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The human prolactin-inducible protein / gross cystic disease fluid protein-15 (hPIP/GCDFP-15) is a secretory glycoprotein found primarily in apocrine tissues including the breast and salivary glands. With largely unknown functions, PIP has been implicated in breast cancer and metastasis, host defense processes and T lymphocyte apoptosis. To begin to address PIP function in vivo, we generated the PIP null mouse (Pip−/−mouse). Additionally, to determine the effect of the loss of PIP on gene expression and to gain insight into some of the molecular mechanisms underlying PIP function, microarray analysis of the submandibular gland was also undertaken. Pip−/−mice developed normally with no overt differences in behaviour or gross morphology and were fertile. However, histological examination of 3-month-old Pip−/−mice sometimes showed enlarged submandibular lymph nodes, lymphocytic aggregations within the prostate lobes, and enlarged medulla in the thymus. Functional analysis of gene expression revealed sets of multiple differentially expressed genes associated with cell death and survival, lipid metabolism, inflammation, immune disease, and cancer, as a consequence of mPIP abrogation. Taken together, these studies lend support to an immunomodulatory role for PIP in vivo and provide further insights into potentially novel signaling pathways and regulatory networks for PIP.

OBJECTIVES To investigate the public's knowledge and attitudes about cardiopulmonary resuscitation in Hong Kong. DESIGN Cross-sectional telephone survey. SETTING Hong Kong. PARTICIPANTS Hong Kong residents aged 15 to 64 years. MAIN OUTCOME MEASURES The knowledge and attitudes towards cardiopulmonary resuscitation. RESULTS Among the 1013 respondents, only 214 (21%) reported that they had received cardiopulmonary resuscitation training. The majority (72%) of these trained respondents had had their latest training more than 2 years earlier. The main reasons for not being involved in cardiopulmonary resuscitation training included lack of time or interest, and "not necessary". People with full-time jobs and higher levels of education were more likely to have such training. Respondents stating they had received cardiopulmonary resuscitation training were more willing to try it if needed at home (odds ratio=3.3; 95% confidence interval, 2.4-4.6; P

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogenbinding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), a and b. In this investigation, we analyzed the expression of decidual ERa and ERb, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERb to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERa and ERb by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERb messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERa expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERa and had no synergistic effect on ERb expression. The use of GG-AD cells, which express only ERb, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERb is expressed in addition to ERa in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERa, but concomitantly increases the expression of a functional ERb that mediates estradiol up-regulation of the decidual progesterone receptor. (Endocrinology 141: 3842‐3851, 2000)

Protein kinase C (PKC) delta is dramatically upregulated in the corpus luteum in the second half of pregnancy in the rat. To gain insight into the hormonal regulation of PKC delta expression, studies were undertaken to analyze the regulation of PKC delta expression in a luteinized rat granulosa cell model. PKC delta protein expression was evaluated in luteinized granulosa cells, isolated from human (h)CG-treated immature female rats 7 h after the injection of an ovulatory dose of hCG and cultured up to 12 days. Cytochrome P450 cholesterol side chain cleavage enzyme expression was observed throughout the culture period, and a majority of the cells expressed steroidogenic acute regulatory protein and responded to rat placental lactogen (rPL)-1 by exhibiting hypertrophy, consistent with maintenance of the luteal phenotype. Both PKC delta protein and mRNA expression increased 3.5-4-fold with time of culture, and PKC delta mRNA expression could be eliminated by treatment of cells with the PKC inhibitor GF109203X. E(2) caused a specific dose- and time-dependent increase in expression of PKC delta protein of twofold, whereas PKC delta mRNA was unaffected by E(2) over a 12-day culture period. Treatment of cells with 500 ng/ml rPL-1 for the final 4 days of a 12-day culture in the absence of E(2) had no effect on PKC delta protein or mRNA expression, while treatment with 500 or 3000 ng/ml rPL-1 in the presence of E(2) significantly enhanced both PKC delta protein and mRNA expression (up to threefold). These results show that two of the major regulators of luteal function in the second half of pregnancy in the rat, E(2) and rPL-1, cooperate to regulate PKC delta expression in luteinized granulosa cells.

The ability of PRL or rat placental lactogen (rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized rat granulosa cell culture model. PRL receptor activation induced relaxin mRNA expression in a concentration- and time-dependent manner. High concentrations of PRL receptor agonist, equivalent to those of the second half of pregnancy in rats, were required to elicit relaxin mRNA expression. A 40-fold induction of relaxin mRNA was observed in cells treated 24 h with 1 microg/ml of rPL-1. Estrogen enhanced relaxin expression induced by PRL but did not affect relaxin expression on its own. PRL/rPL-1 induction of relaxin expression was independent of the extracellular regulated kinase (ERK) members of the mitogen-activated protein kinase (MAPK) pathway, based on the inability of the ERK kinase inhibitor PD98059 to block induction of relaxin expression. PRL/rPL-1 induction of relaxin expression required protein kinase C (PKC) delta, based on the ability of the preferential PKC delta inhibitor rottlerin to abolish induction of relaxin expression. Direct activation of PKC by phorbol myristate acetate, however, was not sufficient to promote induction of relaxin mRNA expression. Stats (signal transducers and activators of transcription) 3 and 5 DNA binding activities were induced by PRL/rPL-1 treatment of luteinized granulosa cells but only Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 treatment of luteinized granulosa cells resulted in increased phosphorylation on tyrosine-705 and serine-727 of Stat 3, and these responses were reduced and blocked, respectively, by rottlerin. Tyrosine and serine phosphorylations of Stat 3 in the corpus luteum were also increased in the second half of pregnancy when PL levels are highest. Stat 3, but not Stat 1 or 5, coimmunoprecipitated with luteal PKC delta during pregnancy; Stat 3 transiently coimmunoprecipitated with PKC delta from luteinized granulosa cells in response to PRL receptor activation; and Stat 3/PKC delta complex formation required PKC delta kinase activity. Taken together, these results show that PKC delta is obligatory for PRL/rPL-1-dependent relaxin expression, that PKC delta complexes with Stat 3 in response to PRL receptor activation, and that PKC delta is involved in the regulation of Stat 3 phosphorylation downstream of the PRL receptor. These results demonstrate that PRL/rPL-1 promotes relaxin expression in luteal cells and that this event is mediated, at least in part, via PKC delta.

The prolactin-inducible protein (PIP/GCPD15) is believed to originate from a limited set of tissues, including breast and salivary glands, and has been applied as a clinical marker for the diagnosis of metastatic tumours of unknown origin. We have investigated the potential role of PIP mRNA as a marker of human breast cancer metastasis. Using reverse transcription polymerase chain reaction and Southern or dot blot analysis, PIP mRNA was detected in 4/6 breast cell lines, independent of oestrogen receptor (ER) status. In breast primary tumours (n = 97), analysed from histologically characterized sections, PIP mRNA was detected in most cases. Higher PIP mRNA levels correlated with ER+ (P = 0.0004), progesterone receptor positive (PR+) (P = 0.0167), low-grade (P = 0.0195) tumours, and also PIP protein levels assessed by immunohistochemistry (n = 19, P = 0.0319). PIP mRNA expression was also detectable in 11/16 (69%) of axillary node metastases. PIP mRNA expression, however, was also detected in normal breast duct epithelium, skin, salivary gland and peripheral blood leucocyte samples from normal individuals. We conclude that PIP mRNA is frequently expressed in both primary human breast tumours and nodal metastases. However, the presence of PIP expression in skin creates a potential source of contamination in venepuncture samples that should be considered in its application as a marker for breast tumour micrometastases. © 1999 Cancer Research Campaign

The possible role of the conceptus in stimulating the onset of maternal behavior through its secretion of placental lactogens and their passage into the brain was investigated in female rats. In the first study, significant mitogenic activity in the Nb2 lymphoma cell bioassay was detected in cerebrospinal fluid (CSF) samples collected by push-pull perfusion from rats on days 12–21 of pregnancy, coincident with the establishment of placental function. In contrast, mitogenic activity was absent from CSF in lactating and gonadectomized, virgin females. In a second study the mitogenic activity in day 12 pregnant samples was neutralized 71% with antibodies to rat placental lactogen-I (rPL-I) and > 90% with a combination of antibodies to rPL-I plus rPL-II. In contrast, activity on day 21 of pregnancy, 1 day prepartum, was reduced by antibodies to rPL-II (>85%), but not by antibodies to rPL-I, indicating that the predominant lactogen in the CSF prepartum is rPL-II. The behavioral actions of placental secretions were assessed in the third experiment by infusing recombinant rPL-I and purified rPL-II directly into the medial preoptic area of the brain of steroid-primed, nulliparous rats. Latencies to respond maternally to foster young were significantly reduced in rPL-I- and rPL-II-treated rats (2- to 3-day latencies) when compared with latencies in control females (5- to 6-day latencies). Thus, the conceptus through its secretion of rPLs which apparently gain access to the CSF helps to prime the pregnant female’s brain to respond maternally at the end of gestation. This endocrine communication between the developing conceptus and pregnant female appears to be an important part of the biological system which helps to establish successful maternal care.

The secretion of peptide 23 by rat pituitary cells is stimulated by growth hormone-releasing hormone and inhibited by somatostatin. Recent cloning of the cognate cDNA for peptide 23 revealed that it is identical to pancreatitis-associated protein (PAP). In the present study, the clearance and tissue uptake of recombinant peptide 23/PAP in normal adult male rats was assessed. The plasma half-life of recombinant peptide 23/PAP was 4·8 ±1·4 (s.d.) min. Maximal accumulation of radiolabelled peptide 23/PAP was observed in the kidney, stomach, small intestine and pancreas whereas negligible uptake was seen in the liver, lung or heart. Peptide 23/PAP was detected in a variety of tissue extracts using a radioimmunoassay. Extracts of ileum contained the highest concentrations of peptide 23/PAP. In situ hybridization analysis showed that peptide 23/PAP mRNA was highly expressed in the columnar epithelial cells of ileum, jejunum and duodenum. These observations demonstrate that peptide 23/PAP, a protein previously thought to be of pituitary origin, is widely expressed in the gastrointestinal tract and that it is rapidly removed from the circulation by the kidney and by tissues which express peptide 23/PAP. Journal of Endocrinology (1995) 145, 461–469

Peptide 23 was first identified in pituitary cell conditioned medium as a secreted protein which was regulated in a similar fashion to growth hormone. It was subsequently found to be a member of the C-type lectin gene superfamily and identical to pancreatis associated protein (PAP). It is widely expressed in the gastrointestinal tract. Our present study demonstrates that peptide 23 gene is also expressed in the uterus. Peptide 23/PAP mRNA was at highest levels during estrus and was not detectable in the immature rat uterus. A single injection of 17 beta-estradiol resulted in a transient induction of peptide 23/PAP mRNA in ovariectomized rats whereas a sustained induction was seen with diethylstilbestrol implants. In situ hybridization localized peptide 23/PAP mRNA to the luminal epithelial cells. During gestation, peptide 23/PAP mRNA was detected only in the uterine samples from day 12 to 18 of pregnancy with maximal expression on day 12. Peptide 23 expression was confined to the uterus itself and not expressed in either the decidua or the fetal tissues. PSP/reg, another closely related member of the C-lectin gene family was not expressed in any of these uterine tissues. These results indicate that estrogen may act as a physiological regulator of peptide 23 in the uterus and suggests that this protein may have some role in estrogen action.

To clarify the roles of the rat placental lactogens in embryogenesis and fetal development, we measured the concentrations of rat placental lactogen-II (rPL-II) in fetal rat serum and examined the distribution and expression of rPL-I- and rPL-II-binding sites in rat uteroplacental and fetal tissues. The concentration of rPL-II in fetal rat serum on day 20 of gestation was 28.3 +/- 0.8 ng/ml (mean +/- SEM; n = 6), approximately 1/14th its concentration in maternal serum (398.3 +/- 45.3 ng/ml; n = 6). In the midgestational uterus and placenta, rat PL-I bound specifically to mesometrial decidua and to a capsular layer of stroma overlying the antimesometrial decidua. The binding of radiolabeled rPL-I to these tissues was inhibited by unlabeled rat PRL and human (h) GH, but not by rat GH, suggesting that the rPL-I-binding sites are lactogenic in nature. In the late gestational fetus, rat PL-II bound specifically to fetal adrenal, kidney, small intestine, liver, and pancreas; its binding, like that of rPL-I, was inhibited by rPRL, but not by rGH. rPL-II-binding sites in fetal adrenal were detected as early as day 16, whereas rPL-II-binding sites in fetal kidney and small intestine were not demonstrable until day 18. Lactogenic binding sites in fetal liver and pancreas did not appear until days 19-20. The relative amounts of specific binding of rPL-II to fetal tissues correlated positively with tissue levels of expression of the 4.2- and 1.8-kilobase PRL receptor mRNA transcripts. Radiolabeled hGH, which interacts with somatogenic receptors as well as lactogenic receptors, bound specifically to mesometrial decidua, fetal adrenal, kidney, small intestine, liver, and pancreas. In addition, radiolabeled hGH bound specifically, but with low intensity, to fetal brain. In mesometrial decidua and fetal adrenal, kidney, and small intestine, the binding of hGH was blocked by rPL-II and rPRL, but not by rGH or ovine GH, suggesting the predominance of lactogenic receptors. In contrast, in fetal brain, the binding of hGH was inhibited by rGH, but not by rPL-II, suggesting that the fetal brain contains somatogenic receptors. The presence of rPL-I-binding sites in maternal decidua suggests a paracrine role for the hormone in decidual function at midgestation. The presence of rPL-II in fetal serum and the widespread distribution of rPL-II-binding sites in fetal tissues indicate a role for rPL-II in fetal development.

BACKGROUND Amplification of the c-myc gene (also known as MYC) occurs in up to 20%-30% of breast cancers and has been associated with poor prognosis. PURPOSE The purpose of this study was to define the relationship between c-myc amplification and breast cancer progression in order to better understand the biological significance of c-myc amplification. METHODS We identified invasive tumors with grossly detectable c-myc amplification by using Southern blot analysis to examine frozen tissue from 135 breast carcinomas and polymerase chain reaction (PCR) analysis to examine archival paraffin-embedded tissue from an additional 19 invasive tumors. These 19 tumors were selected on the basis of histologically identifiable in situ and invasive components within the primary tumor and associated lymph node metastases. Amplification of c-myc in these areas was then assessed by quantitative PCR assay. RESULTS We detected gross c-myc amplification in 10 of the tumors examined--eight of the 135 frozen tissue specimens and two of the 19 archival specimens. We selected five of these 10 invasive tumors for further regional analysis. In all four cases where an in situ component was present, amplification of c-myc was present in both the in situ and the invasive components. However, c-myc amplification was present in the corresponding nodal metastases in only two of the four cases where this could be examined. CONCLUSION These results suggest that c-myc amplification can occur at an early stage in tumor progression and that amplification does not always persist in the nodal metastasis.

The two hormones prolactin and growth hormone exhibit considerable structural homology as well as exerting similar biological effects, especially the primate hormones. One effect of prolactin that deserves greater attention is its action on the immune system including the stimulation of growth of experimental lymphomas, both in vivo and in vitro. One cultured lymphoma cell line has proved to be a very useful model system in which to examine prolactin receptor synthesis and turnover as well as post-receptor mechanisms of action. Prolactin and growth hormone receptors from rabbit mammary gland and liver respectively have been partially purified and characterized. Polyclonal antibodies to prolactin and growth hormone receptors have been generated. The antibodies have been shown to cross-react with prolactin or growth hormone receptors from a number of species, indicating structural homology among receptors as well as hormones. The polyclonal antisera inhibit the action of prolactin in vivo as well as in vitro. In addition, several of the same antisera also mimic the action of prolactin. As yet the presence of autoantibodies to prolactin or growth hormone receptors in human serum samples has not been recognized.

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.

The insulin-like growth factor I receptor (IGF-IR), which mediates the mitogenic action of IGF-I, has been shown to play an essential role in normal growth and development. However, the precise role of IGF-IR in the growth and differentiation of the mammary gland has not been elucidated. This study examines the profile of the IGF-IR gene and protein expression during normal postnatal mammary gland development in order to gain further insight into the role of the IGF-I/IGF-IR during mammary gland morphogenesis. Gene and protein expression were examined in developing mouse mammary glands (virgin, pregnant, lactating, involuting) by real time PCR analysis and Western blotting. Both IGF-IR gene and protein expression levels were high during early pregnancy. Interestingly, the level of gene expression was significantly down-regulated during late pregnancy (5.4 fold) and lactation (9-13 fold) and significantly up-regulated (3.9 fold) during late involution, to the level observed in the virgin mammary gland. By in situ hybridization, the IGF-IR transcripts were localized to the proliferating ductal epithelium of the mammary glands of virgin mice and to the differentiating ductal and alveolar epithelium of the mammary glands during pregnancy and lactation. In the involuting gland, the transcripts were localized to the regressing ductal epithelium. These data are direct evidence that IGF-IR expression is important for alveolar cell proliferation and suggest that the progression of involution may require the down-regulation of IGF-IR gene expression. Altogether, these results demonstrate that a developmental IGF-IR gene expression pattern exists in the mouse mammary gland and that increases in gene expression at specific phases of development may reflect an important role for IGF-I/IGF-IR at those phases of development.

C-myc is implicated in the initiation, progression and estrogen response of breast cancer. To further investigate the role of c-myc in breast cancer, we have developed clonal MCF-7 human breast cancer cell lines harboring a stably-transfected human c-myc gene, whose expression was stringently controlled by the bacterial reverse tetracycline transcription activator protein. The expression of the endogenous genomic c-myc gene in MCF-7 cells was abolished by the potent pure estrogen antagonist, ICI 182,780. Functional c-Myc protein was identified by both Western immunoblotting and by its ability to transactivate a chimeric plasmid consisting of E-box sequences upstream of the luciferase reporter gene. One MCF-7 clone, 35im, was chosen for further characterization. C-myc induction by doxycycline was rapid and dose dependent; c-myc mRNA appeared as early as 30 min after doxycycline addition and stimulation of c-myc expression required as little as 50 ng/ml doxycycline, with c-myc mRNA levels reaching a plateau at 2.5 microg/ml doxycycline. ICI 182,780 or doxycycline (a tetracycline analog) treatment did not alter the mRNA levels of Max, the c-myc binding partner. As in wildtype MCF-7 cells, the growth of clone 35im was inhibited by 1 microM or less of ICI 182,780 and stimulated by 10 nM to 1 microM 17beta-estradiol. When maintained in a complete medium containing 5% normal fetal bovine serum (FBS) and ICI 182,780, doxycycline induced cell growth by 400% in an 8-day assay. A similar level of growth was achieved with doxycycline treatment in cells that were arrested by the use of charcoal-stripped FBS. Doxycycline had no effect on the growth of a control MCF-7 clone (18c). Apoptosis, assessed by caspase-dependent cleavage of poly(ADP-ribose) polymerase, was unchanged in clone 35im cells after treatments with doxycycline or ICI 182,780. The present study demonstrates that c-myc alone is sufficient to confer antiestrogen resistance in human breast cancer. Our novel c-myc-inducible MCF-7 cell model offers a unique opportunity to study the diverse actions of the c-myc proto-oncogene in human breast cancer.

Two proprotein convertase cDNAs, PC1 and furin, were stably transfected into the human breast cancer cell line MCF-7. The PC1 or furin over-expressing cells possessed an altered morphology. When grown in vitro in a serum-free medium, the population doubling time of the convertase-transfected cells was twice that of wild-type (WT) cells. High concentrations of estradiol stimulated the growth of all three cell types to a similar extent; however, at low concentrations of estradiol, the convertase-transfected cells grew more slowly than WT cells. In athymic nude mice implanted with 5 mg estradiol pellets, the growth of tumors of convertase-transfected MCF-7 cells was stimulated to a degree similar to that of WT MCF-7 tumors. However, in mice implanted with lower-dose (1.5 mg) estradiol pellets, the tumors of PC1- or furin-transfected MCF-7 cells grew approximately five times slower than those of WT MCF-7 cells. In mice implanted with tamoxifen pellets, tumors of PC1- or furin-transfected MCF-7 cells regressed approximately five times slower than the WT tumors. This study shows that the over-expression of proprotein convertases confers a greater estrogen dependency and anti-estrogen resistance on human breast cancer cells.

Rat placental lactogen-I (rPL-I), the first prolactin-like hormone expressed in the placenta during pregnancy in the rat, is known to influence maternal functions. In the present study, we have investigated the effects of rPL-I on the growth and development of cultured whole rat embryos. Rat embryos, with or without ectoplacental cone (EPC) attached, were explanted at day 9 of gestation. After 48 h of culture, the embryos, enclosed by the yolk sacs, were assessed by the presence of visible heart contractions ('heart beats'), crown-rump length (CRL) and yolk sac diameter (YSD). When intact embryos with EPC were cultured, the concentrations of rPL-I and rPL-II (products of EPC) in the medium were 850+/-841 and 92+/-181 ng/ml respectively (means+/-s.e.m.). In embryo cultures with the EPC removed, rPL-I levels decreased to

Adult virgin female rats display maternal behavior when continuously exposed to foster young for 5-6 days. Central infusions of PRL or placental lactogens (PLs) together with systemic treatment of progesterone (P) and estradiol (E2) stimulate maternal behavior in 1-2 days. In the present set of studies, it was asked whether the actions of lactogenic hormones are dependent upon both E2 and P and specific to lactogenic molecules. Moreover, we wanted to know whether central infusions of rat (r) PRL and PLs were equally effective in inducing maternal behavior. In the first study, adult virgin rats were ovariectomized (ovx) and stereotaxically fitted with bilateral cannulas directed at the medial preoptic area (MPOA). Rats were then assigned to one of four groups: P plus E2, blank (B) plus E2, P plus B, and B plus B. P-filled or B capsules were implanted sc on treatment day 1 and removed on day 11, whereas E2 or B capsules were implanted on day 11. All groups were infused with rPRL (40 ng/side) five times from days 11-13 and injected with bromocriptine (CB-154) sc (days 11-17) to suppress endogenous PRL release. Behavioral testing was conducted daily from days 12-17. It was found that exposure to both P and E2 was necessary to induce a fast onset of maternal behavior in PRL-infused females; priming with P or E2 alone in PRL-treated rats failed to stimulate a fast onset of behavior relative to that in nonsteroid-treated controls. In the second experiment to determine the biochemical specificity of PRL's action, adult nulliparous rats were ovx, implanted with bilateral cannulas directed at the MPOA, treated with both P and E2, injected with CB-154, and infused centrally (five times) with 40 ng (per side) of bovine GH, ovine LH, or vehicle. Central infusions of either bovine GH or ovine LH failed to stimulate maternal behavior, suggesting that the stimulatory actions of PRL are related to its lactogenic properties. In the final study, rats were ovx, fitted with bilateral cannulas directed at the MPOA; treated with P, E2, and CB-154; and given a single set of bilateral infusions of rPL-I or rPRL (40 ng/side.infusion) on day 11, three sets of infusions of rPL-I or rPRL (days 11 and 12), or vehicle infusions. Rats given three infusions of rPL-I and rPRL responded faster than controls, although the effect was not as robust as that in animals given five infusions in the initial study. rPL-I and rPRL groups did not differ from one another. Together these studies indicate that 1) both P and E2 are required for lactogenic stimulation of maternal behavior; 2) the stimulatory actions of PRL and rPLs on maternal behavior are related to their lactogenic properties; 3) extended treatment of females with lactogenic hormones is more effective in stimulating the onset of maternal behavior; and 4) the neural potencies of rPRL and rPL-I are similar. These findings provide support for the idea that the induction of maternal behavior is stimulated by the central actions of lactogenic hormones.

Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.

Peptide 23 is a newly identified protein secreted by rat pituitary cells in primary culture. Although the secretion of this protein is stimulated by GH-releasing hormone and inhibited by somatostatin, the N-terminal amino acid sequence of peptide 23 shows no homology to rat GH. Using the polymerase chain reaction technique, we cloned and sequenced the peptide 23 complementary DNA (cDNA). By means of the mixed oligonucleotide-primed amplification of cDNA technique, primers corresponding to the NH2-amino acid sequence of peptide 23 were used to amplify, clone, and sequence a 74-basepair cDNA of peptide 23. This polymerase chain reaction product was then used as a primer to amplify the complete peptide 23 cDNA by means of the rapid amplification of cDNA ends procedure. The cDNA of peptide 23 obtained by the rapid amplification of cDNA ends procedure contained 777 nucleotides and encoded a 175-amino acid protein with a 26-amino acid putative signal peptide. The calculated mol wt of the mature protein (16,613 daltons) was in good agreement with that estimated by polyacrylamide gel electrophoresis (16 kilodaltons). Northern blot analysis revealed a major messenger RNA species of about 0.9 kilobase and a minor species of about 1.7 kilobases in cultured rat anterior pituitary cells. In rats, peptide 23 was most abundant in the pancreas and gastrointestinal tract. A GenBank sequence search revealed complete sequence identity between peptide 23 cDNA and pancreatitis-associated protein cDNA, an approximately 73% homology with human hepatocellular carcinoma cDNA from human hepatocellular carcinoma, 64% homology with bovine pancreatic thread protein cDNA, and 55% homology with rat and human reg cDNAs, which have been reported to be expressed in regenerating pancreatic islets. Therefore, peptide 23 is identical to pancreatitis-associated protein and a member of the C-type lectin supergene family.

The human PRL-inducible protein (PIP)/gross cystic disease fluid protein-15 is expressed in pathological conditions of the mammary gland and in several exocrine tissues, such as the lacrimal, salivary, and sweat glands. In human breast cancer cells, the expression of PIP/gross cystic disease fluid protein-15 is stimulated by androgen and PRL, and inhibited by estrogen. However, it is not known whether the expression of PIP in other tissues is under similar hormonal regulation. In the present study we employed reverse transcriptase-polymerase chain reaction followed by rapid amplification of complementary DNA (cDNA) ends to amplify the PIP cDNA homolog, the submaxillary gland protein (SMGP) in the mouse. The mouse PIP/SMGP cDNA encodes a putative secreted peptide of 144 amino acids with a 51% identity with human PIP. Using the mouse PIP/SMGP cDNA as a probe, we examined the tissue- and cell-specific expression of PIP/SMGP messenger RNA by in situ hybridization and Northern blot analysis of mouse and rat tissues. Hormonal regulation was also studied in the rat. PIP/SMGP messenger RNA expression was only detected in the lacrimal and submaxillary glands of the rodents. In the rat submaxillary gland, PIP/SMGP gene expression was confined to the acinar cells. In the male rat lacrimal gland, castration resulted in an increase in expression, and in both male and female rats, androgen replacement abolished PIP/SMGP gene expression. This pattern of regulation was not observed in the submaxillary gland and was actually reversed in human breast cancer cells. PRL had no effect on the regulation of PIP/SMGP in either salivary or lacrimal glands. Our study indicates that tissue-specific factors are important in determining the hormone responsiveness of the PIP/SMGP gene. Regulation of the PIP/SMGP gene in vivo may provide a useful model system to study the mechanism of down-regulation of expression by androgen in a tissue-specific manner.

Regional and temporal patterns of the expression of basic fibroblast growth factor (bFGF), and two of its high affinity receptors (FGFR1 and FGFR2), were examined in the male rat brain during early postnatal development; the reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain mRNA measurements which were expressed relative to mRNA for GAPDH as a constant. In the rat cerebrum, the mRNAs for bFGF and for FGFR2 were relatively low in amount within the first postnatal week, but by 28 days, they were as high as in the 1-year-old rat cerebrum. In contrast, the expression of FGFR1 was biphasic: mRNA levels were higher at postnatal days 1 and 28 than at day 21. Quantitation of mRNA from microdissected regions of 28-day-old rat brain revealed that the expression of bFGF and of FGFR2 showed a marked variation between regions but the expression of FGFR1 appeared less variable between the regions that were analyzed. For all three genes the hippocampus appeared to have high relative amounts of mRNA. The temporal patterns of expression of bFGF, FGFR1 and FGFR2 also differed with brain region during early postnatal development. In the occipital cortex and inferior colliculus, the mRNAs for bFGF and FGFR2 both increased in amount during the first month, unlike that for FGFR1. However, in the cerebellum, the highest expression of bFGF and FGFR1 mRNAs occurred at postnatal day 1; FGFR2 expression apparently showed less change with age. The temporal changes in bFGF, FGFR1 and FGFR2 expression in different brain regions during early postnatal development suggest that receptor regulation may permit different physiological effects of bFGF according to brain region and developmental age.

Rat placental lactogen-I (rPL-I), a member of the PRL/GH gene family, is produced by giant cells in the early trophoblast. The small amount of early placental tissue has limited the purification of rPL-I from this source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (CHO) cells and in these studies have compared the recombinant protein with the native rPL-I. Using an affinity column composed of monoclonal antibody to rPL-I coupled to Sepharose 4B, we have purified rPL-I from four sources: 1) recombinant rPL-I produced and secreted in rPL-I-transfected CHO cells, 2) nonglycosylated recombinant PL-I produced by adding tunicamycin (10 microM/ml medium) to rPL-I-transfected CHO cells, 3) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) serum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed nine subforms with increasing mol wt [approximately 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (approximately 23 kDa) were evident in the nonglycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (approximately 37 kDa) and more acidic pI. When analyzed by the Nb2 lymphoma cell bioassay, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipotent with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as active as ovine PRL in the Nb2 assay. A RIA was established for rPL-I. The variant rPL-Iv, displayed nonparallel displacement of [125I]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum levels of rPL-I by RIA after the injection of recombinant-rPL-I into adult female Sprague-Dawley rats revealed a half-life of 9 min for the recombinant protein compared to 7.8 min for the choriocarcinoma-derived hormone. In conclusion, we have shown that although CHO cells will glycosylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to those of the native, placenta cell-derived hormone.

The prolactin-inducible protein (PIP)/gross cystic disease fluid protein (GCDFP-15) was isolated by us (1, 2) as a glycoprotein secreted by the T47D human breast cancer cell line in response to lactogenic peptide hormones (human prolactin and human growth hormone) and androgen, and independently by Haagensen and co-workers (3) as a protein found in abundance in the fluid of gross cystic disease of the breast. Haagensen and colleagues (3, 4) have measured the clinical profiles of this protein by radioimmunoassay and immunohistochemistry in the blood and tissues of patients having abnormal breast pathology. Based on immunohistochemical data, these investigators concluded that GCDFP-15 is the product of apocrine glands, and that the production of this protein by gross cystic disease and malignant breast tumors is the result of apocrine differentiation. Beside Haagensen’s group, many clinical laboratories are using the GCDFP-15/PIP as a marker for abnormal breast functions and have observed, for instance, that the contents of GCDFP-15 in gross cystic disease fluid is correlated with that of androgens, prolactin and epidermal growth factor (5). PIP/GCDFP-15 protein is abundant in gross cystic disease and malignant breast carcinomas (2 –4, 6). However, little, if any, of this protein is found in normal human mammary tissue (4). Nevertheless, the protein has been detected by immunocytochemistry in normal apocrine tissues such as the serous cells of the salivary gland, Moll’s glands of eyelids, ceruminous glands of the ear canal, sweat glands, perineum and some bronchial glands (3,4). We have also employed Western immunoblot to demonstrate the presence of PIP/GCDFP-15 protein in a variety of physiological fluids such as blood, saliva, tear, sweat, amniotic fluid and breast milk (7). While the studies on PIP/GCDFP-15 protein distribution and tissue contents are informative, they have provided little insight into the cellular origin (site of synthesis) and functional significance of this protein. Also, the genetic mechanisms governing the expression of the PIP/GCDFP-15 gene (PIP*) in normal and neoplastic tissues remain to be explored. For these reasons, we have directed our research into the molecular genetics of PIP gene. [*Since PIP is the official name used in the Genome Data Base and in both Genbank Data Bank and the EMBL Data base, the GCDFP-15 designation will not be used. Throughout this article, PIP is used to denote the gene.

This study compares the molecular characteristics of the insulin-like growth factor II (IGF-II) peptides synthesized and secreted by explants of neonatal brain and adult pituitary of rat to those produced by the Buffalo rat liver cell line (BRL-3A). Metabolic labeling, followed by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the rat brain and liver cells synthesized and secreted the following immunoreactive IGF-II peptides: 19, 11, 10, and 8.7 kilodaltons (kd), whereas the rat pituitary secreted the 10 and 8.7 kd peptides. However, the brain and pituitary explants failed to secrete the mature 7.5 kd IGF-II peptide which was a major species secreted by the liver cells. In the brain and pituitary, the predominant form of IGF-II peptide secreted was the 8.7 kd. This result suggests that 1) different mechanisms of processing of the IGF-II precursor and/or the preferential translation of different messenger RNA (mRNA) species may exist in different cell types, and 2) the 8.7 kd IGF-II peptide may be the biologically relevant molecule in the central nervous system of the rat.

The specific binding of 125I-labeled insulin, hGH and oPRL was surveyed in crude membrane preparations of various tissues of the monkey, rat, guinea pig, rabbit, sheep, pigeon, and frog. Binding varied greatly with the species examined. The highest binding of 125I-insulin was seen in guinea pig tissues, especially kidney, fetal placenta and liver. Significant binding of 125I-insulin was seen in many but not all known target tissues, and in other tissues as well (viz. placental, adrenal, brain). The binding of 125I-hGH and 125I-oPRL paralleled each other, except in rabbit liver, and was highest in rabbit liver and adrenal, female rat liver, frog kidney, and several sheep tissues. There was no relation between phylogenetic proximity to man and the level of specific binding of 125I-hGH observed. The lowest binding of 125I-hGH was in male rat liver and various tissues of the mpnkey and guinea pig, a high proportion of which showed significant binding of 125I-insulin. 125I-insulin was displaced from the membra...

Normal growth, differentiation, and function of the mammary gland are under the influence of hormones-insulin, estrogen, progesterone, thyroid hormone as well as growth hormone, prolactin (PRL), and placental lacto· gens. While some of these hormones may affect primarily growth and development of the mammary gland, others, such as PRL, are of major importance in stimulating mammary gland function. A comprehensive treatment of endocrine influences on the mammary gland may be found in (12). Here we highlight progress in understanding the mechanism of action of PRL on mammary gland function. Since large gaps in this understanding remain, we indicate areas where research may be fruitful. The role played by PRL on mammary gland function varies from one species to another. For example, lactational changes in the mammary gland can be induced in rabbits by administering PRL alone, while in rats other hormones are required. In the cow the peripartum increase in serum PRL is crucial for successful lactation; but if PRL secretion is inhibited at later periods postpartum, milk secretion is hardly affected. In women, however, inhibition of PRL secretion by similar pharmacological means at any time postpartum causes prompt cessation of lactation. Generalizations and ex· trapolations from one species to another must thus be made with consider· able caution. The possible role of PRL in the etiology of breast cancer, which is not covered here, has been reviewed extensively (29, 33, 7 1).

PRL and other lactogenic hormones are potent mitogens in a lymphoma cell line derived from a lymph node of an estrogenized Noble (Nb) rat. The present study demonstrates that these cells (designated Nb2 node) possess receptors that bind only lactogenic hormones. There are approximately 12,000 receptor sites per cell. The kinetics of binding of [125I]iodo-PRL exhibited by Nb2 lymphoma cells is unusual in that PRL binding follows a biphasic pattern. Binding of [125I]iodo-PRL reaches a maximum in 1 h at 37 C, followed by a rapid decline. This pattern was not observed if binding was carried out in the presence of chloroquine, a lysosomotropic agent, or if cell homogenate was used for binding. We also examined the relationship between receptor occupancy and the magnitude of PRL response in these cells. Maximal growth stimulation by PRL occurs when only 35% of the maximal binding of PRL is achieved, suggesting that a majority of the PRL binding sites may be spare receptors. This study also revealed that the dissociation constant (Kd) of the PRL receptors in Nb2 cells is 75 pM, approximately 20-fold higher than that of the receptors in other cell types. PRL at 6 pM produces a half-maximal growth response in the Nb2 cells. Antibodies against the PRL receptors are able to abolish the PRL-induced proliferation of Nb2 cells. In the absence of PRL, these antibodies alone can induce proliferation of Nb2 cells, mimicking the action of PRL. Divalent (Fab)2 fragments, but not monovalent Fab, were also effective. These observations suggest that antibodies to the receptor, or the hormone itself, initiate a biological response possibly by cross-linking PRL receptors on the cell membrane, and that the entry of the PRL molecule, or fragments of it, into the intracellular compartment is not necessary for the biological action of PRL.

Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of (125)I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37 degrees C. (125)I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of (125)I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of (125)I-labelled prolactin. The binding of (125)I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver-Burk analyses suggest that (125)I-labelled prolactin has a high affinity for its receptor. Binding of (125)I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.

Chronic excess of glucocorticoids results in Cushing's syndrome in humans. A common cause of excess cortisol secretion is the presence of an adrenocorticotropin secreting pituitary tumor which stimulates the adrenal cortex to produce excess glucocorticoids. ACTH-secreting AtT-20 mouse pituitary cells transplanted subcutaneously in oestrogenized athymic nude mice form tumors rapidly. Six weeks after receiving the tumor transplants, the mice weighed 45% more than normal mice due to the increase in body fat. The tumor-bearing mice exhibit the familial "buffalo hump" appeaance due to the abnormal distribution of body fat. The adrenal glands of the tumor-bearing animals are enlarged due to hypertrophy of the zona fasciculata. The foamy looking fasciculata cells in normal mice were converted to dense, eosinophilic cells in the tumor-bearing mice. Transplantation of normal pituitary glands to athymic nude mice with or without oestrogen treatment did not produce these morphological changes. The experimental model described here may be useful for future studies of Cushing's syndrome.

Extracts of frozen human pituitaries were mitogenic in a fetal rabbit chondrocyte bioassay. In the presence of 10% fetal bovine serum, a 10-fold increase in chondrocyte cell number was observed upon addition of the pituitary factor to the culture medium. After gel filtration, the chondrocyte growth factor eluted with proteins of approximately 40,000 molecular weight. These fractions were pooled and purified further upon ion exchange chromatography using DEAE-cellulose. The most active fraction stimulated cell proliferation in a dose-dependent manner down to 10 ng/ml. The chondrocyte growth factor was trypsin- and heat-sensitive (100 degrees C, 10-15 min). Its isoelectric point (pI 7.9) was different from bovine brain and pituitary fibroblast growth factor (pI 4.8-5.8 and pI 9.5, respectively. Unlike the somatomedins and epidermal growth factor, it was acid-labile. Preparations of human growth hormone, prolactin, luteinizing hormone, and follicle-stimulating hormone, prolactin, luteinizing hormone, and follicle-stimulating hormone, as well as vasopressin and oxytocin, were inactive in the bioassay, demonstrating that the human pituitary contains a chondrocyte growth factor which appears to be distinct from these anterior and posterior pituitary hormones.

Two human breast cancer cell lines (T-47D and MCF-7) and one cell line derived from normal human milk (HBL-100) not only specifically bound but also degraded prolactin. Quantitative differences in the ability to bind and degrade prolactin among the cell lines exist, although there was a good correlation between the number of prolactin receptor sites and prolactin degradative activity. Iodo-prolactin as well as native prolactin were degraded. The prolactin molecule was processed to yield at least three small molecular weight peptides which were released into the incubation medium. These peptides neither bound to fresh receptors nor to anti-prolactin antibodies. The protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone, lysosomotropic agents such as chloroquine and ammonium chloride, and metabolic inhibitors 2,4-dinitrophenol and sodium azide, all abolished prolactin degradation by the breast cancer cells. When prolactin degradation was inhibited, specific binding and the subsequent release of intact 125I-prolactin was still observable, suggesting that hormonal degradation was not a prerequisite to dissociation of prolactin. However, prolactin degradation did account for the accelerated rate of dissociation of prolactin. Studies utilizing inhibitors suggest that the receptor-bound 125I-prolactin was degraded by an energy-dependent internalization process such as pinocytosis; lysosomal enzymes are probably involved in the degradation of prolactin by human breast cancer cells.

Antisera generated in guinea pigs against partially purified PRL receptors derived from rabbit mammary glands were tested for their effect on PRL binding and the biological effects of PRL in vitro and in vivo. Several dilutions of either normal guinea pig serum or anti-PRL receptor serum were incubated in vitro with homogenates of rat ovaries. Although specific binding of PRL was inhibited as much as 90% by the anti-serum, normal guinea pig serum had little effect. There was no inhibition of binding of LH and FSH to ovaries by these antisera. When these antisera were administered to normal cycling rats, the most pronounced effect was an increase in the number of corpora lutea, presumably due to the prevention of the luteolytic effect of PRL. When one of these antisera was given to adult rats immediately after parturition, weight gain of their pups decreased significantly, possibly reflecting a decrease of milk yield. Thus, passive immunization of animals with anti-PRL receptor sera modifies the actions of PRL on some of its target tissues.

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.

A placental lactogen or chorionic mammotropin (rCM) has been identified in the serum of pregnant rats by radioreceptor assay (RRA). Two peaks of activity were found, the first between days 11-13 and the second between days 17-21 of pregnancy. Gel filtration of day 12 serum or placental extracts revealed two peaks of lactogenic activity, the first eluting ahead of and the second appearing immediately after 125I-hPRL (human prolactin). On the other hand, when serum or placental extracts from day 17-21 pregnant rats were fractionated in the same manner, only a single peak of lactogenic activity was eluted after 125I-hPROL. The placental concentration of rCM increased from 11 mug/g wet weight at day 12 to 74 mug/g at days 14-15, after which the concentration declined to 29 mug/g on day 21. Electrophoresis of a fraction from G-100 column of a day 12 0r day 20 placental extract or serum with a V-e/V-o ratio of 2.08 revealed a single peak of lactogenic activity by RRA with an Rf equal to 0.43. Large MW rCM (V-e/V-o ratio of 1.35) from serum had an Rf equal to 0.52 whereas the large MW species from placental extracts had a mobility similar to that of small MW fraction. The half-time disappearance rate of serum rCM on day 12 is 19.5 min compared to 1.2 min on days 17-21 of pregnancy.

The replication of Nb 2 Node rat lymphoma cells in suspension culture is specifically stimulated by lactogenic hormones. Human (hPRL), ovine, bovine, and rat PRLs stimulated replication in a dose-dependent manner in the concentration range of 10 pg/ml to 1 ng/ml. Human, ovine, and bovine placental lactogens were similarly active. In addition, cell replication was stimulated by human GH (hGH), which is known to have lactogenic activity. Other hormones and growth factors examined were inactive. The growth stimulatory effects of hPRL and hGH were completely inhibited when excess anti-hPRL and anti-hGH, respectively, were added to the medium. A bioassay based on the response of the Nb 2 Node lymphoma cells to lactogenic hormones has been developed. Human serum stimulated cell replication. The effect was completely abolished if excess antibodies to both hPRL and hGH were present. The stimulation obtained with a number of human serum samples correlated very well with the sum of the hPRL and hGH concentrations in the sera, as determined by RIA (r = 0.95; P < 0.001). The concentrations of either hPRL or hGH in human serum could be individually determined by specifically blocking the growth stimulatory effect of the other hormone by adding excess anti-hGH or anti-hPRl. The sensitivity of this bioassay for PRL and hGH in serum exceeds that of RIAs.

The pituitary gland plays a central role in the regulation of mammary gland development. It secretes a variety of hormones that directly or indirectly influence the mammary gland. The best known mammogenic hormone of the pituitary gland is prolactin which directly promotes development and differentiated function (e.g., milk synthesis) of the normal mammary gland. Also, the pituitary gland can indirectly influence mammary gland development via the production of gonadotropins which stimulate the ovary to produce steroids such as estrogen and progesterone; these ovarian steroids are indispensible in mammary gland development. Furthermore, pituitary hormones regulate the production of thyroid hormones and adrenal glucocorticoids which are important in the performance of the mammary gland.

Incubation of T-47D human breast cancer cells with the synthetic progestin, medroxyprogesterone acetate (MPA), resulted in a time and dose-dependent increase in epidermal growth factor-receptor (EGF-R) mRNA. Concentrations of MPA as low as 1 nM resulted in a greater than five fold increase in EGF-R mRNA. A significant increase (2-3 fold) in EGF-R mRNA was apparent 12 hr after exposure to MPA and a further increase was seen 12-48 hr after addition of MPA to the cultures. From these studies it is concluded that the increased EGF binding in progestin-treated T-47D cells results at least in part from increased EGF-R gene expression. We believe this is the first report of a steroid hormone modulating expression of this growth factor receptor gene.

Although polyamines are important in regulating proliferation of mammalian cells, their role in hormone induction of cell growth has not been delineated. In the estradiol-responsive human breast cancer cell line, T-47D clone 11, estradiol (10 −10 M) was able to stimulate cell proliferation and the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the biosynthesis of polyamines. α-Difluoromethylornithine (DFMO), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Exogenous addition of putrescine, the natural product of ODC, rescued the inhibitory effect of DFMO. In addition, DFMO abolished the estradiol-induced growth of several other estrogen-responsive human breast cancer cell lines but did not affect the growth of hormoneindependent cell lines. Further, a serum factor was found to be required for estradiol to exert its effect. To gain insight into the nature of this and possibly other extrinsic factors involved, the effect of estradiol on the proliferation of T-47D cells transplanted into athymic nude mouse was evaluated. In this in vivo system, estradiol alone produced only moderate growth of the human breast tumor. The simultaneous transplantation of a prolactin (PRL)- and growth hormone (GH)-secreting rat pituitary tumor or normal rat pituitary glands at a different site dramatically potentiated the effect of estradiol on the growth of the breast tumor xenograft. Purified PRL or GH were without effect, indicating that the active pituitary factor is neither PRL nor GH. Further, conditioned medium from rat pituitary tumor cells potentiated the mitogenic effect of estradiol on T-47D and several other estrogen receptor-positive human breast cancer cell lines in vitro under serum-free condition. In conclusion, we have identified both intrinsic (polyamines) and extrinsic (pituitary/serum) factors that are importance for estrogen to exert its mitogenic action. The next goal will be to elucidate the mechanisms of action of these molecules in the modulation of estrogen action.

The diverse actions of prolactin have been reviewed by Bern and Nicoll (1968) and Horrobin (1978). Besides its well-known mammogenic role, prolactin is important in regulating the functions of many organs in many species. In the mammals, the mammary gland is undoubtedly one of the principal target organs of prolactin. In this tissue, prolactin is a lactogenic and a mitogenic hormone (Cowie and Tindal, 1971; Ceriani, 1974). The biological actions of prolactin in other organs such as liver, kidney and the reproductive organs are less well defined. Nevertheless, prolactin has been reported to stimulate somatomedin production (Francis and Hill, 1970) and ornithine decarboxylase activity in rat liver (Richards, 1975) and to affect kidney’s ability to retain Na+ ions (Nicoll, 1975). The luteotropic and luteolytic action in the ovary is well documented (Nicoll, 1975; Wuttke and Meites, 1971). Prolactin also stimulates prostatic growth (Moger and Geschwind, 1972) and uptake of testosterone by the prostate (Farnsworth, 1977).

A guinea pig antiserum to prolactin receptors selectively inhibited the binding of [125I] prolactin to its membrane receptors as well as prolactin-mediated incorporation of [3H] leucine into casein and transport of [14C] aminoisobutyric acid, but was without effect on the binding of [125I] insulin and insulin-mediated events in explants of rabbit mammary glands maintained in culture. These findings provide direct evidence for an obligatory functional role of a membrane receptor in mediating the action of a polypeptide hormone.

Prolactin and estrogen play key roles in influencing the growth behaviour of experimental breast tumors of rodents (7), (11), (17), (23), (43). However, the involvements of these two hormones, especially that of prolactin, in the tumorigenesis of the human breast, is still unclear (20). Whether or not trophic factors other than these two hormones are also important in growth regulation of breast cancer remains to be elucidated. Clinical investigations have indicated that pituitary hormones are important in the etiology of human breast cancer although the identity of the pituitary factors have not been elucidated. In view of the important role that prolactin plays in the tumorigenesis of rodent breast cancer, the notion that this hormone may be involved in the etiology of human breast cancer has been formulated (23). Evidence, mainly derived from clinical observations, has so far failed to establish an importance of prolactin in the breast disease of man (20). However, studies with human breast tumor biopsies maintained in organ culture using a pentose pathway histochemical effect suggest that some tumors show some form of prolactin dependency (26). Further, it has been reported that human breast biopsies maintained in organ culture (42) or transplanted in athymic nude mice (16) respond to prolactin and placental lactogen with an increase in DNA synthesis. One of the recent advances in human breast cancer research has been the development of a considerable number of cell lines derived from breast cancer specimens (4). The use of these human breast cancer cell lines maintained in tissue culture has provided new insight into the hormonal control of breast cancer in man. The proliferation of some human breast tumor cell lines in culture are affected by hormones such as insulin (1), (22) and epidermal growth factor (4).

Mitogenic stimulation of Nb2 lymphoma cells by lactogenic hormones (prolactin, human growth hormone) caused a dramatic early increase in ornithine decarboxylase (ODC) activity that achieved a maximal level by 6–8 h. A marked increase in ODC activity was also generated when cells which had reached a growth plateau were transferred to fresh medium that did not stimulate growth. Furthermore, low concentrations of human growth hormone (20 pg/mL) elicited a proliferative response, but did not cause a detectable early increase in ODC activity. The early peak of ODC activity thus appeared not to be directly involved in mediating lactogen-stimulated growth nor was it required to support the mitogenic response. However, prolonged suppression of ODC activity by DL-α-difluoromethylornithine (DFMO) (200 μM) attenuated the growth of Nb2 cells (50–60% inhibition), indicating that normal cell growth was dependent on ODC and polyamine biosynthesis. Under these conditions, putrescine, the enzyme product, or the polyamines spermidine and spermine restored normal cell growth when added at a concentration of 1 μM or greater. Nb2-SP cells, variants which proliferate in the absence of prolactin, were about two times more resistant to the growth suppressive effects of DFMO than prolactin-responsive Nb2 cells.

The developmental pattern of prolactin receptors was examined in intact interstitial cells isolated from testes of 19-day old foetuses (F19), 1 to 45-day immature (N1 to N45) and adult animals. The prolactin binding activity in F19 interstitial cells is low, being 27% of that found in cells of the adult. There is an abrupt 60% increase in prolactin binding immediately after birth (N1), followed by a slow, but gradual, increase for the next 34 days. A second rapid increase in prolactin binding occurs between days 34 and adulthood which follows an increment in serum prolactin concentrations after day 24. Equilibrium analysis revealed that the receptors in interstitial cells from N1, N34 and adults have similar affinity (Kd= 2.7 to 3.3 times 10-10M) for prolactin. However, cells from adults contain twice as many prolactin receptors per cell as that of immature animals. This increase in prolactin receptors may play a role in the modulation of interstitial (Leydig) cell function during sexual maturation.

Publisher Summary In birds, prolactin promotes nesting behavior and the development of the crop sac. In mammals, the most widely studied effects of prolactin are those related to the reproductive organs. In the male, prolactin affects the prostate and the testis; in the female, prolactin is both luteotropic and luteolytic for the ovary. The best known effect of prolactin in mammals is on the mammary gland: prolactin promotes both growth and differentiation in this organ and is the principal hormone that influences the production of milk. Apart from its ability to stimulate lipid synthesis and alter growth behavior, prolactin also stimulates the synthesis of unique proteins in the T-47D HBC cells. This chapter discusses an experiment in which [ 35 S]methionine-labeled proteins secreted by T-47D cells under the influence of various hormone combinations were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. The chapter discusses relationship between different forms of prolactin-inducible proteins (PIPs), and PIP in tumors and serum of breast cancer patients.

A radioreceptor assay with a sensitivity of 5 nanograms per milliliter has been developed for mammalian and avian pituitary prolactin, placental lactogenic hormones, and humnan growth hormone, using a membrane receptor preparation isolated from rabbit mammary glands. Prolactin preparations inhibited the binding of [(125)I]prolactin to receptors in direct proportion to the biological potency of these preparations. Thus, the radioreceptor assay provides a convenient and simnple assay for hormones which have lactogenic activity.

Excerpt Dr. Leo G. Rigler: This case conference will concern itself with one of the most fascinating chapters in medical history, the development of the use of artificial contrast in roentgen diagn...