRL specifically blocked IRF-1 but not NF-kappaB signaling to the iNOS promoter. We found that IL-1beta regulated IRF-1 gene expression through stimulation of p38 mitogen-activated protein kinase (MAPK), which mediated signal transducer and activator of transcription 1 (Stat1) serine phosphorylation and Stat1 nuclear translocation to activate the IRF-1 promoter. 16-kDa PRL effectively inhibited IL-1beta-inducible p38 MAPK phosphorylation, resulting in blocking Stat1 serine phosphorylation, its subsequent nuclear translocation and activation of the Stat1 target gene IRF-1. Thus, 16-kDa PRL inhibits the p38 MAPK/Stat1/IRF-1 pathway to attenuate iNOS/NO production in endothelial cells.

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

Others have demonstrated the presence of an autocrine prolactin (PRL) growth loop in the normal human prostate. In this study we have used three human prostate cancer cell lines but have focused on the androgen-independent human prostate cancer cell line, DU145, to ask: (a) whether this autocrine growth loop is maintained beyond the loss of androgen sensitivity in the progression of prostate cancer; and (b) whether interruption of this growth loop by a PRL receptor antagonist, an S179D mutant PRL, could inhibit the formation of DU145-derived tumors. The autocrine loop was examined in most detail in the DU145 cell line but was demonstrated to be functional in all three of the lines by the reversible inhibition of growth in vitro by the S179D PRL receptor antagonist. To investigate the effect of S179D PRL on the growth of DU145 tumors in nude mice two sets of experiments were performed. In the first set, Alzet minipumps containing no PRL, wild-type (WT) PRL, or the S179D PRL (the last two delivering 4.56 microg/24 h and 4.26 microg/24 h, respectively), were implanted s.c. on day 1. On day 4, 5 x 10(6) DU145 cells were injected s.c. in the hindquarter. On day 22, the animals were killed, tumors were removed, measured, and subsequently fixed and processed for histological confirmation of tumor formation. The incidence of tumors in the no-PRL control group was 9/11 animals (82%). In the animals treated with WT PRL, the incidence was 8/10 (80%), whereas in the animals treated with the S179D PRL, the incidence was markedly reduced to 3/11 (27%). Although WT PRL had no effect on the incidence of tumors, the average size of the tumors increased from 25.8 +/- 5.99 mm(3) in controls to 66.66 +/- 18.06 mm(3) in WT PRL-treated animals. In the second set of experiments, 5 x 10(6) DU145 cells were injected on day 1. On day 18, Alzet minipumps containing no PRL, WT PRL, or S179D PRL were implanted. On day 42, the animals were killed and the tumors processed as before. S179D PRL caused a reduction in tumor size from 1731 +/- 283 mm(3) in the no-PRL controls to 1031 +/- 295 mm(3), whereas WT PRL slightly increased the size to 2118 +/- 630 mm(3). We conclude that PRL is used as an autocrine growth factor by human prostate cancer cells both in vitro and in vivo and that interruption of this growth loop in vivo inhibits tumor initiation and the growth of well-established tumors.

In the present study, we examined the role of prolactin and glucocorticoids in regulating bcl-x transcription in mammary epithelial cells. We report that dexamethasone, but not prolactin, induced native bcl-x gene expression in a dose-dependent manner in HC11 cells and enhanced serum-starved HC11 cell survival. This effect was mediated through the glucocorticoid receptor and independent of STAT-5 activity. We propose that the mechanism through which glucocorticoids enhance mammary epithelial cell survival is by increasing steady-state levels of bcl-xL, RNA.

Type I interferons (IFN alpha and IFN beta) are presently used in the adjuvant treatment of several human cancers. However, these cytokines have demonstrated only modest success in breast cancer therapy, and research efforts have focused on improving their efficacy. Recent progress in understanding the molecular mechanisms underlying the antiproliferative effects of IFNs has identified the cytoplasmic transcription factor Stat1 as a critical mediator. It is, therefore, possible that IFN-induced growth inhibition of mammary epithelial cells is counteracted by other cytokines that also use Stat1. One such candidate IFN-antagonist with particular relevance to breast cancer is the mammotropic hormone prolactin (PRL). The main goal of this study was to examine whether PRL would interfere with type I IFN (IFN alpha/beta) signal transduction by competing for limited cytoplasmic Stat factors. A second aim was to test whether pretreatment of mammary tumor cell lines with IFN gamma could enhance the effect of IFN alpha/beta. By analyzing the effect of PRL on IFN alpha/beta-induced tyrosine phosphorylation of Stat proteins and their binding to IFN-regulated genes, we now report that costimulation of PRL receptors did not interfere with IFN alpha/beta signals in several human breast cancer cell lines, including T47D, MCF-7, and BT-20. Specifically, PRL did not affect IFN alpha/beta-induced tyrosine phosphorylation or heterodimerization of Stat1 and Stat2 in any cell line. Instead, IFN alpha/beta- and PRL-induced tyrosine phosphorylation of Stat1 was additive and occurred without evidence of competition for limited concentrations of cytoplasmic Stat1. A similar additive relationship was observed on IFN alpha/beta- and PRL-induced Stat3 tyrosine phosphorylation. Furthermore, electrophoretic mobility shift assays showed that type I IFNs induced predominantly Stat1-Stat2 or Stat1-Stat3 heteromeric complexes with various IFN-response elements of IFN-stimulated genes, whereas PRL induced Stat1 homodimers. Despite significant mutual use of Stats by IFNs and PRL, these results indicated a high degree of signaling specificity in the two receptor systems, and that cytoplasmic levels of Stat proteins were not limiting. Similarly, PRL did not interfere with the growth-inhibitory effect of IFN beta. On the other hand, the study indicated that pretreatment of human breast cancer cell lines with IFN gamma enhanced the growth-inhibitory action of type I IFNs, suggesting a possible avenue for improving the effect of type I IFNs in the treatment of breast cancer patients.

We have previously shown that transforming growth factor beta 1 (TGF-beta 1) receptor and TGF-beta type II receptor (T beta R-II) are produced in lactotropes, and that TGF-beta 1 inhibits the growth of these anterior pituitary cells by an autocrine mechanism. To study the changes of the expression and function of this growth factor during tumorigenesis, we have measured the levels of TGF-beta 1 and T beta R-II mRNAs and proteins in the normal and tumor anterior pituitary cells in vivo and in vitro and have compared the cell growth responses to TGF-beta 1 in normal and tumor pituitary cells in vitro. Treatment with estradiol-17 beta for 1, 2, 4, and 8 weeks caused a time-dependent increase in pituitary protein, prolactin, and prolactin mRNA levels and in plasma prolactin levels, suggesting that estrogen enhanced lactotropic proliferation in anterior pituitary glands. The levels of TGF-beta 1 protein and mRNA in anterior pituitary tissues were reduced over time after estrogen treatment during the development of pituitary tumors. The mRNA and protein levels of T beta R-II decreased markedly during the development of pituitary tumors. In addition, two transformed lactotropes, GH3 and PR1 cell lines, showed markedly reduced levels of TGF-beta 1 as well as T beta R-II mRNA. Comparison of the antiproliferative effects of TGF-beta in transformed and normal lactotropes in cultures revealed that the sensitivity of GH3 cells is reduced, and that PR1 cells are virtually resistant to TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

A model for the evolution of hormone-independent tumors from a pregnancy-dependent precursor is exemplified by the TPDMT-4[pregnancy-dependent (PD)] and T4OI96 [ovarian-independent (OI)] in vivo tumor lines developed in DDD mice. In vivo, the OI tumor grows rapidly in virgin mice and is more anaplastic than the PD tumor which, in virgin mice, grows as a hyperplastic alveolar gland from which tumors arise only during pregnancy. The regulation of the proliferation of these two tumors was compared in primary culture using a three-dimensional, serum-free, collagen gel cell culture system. In medium containing insulin, the growth of cell organoids from PD tumors was stimulated by the same factors that stimulate the growth of normal mammary epithelial cells from virgin or pregnant mice. These factors include progesterone and prolactin (but not estrogen), epidermal growth factor, basic fibroblast growth factor, linoleic acid, cyclic AMP, prostaglandin E2, phosphatidic acid, and lithium. Most of these tumors (about 80%) could not grow in medium with only insulin present; of those that did, growth was slow and was stimulated further by the above agents. PD tumor cells formed stellate colonies in the collagen matrix similar to those of normal cells. These findings show that the growth regulation of PD tumor cells is, in all aspects examined, similar to that of normal cells. In addition, the capacity for growth in the presence of only insulin (more autonomous growth) is not necessarily accompanied by deletions in responses to growth factors or hormones, or in lipid response pathways. In contrast, organoids from OI tumors needed only insulin for growth. The growth of some of these tumors was stimulated further by only basic fibroblast growth factor and phosphatidic acid. Cyclic AMP and agents that elevate intracellular cyclic AMP inhibited growth, the opposite of the response seen in normal or PD cells. OI organoids grew as cell masses rather than as stellate structures. These data show that while PD tumors are remarkably similar to normal cells in the regulation of their proliferation, OI tumors have incurred multiple defects in growth-regulatory pathways possibly including the production of autocrine growth factor(s). In addition, the ability of OI tumor cells to adhere to the collagen gel matrix and undergo normal morphogenesis is reduced. These results may highlight specific events required for the progression from ovarian dependency to ovarian independency related to the hormonal regulation of growth.

The effect of a number of steroids, growth factors, and peptides on aromatase activity in two estrogen receptor positive breast cancer cell lines (MCF7 and T47D) was investigated. The cells were incubated in Dulbecco's minimum essential medium containing phenol red and 10% fetal calf serum. Pronounced differences in basal aromatase activity and different responses to the addition of experimental agents were found in the two cell lines. Aromatase activity in MCF7 cells was significantly stimulated by phorbol 12,13-diacetate [PDA], dibutyryl cyclic AMP [(Bu)2cAMP], transforming growth factor alpha, and epidermal growth factor individually and PDA and (Bu)2cAMP in combination, while it was inhibited by dexamethasone and unaffected by transforming growth factor beta, fibroblast growth factor, platelet-derived growth factor, prolactin, and tamoxifen. Addition of cortisol to MCF7 cells had no effect on aromatase activity at 1 nM, caused suppression of activity at 10 nM and stimulated activity at 100 nM. Aromatase activity in T47D cells was stimulated by transforming growth factor alpha, epidermal growth factor, platelet-derived growth factor, prolactin, dexamethasone, and cortisol individually and PDA and (Bu)2cAMP in combination. It was unaffected by transforming growth factor beta, PDA, (Bu)2cAMP, and fibroblast growth factor. These findings suggest that aromatase activity is induced by agents which stimulate cyclic AMP-dependent protein kinases [e.g., (Bu)2cAMP] and that this effect is potentiated by factors which stimulate protein kinase C [e.g., PDA]. The effect on aromatase activity of growth factors, the actions of which are believed to be mediated by receptors linked to tyrosine kinase activity, is not as clearly defined, with a factor causing stimulation, inhibition, and no change in activity depending on the tissue concerned. Further insight into these differences will require resolution of the molecular mechanisms that mediate the actions of stimulatory and repressive growth factors on aromatase activity of oestrogen-producing cells.

The present experiments were designed to test the role of insulin-like growth factor-related peptides (IGF-RPs) in hormonally stimulated N-nitrosomethylurea (NMU)-induced mammary tumor colony formation in soft agar. To evaluate production of IGF-RP by NMU cells, we tested the abilities of a monoclonal antibody directed against insulin-like growth factor I (alpha sm 1.20B) and of a polyclonal antibody raised against the insulin-like growth factor I (Ab 134) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from estradiol (E2)-, prolactin (PRL)-, and progesterone (Pg)-treated NMU-induced rat mammary tumors. Both Abs abolished the colony-stimulating action of genuine insulin-like growth factor I while having no effect when added alone or with control CM. The addition of either alpha sm 1.20B or Ab 134 (but not that of an irrelevant Ab) consistently blocked the colony-stimulating action of E2-CM, PRL-CM, and Pg-CM, suggesting that IGF-RPs are produced by NMU mammary tumor cells exposed to these hormones. Next, we directly tested the role of IGF-RPs as mediators of hormonally stimulated growth. We indeed observed that the addition of alpha sm 1.20B markedly inhibited the colony-stimulating actions of E2, PRL, and Pg added to NMU mammary tumor cells in soft agar in the absence of serum. We conclude that, in our experimental system, IGF-RPs not only are produced upon exposure to E2, PRL, and Pg, but also are important mediators of hormonally stimulated growth.

To determine whether lactalbumin production by normal and neoplastic human mammary tissue is under the same control, the effect of prolactin treatment was studied in organ culture. Of 9 premenopausal normal breast samples, 6 produced lactalbumin in culture, and all 6 responded to prolactin treatment over 4 days. One biopsy of pregnant breast tested also responded to prolactin treatment, producing 200 times more lactalbumin in culture than did normal breast. Two of 4 normal postmenopausal biopsies produced lactalbumin, and one increased synthesis and release after prolonged exposure to prolactin. Of 10 scirrhous carcinomas, 6 produced lactalbumin, but none responded to prolactin treatment. In 2 premenopausal patients, normal breast tissue responded to different concentrations of prolactin, which were without effect on malignant tissue from the same breast. In summary, lactalbumin production in the samples that we have studied can be stimulated in normal but not in malignant breast tissue. This may indicate an absence or deficiency of prolactin receptors in malignant tissue.

Huggins and Morii (J. Exp. Med., 114: 741, 1961) reported that massive adrenal necrosis occurs in 79 and 100% of female Sprague-Dawley rats receiving 20 and 30 mg, respectively, of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Here, adrenal necrosis and regeneration were studied in 158 rats for up to 21 days post-DMBA by radioautography of the adrenals of animals given 50 microCi [3H]thymidine 30 min before sacrifice. Adrenal cell proliferation was markedly inhibited 21 days post-DMBA. Regenerated adrenals were more susceptible to this adrenocorticolytic effect. To investigate if alterations in adrenal function modify tumorigenesis, animals underwent adrenalectomies (ADX), hypophysectomies, ovariectomies, and pituitary transplants alone or in combination 6 days after receiving DMBA (20 mg/100 g intragastrically) at 50 days of age. To prevent adrenal necrosis, 24 animals were pretreated with metyrapone. Methylprednisolone acetate, 1 mg i.m., was given to 40 animals every 5 days beginning 6 days post-DMBA. There were 50 non-DMBA-treated intact and surgical controls. DMBA was necessary but not sufficient to induce mammary tumors. No tumors developed in controls or in 46 animals hypophysectomized 6 days after DMBA. Metyrapone reduced tumor incidence and yield. ADX after DMBA treatment increased the tumorigenic response and eliminated resistance to tumorigenesis in older rats. Only three tumors developed in DMBA-treated rats receiving methylprednisolone acetate. Mammary tumorigenesis was increased by pituitary transplant 6 days after DMBA to intact and ADX animals. Ovariectomy 6 days after DMBA was as effective as methylprednisolone acetate in preventing tumorigenesis; ADX did not overcome either inhibition. We conclude that adrenal hormones inhibit proliferation of initiated mammary cells.

A transplantable rat mammary carcinoma (R3230AC) was previously shown to contain prolactin receptors. Our objective was to determine whether these receptors were functional by measuring specific markers of prolactin action: casein and alpha-lactalbumin (alphaLA) messenger RNA's (mRNA's). Total RNA exacts were translated in a wheat germ cell-free system. Newly synthesized 3H-casein and 3H-alphaLA were separately precipitated with specific antibodies and identified by their mobilities on sodium dodecyl sulfate-acrylamide gels and by competition with nonradioactive casein and alphaLA. Casein and alphaLA mRNA's were both present in unstimulated tumors grown in virgin rats. Casein mRNA but not alphaLA mRNA was markedly stimulated by injections of exogenous ovine prolactin or perphenazine, an agent that stimulates endogenous prolactin. This response was selective in that total mRNA was not significantly altered by prolactin.

The role of progesterone either alone or in combination with other hormones in breast cancer growth is not well established. In these experiments, using the hormone-responsive N-nitrosomethylurea-induced rat mammary tumor grown in the soft agar clonogenic assay, we tested the colony-stimulating effect of progesterone and the synthetic progestin R5020 over a wide range of physiological and pharmacological concentrations (from 0.1 nM to 10 microM). Both progesterone and R5020 were found to have a significant colony-stimulating effect which was more pronounced in the absence of serum. The action of progesterone appeared to plateau at concentrations of 10 or 100 nM, whereas R5020 was maximally effective at lower concentrations (approximately 1 nM). A biphasic dose-dependent effect was occasionally seen both with progesterone and R5020 with a loss of colony-stimulating effect at high concentrations. The combined administration of varying doses of progesterone (0.1, 1, 10, and 100 nM) and estradiol (10(-10) M and 10(-9) M) was found at times to potentiate and at times to decrease colony formation over that observed with the individual treatments. The former effect, when present, was usually seen with low doses of progesterone, while the latter was frequently observed with high concentrations (100 nM). No major potentiation or suppression of colony formation over individual treatments was observed when varying doses of progesterone (1, 10, and 100 nM) were added together with prolactin (50 ng/ml). The administration of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine completely blocked the colony-stimulating effect of progesterone. The inhibitory effect of alpha-difluoromethylornithine was completely reversed in a dose-dependent fashion by exogenous administration of spermidine, thus implying a critical involvement of the polyamine pathway in progesterone action.

The role of prolactin (PRL) in supporting the growth of human breast cancer is still unclear. The ability to grow primary breast cancer specimens in the soft agar clonogenic assay in the absence of serum gave us the opportunity to evaluate the growth-promoting effect of PRL and to compare it to that of estradiol in the same tumor samples. PRL was tested both at physiological concentrations (20 ng/ml) as well as in pharmacological amounts (200 ng/ml) comparable to circulating blood levels in hyperprolactinemic states. Estradiol was simultaneously tested in physiological amounts (10(-8)M). In 17 infiltrating ductal carcinomas, the lower dose of PRL stimulated colony formation to 126 +/- 5.2% (SE) of control, while the higher dose increased colony number to 159 +/- 10.4% of control. This latter effect was comparable to that observed with estradiol (159 +/- 8.5% of control). The effect of PRL was more pronounced in estrogen receptor-positive tumors. Nine of ten estrogen receptor-positive tumors were PRL sensitive, while three of seven estrogen receptor-negative tumors exhibited a clear response to PRL administration. PRL did not stimulate colony formation in a malignant cystosarcoma phylloides and in two benign lesions (fibroadenoma and fibrocystic disease). We conclude that, at least under the conditions of the soft agar clonogenic assay, PRL exerts a dose-dependent growth-promoting effect on human breast cancer. Such effect is comparable to that of estradiol when PRL is added in concentrations similar to circulating blood levels in hyperprolactinemic patients.

In a preliminary report we described the effects of rat prolactin on the incorporation of [14C]acetate into lipids by a cell line from a dimethylbenz(a)anthracene-induced rat mammary tumor. The characteristics of the response to prolactin were very similar to those described for the normal rat mammary gland; namely, insulin was required for full expression of the response, maximal activity was not seen until 36 hr after the addition of the hormones, and growth hormone was able to elicit the same response. However, we were unable to detect binding of 125I-labeled prolactin to these cells, and furthermore, other more purified prolactin preparations were inactive. Upon further investigation we discovered that the activity resided in a low-molecular-weight fraction of the rat prolactin B-1 preparation and was probably either vasopressin or oxytocin or both. These data suggest the possibility that vasopressin may play a role in rodent mammary tumorigenesis.

Cells obtained from N-nitrosomethylurea-induced rat mammary tumors were grown in vitro using the soft-agar clonogenic assay technique. Their growth was studied in regular media containing serum as well as in media lacking serum, but to which insulin was added. Deletion of serum from the media resulted in a mean decrease of 49% in the number of colonies formed in vitro in 13 of 18 tumors and was without effect in the remaining 5 tumors. The addition of either 17 beta-estradiol (10(-8) M) or ovine prolactin (OPRL, 100 ng/ml) to the defined media resulted in an increase in the number of colonies formed in 12 of 18 tumors. The mean numbers of colonies per Petri dish in 17 beta-estradiol- and OPRL-treated Petri dishes were 95 +/- 5.4 (S.E.) and 92 +/- 6.2% of the values seen in serum-containing media. Simultaneous addition of both hormones to the defined media resulted in a significant increase in the number of colonies formed which was greater than that seen when either hormone was added separately. Of four tumors where neither hormone influenced colony formation, the addition of both 17 beta-estradiol and OPRL resulted in an increase in the number of colonies formed in three tumors. We conclude that the N-nitrosomethylurea-induced mammary tumors can be grown in soft agar using defined media and that their growth can be enhanced by either 17 beta-estradiol or OPRL. These hormones have a synergistic effect on the growth of some of these tumors in vitro. These data are consistent with the known in vivo effects of these hormones on the N-nitrosomethylurea-induced rat mammary tumors.

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.

Biochemical parameters characterizing growth, functional activity, and hormone dependence were compared in the normal mammary gland, the 7,12-dimethylbenz(a)anthracene-induced hyperplastic aveolar nodule (HAN), and the mammary tumor in the rat. The rate of DNA synthesis in HAN was significantly lower than the rates in either the normal mammary gland cells or the mammary tumor cells cultured in medium containing identical hormone supplement for the same duration of time. The rate of casein synthesis in the explants of pregnant rat mammary gland and HAN was greatly stimulated when cultured in medium containing insulin, cortisol, and prolactin. The mammary tumor, however, failed to produce an increase in casein synthesis under the same experimental conditions. The specific estradiol-binding proteins were either absent or present in very low concentrations in HAN. In contrast, mammary tumor cytosol contained high concentrations of specific estrogen-binding proteins. The normal mammary gland, in spite of its low cellularity, had twice as many estradiol-binding macromolecules as did HAN. Altogether, these results show that HAN cell populations differ from normal mammary gland and mammary tumor cells by their nonresponsiveness to hormonal stimulation for growth and by their lack of specific receptors for extradiol. They retain, however, the functional capacity to synthesize casein, a biochemical property that the mammary tumor does not posses.

The effect of adrenalectomy on 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth was studied in Sprague-Dawley female rats. Weekly measurements revealed that adrenalectomy significantly increased both mammary tumor size and number and elevated serum prolactin levels as compared to the intact controls. Daily injection of 1 mg hydrocortisone acetate into the intact 7,12-dimethylbenz(a)anthracene-tumor bearing rats did not significantly alter tumor size, number, or serum prolactin levels but, when injected into adrenalectomized rats, it prevented increased tumor growth and prolactin release. Daily injection of ovine prolactin and hydrocortisone suppressed endogenous prolactin release but significantly increased tumore size and number. Ergocornine, a prolactin-inhibiting drug, blocked adrenalectomy-induced tumor growth and partially blocked prolactin release. These results indicate that adrenalectomy in rats stimulates tumor growth by increasing prolactin release.

Regression of MTW9 mammary carcinoma, which consistently follows withdrawal of mammotropic hormones, was characterized by a rapid decrease of thymidine incorporation into DNA but only a slight reduction or uridine incorporation into RNA and amino acid incorporation into proteins. Within 24 hr of hormone withdrawal, cytosol proteins of MTW9 became more easily degraded by trypsin, alpha-chymotrypsin, or subtilisin BPN'. Labilization of cytosol proteins occurred much earlier than any change in the level of protein synthesis or lysosomal enzyme activity. The data showing increased susceptibility to proteolysis could not be explained either by the presence of endogenous proteases, by the destruction of the exogenous proteases used in the assay, or by the existence of protease inhibitors. Nor were any differences detected either in the distribution of radioactive precursor among the cytosol proteins from growing or regressing tumors or in the electrophoretic pattern of the same proteins. Preincubation of the cytosol proteins with dithiothreitol or with prolactin, 17 beta-estradiol, progesterone, and hydrocortisone did not modify the susceptibility to proteolysis. However, after heat denaturation, cytosol proteins of regressing and growing tumors became equally susceptible to proteolysis. It is suggested that regression of MTW9 mammary carcinoma occurs not only because cell reproduction is arrested, but also because susceptibility of cytosol proteins to proteolysis is increased.

Twenty biopsy specimens of benign human breast tumors obtained from 20 patients were processed into small slices and individually cultured for 2 days in Medium 199. The medium was supplemented with bovine insulin (5.0 microgram/ml), human placental lactogen (HPL) (10.0 microgram/ml), or ovine prolactin (10.0 microgram/ml). Four hr prior to termination, [3H]thymidine was added to the culture medium to determine DNA synthesis. The addition of insulin to the culture medium consistently increased: (a) the mean incorporation of [3H]thymidine into chemically extracted DNA (p less than 0.05); (b) the mean number of [3H]thymidine-radiolabeled epithelial cells (p less than 0.05), and (c) the mean number of epithelial cells bearing mitotic figures. The addition of HPL increased the mean number of [3H]thymidine-radiolabeled epithelial cells (p less than 0.05) and the mean number of epithelial cells bearing mitotic figures (p less than 0.05). [3H]Thymidine incorporation into chemically extracted DNA was also increased when HPL was added to the medium, although this increase did not quite achieve the 5% level of significance. The addition of ovine prolactin to the culture medium did not have any significant effect on DNA synthesis. This study provides evidence that insulin and HPL are direct stimulants of DNA synthesis of the epithelium contained in benign human breast tumors.

We have demonstrated recently that the local metastatic growth of Morris hepatoma 44 is thyroid dependent ( Mishkin , S., Morris, H. P., Yalovsky , M., and Murthy , P. V. N. Gastroenterology, 77; 547-555, 1979; Mishkin , S. Y., Pollack , R., Morris, H. P., Yalovsky , M., and Mishkin , S. Cancer Res., 41: 3040-3045, 1981) and that exogenous thyroxine (8 micrograms/kg/day) and prolactin (100 micrograms/day) significantly stimulated tumor growth, while growth hormone (100 micrograms/day) failed to do so ( Pollack , R., Mishkin , S. Y., Morris, H. P., and Mishkin , S. Hepatology, 2: 836-842, 1982). In the present study, thyroid ablation (hypothyroidism) and hypophysectomy inhibited tumor growth significantly. These effects were almost totally reversed by administration of exogenous thyroxine to hypothyroid rats. While prolactin or growth hormone or thyroxine alone failed to restore tumor growth in hypophysectomized animals, administration of all three hormones partially but significantly reversed the inhibition of tumor growth. The number and size of pulmonary metastases paralleled local growth in all the above-mentioned conditions. Plasma membrane lactogenic receptors, measured using human growth hormone, were decreased in hypothyroidism and hypophysectomy groups. Binding levels were restored in those groups in which tumor growth was stimulated. In summary, the local and metastatic growth of Morris hepatoma 44 is affected by anterior pituitary hormones. Plasma membrane lactogenic receptors may mediate these effects.

We have previously reported the identification and characterization of a prolactin-inducible protein (PIP) as well as the cloning of the gene encoding PIP in cultured human breast cancer cells. We now present three lines of evidence that the gene encoding PIP is also expressed by some human breast cancers in vivo: detection of PIP immunoreactivity in the serum of some breast cancer patients; immunohistochemical detection of PIP in breast cancer sections; and the presence of PIP mRNA, detected by complementary DNA hybridization in human breast biopsy samples. In a preliminary study using Western blot analysis authentic PIP was detected in the serum of some patients with breast cancer. Subsequently the sera of 234 unselected patients with breast cancer were assayed for the presence of PIP using a specific radioimmunoassay. Thirty-five % of these sera contained detectable PIP (i.e., greater than 3 ng/ml). As well as were able to show by immunohistochemical techniques that PIP immunoreactivity was present in some human breast biopsy specimens. Levels of estrogen receptor, progesterone receptor, and PIP mRNA were determined in an unselected population of 51 human breast tumor biopsies. Sixty-one % of these tumors had detectable PIP mRNA; a positive correlation (r = 0.52; P less than 0.01) was found between PIP mRNA levels in breast biopsy samples and estrogen receptor content, a known prognostic indicator in human breast cancer.

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.

The intracellular specific 17beta-estradiol binding in the human breast cancer cell line, MCF-7, was shown to be modified by prolactin. Both ovine and human prolactin doubled the estradiol receptor (E2R) level, but the latter was at least 10 times more stimulatory on a concentration basis. Most of the E2R complex (approximately 80%) was transported to the nucleus, and the prolactin stimulation was reflected in an elevated nuclear uptake of the tritiated 17beta-estradiol. Neither ovine nor human prolactin altered the growth rate of the cells when E2R stimulation was maximal. Insulin (10 mug/ml) stimulated tritiated thymidine incorporation and total DNA content but had no apparent effect on E2R concentration. At 10(-4) M, N6,O2'-dibutyrylcyclicadenosine 3':5'-monophosphate increased insulin stimulation of tritiated thymidine incorporation and brought about a prolactin stimulation of apparent DNA synthesis. Theophylline (10(-3) M) blocked both of these effects of N6,O2'-dibutyrylcyclicadenosine 3':5'-monophosphate. The possible mechanism implicating prolactin as an effector of differentiation and growth of MCF-7 cells is discussed.

The human breast cancer (Br-10) serially transplanted to nude mice (BALB/c-nu/nu) grew well in female mice but very slowly or not at all in untreated male mice and female mice treated with 1 mg of testosterone i.m. twice a week. The growth in female mice was arrested by ovariectomy, and that in male mice was accelerated by 0.1 mg of estradiol i.m. once a week. Tumors in female and estrogenized male mice retained the original histology of duct carcinoma. Tumors in ovariectomized female, androgenized female, and male mice consisted of cells with smaller and more uniform nuclei, forming markedly dilated lumina in the first group and arranged in lobular patterns in the latter two groups. High-affinity 8 S and 4 S estrogen receptors were present in tumors transplanted to female nude mice, but no progesterone receptors were detected. These results provide experimental evidence for the hormone dependency of a human breast cancer in vivo and strongly suggest the important role of estrogen and androgen in the growth regulation of some estrogen receptor-positive human breast cancers.

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.

The production of plasminogen activator (PA) and its regulation by hormones and other effectors were studied in organ cultures of primary rat and mouse mammary tumors. PA was quantitated using the radioiodinated fibrin plate method. The level of PA in tumor tissue was 10- to 100-fold higher than that in normal rat or mouse mammary glands; the rates of PA secretion were 10- to 1000-fold higher in the tumor cultures. PA production was stimulated by prolactin and pituitary extracts in N-nitrosomethylurea- and 7,12-dimethylbenz(a)anthracene-induced rat tumors but not in mammary tumor virus-induced mouse tumors; hydrocortisone inhibited PA production in all three tumor categories. Sex hormones and agents such as cholera toxin and retinoic acid effectively modulated enzyme production by some tumors. Three major points of interest emerge from our findings: (a) the pattern of tumor PA response to hormones differs qualitatively and quantitatively from that previously determined for the normal mammary; (b) the profile of responses of tumor PA and tumor growth to hormones shows numerous correlations suggesting that these two parameters may be coordinately regulated; (c) pituitary extracts contain an apparently novel factor that stimulates rat mammary tumor PA synthesis.

Dimethylbenzanthracene-induced rat mammary tumors were defined as either prolactin responsive or prolactin independent on the basis of growth response to prolactin administration. There was no difference in tumor binding of prolactin between the two groups when tumors were biopsied before treatment. Prolactin binding was, however, significantly higher in responding tumors when biopsies were obtained following treatment. By contrast, when tumors were defined as responsive or independent on the basis of response to suppression of serum prolactin with bromoergocryptine, there was significantly higher prolactin binding in the responsive than in the independent group both before and after treatment. During serial treatment with prolactin followed by bromoergocryptine, there was a progressive decline in prolactin binding to tumor biopsies, particularly in prolactin-independent tumors. Prolactin binding to pretreatment tumor biopsies thus did not predict which tumors would respond to administration of prolactin but, for the total group, did indicate tumors likely to regress with prolactin withdrawal. However, the correlation between prolactin binding and tumor regression following hormone withdrawal was not sufficiently strong to permit reliable prediction of behavior for individual tumors. Prolactin-independent growth was associated with decreased prolactin binding to tumor tissue, particularly following manipulation of serum prolactin levels.

Estrogen, prolactin, and other pituitary factors are implicated in the etiology of human breast cancer. In the present study, the effects of estrogen and factors from GH3 rat pituitary tumor cells on the growth of T-47D human breast cancer cells were tested in athymic nude mice. Four groups of nude mice were used: Group 1 (T) received s.c. injection of T-47D cells only; Group 2 (TE) mice were given injections of estradiol valerate and T-47D cells; Group 3 mice were given injections of T-47D and GH3 cells (TG), one cell type on each flank; and Group 4 received estradiol valerate and T-47D and GH3 cells (TEG). We found that the human breast cancer cells, T-47D, did not proliferate in Groups 1 and 3 despite the presence of high circulating levels of prolactin and growth hormone produced by the growing GH3 pituitary tumors in Group 3. This suggested that prolactin and growth hormone were not sufficient to stimulate the growth of human breast cancer cells. T-47D cells exhibited only moderate growth in Group 2 but proliferated rapidly in Group 4. The T-47D tumors of Group 4 were 8 times larger than those in Group 2 after 42 days of growth. These results demonstrate that the simultaneous presence of estrogen and pituitary growth factors are required for maximal growth of T-47D human breast cancer cells in nude mice. The identity of the pituitary-dependent growth factor(S) that stimulates the growth in vivo of human breast cancer cells remains to be elucidated.

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.

We have demonstrated previously that many human cancer cell lines maintained in tissue culture possess specific cell surface receptors for human prolactin (HPRL) and human growth hormone (HGH). In the present studies, the biological response in vitro of one human breast cancer cell line, T-47D, to the two pituitary hormones was examined. T-47D cells, when grown on tissue culture dishes, display typical epithelioid characteristics; cells are flat and polygonal in shape and are very adhesive to the plastic substratum. Upon the addition of HPRL or HGH (10 to 1000 ng/ml), in the presence of hydrocortisone, insulin, and triiodothyronine, each at 1 microgram/ml, the T-47D cells became round and refractile. In addition, there was a dramatic reduction in the adhesiveness of the cells to the substratum; 80% of the hormone-treated cells were detached by trypsin (25 micrograms/ml) in 30 min at 37 degrees, as compared with 5% for cells not treated with hormones. These prolactin-induced changes could be abolished upon the addition of antiserum to prolactin. Neither HPRL nor the combination of hydrocortisone, insulin, and triiodothyronine alone was active, indicating a synergism between HPRL and hydrocortisone, insulin, and triiodothyronine. It was subsequently found that only hydrocortisone was required for the action of HPRL, and that human luteininzing hormone and ovine growth hormone were inactive, whereas ovine prolactin exerted a very weak effect. In addition, in the presence of hydrocortisone (or hydrocortisone, insulin, and triiodothyronine), HPRL (or HGH) retarded cell proliferation by 30%, whereas HPRL or hydrocortisone by itself had no effect on cell growth. Ultrastructural studies revealed that, accompanying cell rounding and reduced adhesion, HPRL and HGH increased the formation of intracytoplasmic lipid droplets in the T-47D cells. The increase in lipid synthesis was confirmed by the staining of cells with Oil Red O, and by monitoring the incorporation of [14C]acetate into lipid; HPRL stimulated lipid synthesis and accumulation by approximately 2-fold. Thus, receptor-positive human breast cancer cells are biologically responsive in vitro to HPRL and HGH.

The metabolism of both testosterone and dehydroepiandrosterone by 10 adenocarcinomas induced and grown in the presence of high prolactin (plasma prolactin greater than 220 ng/ml) was compared with the metabolism of 10 adenocarcinomas induced and grown in the presence of normal levels (plasma prolactin less than 60 ng/ml). The tumors associated with high prolactin significantly metabolized more testosterone to both 5alpha-dihydrotestosterone and 5alpha-androstanediol. The metabolism of dehydroepiandrosterone was similar in both groups of tumors. It is concluded that prolactin may differentially influence the metabolism of C19 steroids by rat mammary adenocarcinomas.

The rat lymphoma cell line Nb2 is highly prolactin responsive in terms of growth. Estrogens are without effect in these cells that lack the estrogen receptor. The growth stimulation by lactogenic hormones is effectively inhibited by antiestrogens, such as tamoxifen and nafoxidine, at concentrations as low as 10(-10) M. The growth inhibition is partially reversed by replacement of the antiestrogens by prolactin. Nb2 cells contain estrogen-noncompetitive specific antiestrogen binding sites on their membranes that bind tamoxifen with an average Kd of 3.1 X 10(-10) M. Addition of antiestrogens to the binding reaction inhibits lactogenic hormone binding to membrane-bound receptors. The order of affinities of various antiestrogens (tamoxifen, nafoxidine, 2-(4-tert-butyl-phenoxy)ethyl diethylamine hydrochloride, and LY117018) for the antiestrogen binding sites parallels the order of their potencies as growth and lactogen binding inhibitors. These data suggest that antiestrogens, possibly acting through the antiestrogen binding sites, may function as antilactogens.

Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.

The effects of hypophysectomy and prolactin-suppressing drugs on the growth of mammary tumors induced in Sprague-Dawley rats by N-nitrosomethylurea and dimethylbenz(a)anthracene were compared. The influence of ovine prolactin and growth hormone administration on N-nitrosomethylurea-induced tumors was also studied in hypophysectomized animals. After hypophysectomy, all 13 tumors induced in 13 rats by N-nitrosomethylurea underwent regression, as did ten of 12 induced by dimethylbenz(a)anthracene. There were no new tumors. Pergolide mesylate, a long-acting ergoline derivative, was given in a dose of 80 micrograms twice daily by s.c. injection for 28 days. Only three of 12 N-nitrosomethylurea-induced tumors regressed, while four became static. However, only two new tumors developed in the 12 pergolide-treated rats, compared to 11 in the 12 untreated controls. Bromocriptine mesylate, at ten times the pergolide dose, was even less effective; one of 16 tumors regressed, two became static, and eight new tumors appeared in the 16 rats. In contrast, eight of 12 dimethylbenz(a)anthracene-induced tumors regressed during pergolide therapy, two became static, and there was only one new tumor among the 12 rats. Prolactin, 1 mg twice daily for 7 days by s.c. injection, was given to another eight rats bearing 11 N-nitrosomethylurea-induced tumors, commencing 7 days after hypophysectomy. Regression of five tumors borne by four rats was reversed but resumed when treatment was stopped. Regression of five tumors in the other four animals was arrested without regrowth; the sixth became inpalpable. All of these six grew rapidly when growth hormone, 2 mg twice daily, was administered in addition to prolactin.

MRMT-1 is a mammary carcinoma induced in immunologically impaired female Sprague-Dawley rats fed 3-methylcholanthrene. Its biological characteristics include transplantability to syngeneic normal rats and spontaneous metastases to various organs. Hypophysectomy performed 48 hr after tumor inoculation resulted in tumor regression accompanied by the disappearance of the metastasis in lungs of all the animals. The hypophysectomized animals were given replacement treatments, such as transplantation of pituitary homogenates, pituitary homograft underneath the renal capsule, or prolactin administration, and the inhibited tumor growth was markedly reactivated, with the reappearance of lung metastasis. These results indicate that MRMT-1 mammary carcinoma is prolactin dependent.

An organ culture method suitable for the maintenance of viable human breast cancer for at least 14 days has been described. This method was applied to a total of 94 breast cancer specimens. It allowed good survival of "soft" tumors of various histological types, with loose connective stroma even in hormone-free medium. In contrast, "scirrhous" cancers showed poor survival in hormone-free medium; viable cells were maintained only at the very periphery of the explants. Supplementation of the medium with insulin (10 mug/ml), ovine prolactin (5 mug/ml), and hydrocortisone (1 mug/ml) in various combinations seemed to induce enlargement of viable cancer cells and moderate loosening of the stroma in some cases. However, it did not improve the survival of central tumor cords in scirrhous explants. Further supplementation of the medium with 17 beta-estradiol (minimum effective dose, 0.1 to 10 ng/ml), although it did not affect soft tumors, markedly improved survival of the cancer cells of scirrhous tumors throughout the whole explants, with evidence of collagen digestion around the neoplastic cells. This was observed in 18 of 20 scirrhous cancers subjected to this treatment. Estradiol need not be present during the whole culture period; the results at 14 days were identical in explants treated with estradiol for the first 7 days only or for the entire period. Addition of purified collagenase during the first 24 or 48 hr of culture resulted in complete dissolution of the collage. After such treatment, culture under the usual conditions resulted in excellent survival of the explants without improvement from hormone supplementation; thus, while estradiol was necessary when collagen was present, it was not longer required after collagen digestion. It can be concluded that breast cancer cells in organ culture are only slightly, or not at all, hormone dependent for survival, provided that they are not restrained by a dense collagen barrier. The estrogen-induced changes allowing survival inside the scirrhous explants strongly suggest the presence of an estrogen-dependent collagenolytic enzyme system in the collagen-rich breast cancers. This system could represent an important component of the hormone dependency of human breast cancer growth.

DNA synthesis in mammary gland estimated by [3H]thymidine incorporation was significantly higher on the day of proestrus than on the second day of diestrus in 50-day-old female Sprague-Dawley rats. The percentage of progressive mammary tumors, tumor growth rate, and the number and the weight of tumors per tumor-bearing rat were significantly higher in the animals given a single i.v. injection of 5 mg 7,12-dimethylbenz(a)anthracene at proestrus than in the animals given it at diestrus. Inhibition of DNA synthesis at proestrus by 2-bromo-alpha-ergocryptine also suppressed mammary tumorigenesis by the carcinogen. In 90-day-old rats in which little difference was found in mammary gland DNA synthesis between proestrus and diestrus, there was no difference in mammary tumorigenesis between animals given the carcinogen at proestrus and animals given it at diestrus. On the other hand, the prestimulation of mammary gland DNA synthesis by prolactin increased the growth, the number, and the weight of carcinogen-induced mammary tumors. These results demonstrate the importance of mammary DNA synthesis at the time when a carcinogen acts on the glands in mammary tumorigenesis.

A previous study described the development of cultures of Nb2 node rat lymphoma cells which specifically require a lactogenic hormone, e.g., prolactin (PRL), as a growth factor. In the present study, sublines of these cultures have been established, including clones, which do not depend on exogenous lactogens for growth; the cells do not appear to produce autocrine, PRL-like substances. Although these autonomous cells grew readily in the complete absence of lactogens, their growth rate was stimulated (up to approximately 30%) by PRL concentrations greater than or equal to 0.1 ng/ml. When cultures of the lactogen-dependent and of the cloned, autonomous lymphoma cells were incubated for 3 days with sodium n-butyrate (NaBT; 2 mM), cells were arrested in the G1 phase of the cell cycle, as shown by flow cytometric analysis. A substantial proportion of the G1-arrested cells was viable and readily resumed growth, within 1 day, when transferred to NaBT-free medium containing PRL (1-100 ng/ml). The resumption of growth of the cells from the lactogen-dependent cultures was critically dependent on PRL; in its absence cells lysed. Surprisingly, the rapid recovery of the cells from the lactogen-independent cultures also depended on the presence of PRL in the medium; in its absence growth did not resume for at least 2.3 days. The acquired need of the NaBT-treated, autonomous cells for PRL was only transient, since such cells reverted fully to the PRL-independent state within about 3 days of culturing in PRL-containing, NaBT-free medium. It is proposed, as a working hypothesis, that the autonomous lymphoma cells can be mitogenically activated by two different pathways, one requiring exogenous lactogens and another which is independent of lactogens; the latter pathway recovers much more slowly from the treatment with NaBT than the lactogen-dependent pathway. This model could explain the sensitivity of the autonomous lymphoma cells to PRL and their transient dependency on exogenous lactogens during their recovery from NaBT treatment. NaBT would appear to be a useful agent for studying the mechanism(s) by which the autonomous lymphoma cells circumvent the need for mitogenic lactogens.

Selective nutritive conditions were used to isolate normal epithelial cells from fibroblasts in primary cell cultures prepared from adult rat prostate. The pure population of normal epithelial cells proliferated at an exponential rate on a simple polystyrene substratum with doubling times of 35 to 50 hr for 10 to 12 days in the absence of high epithelial cell density, other cell types, or added extracellular matrix elements. Optimization of the nutritive environment allowed direct analysis of the hormone:growth factor requirements for sustained proliferation of the isolated epithelial cells in serum-free medium. An in situ videometric method was used to assay the effect of over 30 known hormones and growth factors on proliferation of the prostate epithelial cell population. The results revealed direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, one or more unidentified factors from bovine pituitary, and possibly prolactin. No direct mitogenic effect of androgen on isolated prostate epithelial cells could be demonstrated. Radioimmunoassay of androgen in the primary cultures showed that endogenous androgen was about 34 pM on Day 1 of culture and thus probably too low to mask a response to exogenous androgen. Deletion of any single active growth factor did not reveal an androgen response. The results demonstrate a multihormonal control of normal prostate epithelial cell maintenance and proliferation without the direct participation of androgen.