Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion.     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Prolactin sensitivity of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol

We examined the responsiveness to prolactin and growth hormone of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol (DES) and from control mice. The mammary epithelial cells were cultured inside collagen gels with serum-free medium containing insulin, epidermal growth factor, and linoleic acid. This produces prolactin-sensitive cells with low levels of casein production, as measured in cellular homogenates with a specific enzyme-linked immunosorbent assay for alpha-casein. The collagen gels containing these cells were then released and the medium supplements changed to insulin, linoleic acid, and prolactin at concentrations from 10 to 1000 ng/ml and growth hormone at 0, 10, or 100 ng/ml. This second phase of the culture, the differentiation phase, allows the cells to accumulate casein if they have this capacity. When cultured with prolactin only (no growth hormone), the cells from DES-exposed mice consistently accumulated 50-100% of the casein content of normal cells, but never more. Growth hormone, when added to prolactin-containing medium, increased casein accumulation above the levels seen with prolactin alone. Combinations of prolactin and growth hormone enhanced the difference between casein accumulation in DES-exposed and control cells, and DES-exposed cells were much less responsive to growth hormone. In our studies, the isolated mammary epithelial cells of estrogen-exposed mice are not more sensitive to prolactin than cells from normal animals as previous reports reports had suggested, but rather are generally less sensitive to hormonal stimulants.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Two monoclonal antibodies against prolactin-receptor are internalized in epithelial mammary cells without mimetic prolactin effect on casein secretion*

Summary— Prolactin exerts an early stimulatory effect on casein secretion which was qualified as a secretagogue effect. After binding to its receptor, the hormone transits intracellularly through the mammary epithelial cell. When this transit is slowed down the secretagogue effect does not occur. Different monoclonal antibodies which bind to the rabbit prolactin receptor have been previously developed. One of them (A917) mimics prolactin effect on casein gene expression. Another (M110) blocks this prolactin effect. In order to study the respective role of the hormone and its receptor, we have examined the binding of the two monoclonal antibodies (M110 and A917), labeled with biotin or colloidal gold, to the receptor of lactating rabbit mammary epithelial cells in incubation. Subsequently, the intracellular movement of these antibodies and the secretory response have been measured. Irrespective of the labeling (biotin or colloidal gold) or the preparation of tissues (fragments or enzymatically dissociated cells), Ml 10 and A917 bound to the basal membrane of mammary epithelial cells. However, only M110 bound to apical membrane of dissociated cell when this membrane was in direct contact with the incubation medium, showing that the two antibodies discriminate the receptor located on the apical membrane. Following internalization, each antibody was carried via a peculiar pathway. M110 remained associated with the cells during a 1-h incubation, mainly in endosomes, multivesicular bodies and lysosomes like vesicles. In contrast, A917 was very quickly detectable in endosomes, multivesicular bodies and vesicles of the Golgi region and was carried throughout the cell to the lumen of the acini. M110 and A917 were extremely rare in secretory vesicles containing casein micelles. When mammary fragments pulse labeled for 3 mm with ³H-leucine were chased for 60 mm in the presence of prolactin, M110 or A917, only prolactin was able to increase casein secretion. These results show that two monoclonal antibodies against prolactin receptor are internalized after binding to the surface of the mammary cell. They are carried intracellularly by different routes. Internalization of these antibodies is not sufficient to mimic the secretagogue effect of prolactin.     

Lactogenic response of mouse mammary explants from different days of pregnancy to placental lactogen and pituitary prolactin

Summary The lactogenic response of mouse mammary gland explants to human placental lactogen (hPL) and ovine pituitary prolactin (oPRL) was examined on days 10 to 18 of pregnancy by measuring³H-amino acid incorporation into calcium-rennin precipitable casein. To determine the lactogenic response of the explants, the mean slopes of dose-response curves were calculated for each hormone treatment. Slope means of dose-response curves for oPRL and hPL did not differ from each other on any day of pregnancy examined. A triphasic pattern of response was suggested when slope means of dose-response curves for both hormones were plotted as a function of day of gestation. Peak responses were observed on days 10, 13 and 17–18. Combinations of oPRL and hPL, in ratios of oPRL:hPL=2∶1 and oPRL:hPL=1∶2, also produced a triphasic pattern of sensitivity very similar to that produced by either hormone alone. These results suggest that mouse mammary explants may be more sensitive to oPRL and hPL on days 10, 13 and 17–18 of pregnancy. This work was supported by grants from the National Science Foundation (76-01928) and the National Institutes of Health (5 S06RR08132-0251) to F. Talamantes.     

Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland

Summary Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 μg/ml), aldosterone (1 μg/ml), growth hormone (5 μg/ml), cortisol (5 μg/ml), and prolactin (80 ng/ml, present as a contaminant in 5 μg/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNA_csn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo. This work was supported by Department of Health, Education and Welfare Grants CA11058 and CA25304 from the National Cancer Institute.     

Placental growth hormone and lactogen production by perifused ovine placental explants: regulation by growth hormone-releasing hormone and glucose

The factors controlling normal placental development are poorly understood. We have previously reported the presence of ovine placental growth hormone (oPGH) and growth hormone receptors in ovine placenta, and oPGH production by the trophectoderm and syncitium during the second month of pregnancy. To identify factors regulating oPGH production, we developed a perifusion system to measure oPGH and ovine placental lactogen (oPL) production by Day 45 ovine placental explants. The mRNAs for both hormones were quantitated by real-time polymerase chain reaction in explants collected after perifusion periods of up to 8 h. Ovine PGH and oPL were released into the medium at mean rates of 2.45 +/- 0.2 and 353.6 +/- 13.6 ng/g/h, respectively. Ovine placenta produces growth hormone-releasing hormone (GHRH), but addition of GHRH to the perifusion medium did not modify either oPGH or oPL production. In vivo, oPGH production occurs between Days 30 and 60 of pregnancy. Because modulation of the maternal diet during this period affects placental development, the potential regulation of oPGH and oPL production by glucose was evaluated. Glucose supplementation of the perifusion medium resulted in a concentration-dependent decrease in oPGH release after 4 h, but oPGH mRNA levels were not affected. Production of oPL was not affected by glucose. Thus, oPGH and oPL belong to the same growth hormone/prolactin family but are differentially regulated by glucose. Ovine PGH modulations should be taken into account in metabolic experiments performed during the first trimester of pregnancy in sheep.     

Plasticity of the response of fetal mouse fibroblast to lactation hormones

In the work described in this article, mouse fetal fibroblasts were treated with three lactation hormones (insulin, progesterone and oxytocin) and the cellular changes were analyzed by RT-PCR-Southern hybridization. A gene-expression pattern characteristic of mammary epithelioid cells was induced by the hormones, as indicated by expression of the marker genes alpha-casein and beta-casein. Two mammary epithelial cell-specific gene expression vectors were constructed with bovine alpha-s1-casein or ovine beta-casein gene promoters directing an EGFP reporter gene. Transient expression of the EGFP gene was observed in cells treated by the hormones but not in control cells. Cell morphology also changed after insulin and oxytocin treatments; the cells resembled epithelial cells rather than fibroblasts. Our results suggest that mouse fetus fibroblasts can be partially induced by lactation hormones to resemble mammary epithelial cells. This procedure might help to increase the efficiency of gene targeting in studies of mammary gland bioreactors.     

Lactogenic response of mouse mammary explants from different days of pregnancy to placental lactogen and pituitary prolactin

The lactogenic response of mouse mammary gland explants to human placental lactogen (hPL) and ovine pituitary prolactin (oPRL) was examined on days 10 to 18 of pregnancy by measuring 3H-amino acid incorporation into calcium-rennin precipitable casein. To determine the lactogenic response of the explants, the mean slopes of dose-response curves were calculated for each hormone treatment. Slope means of dose-response curves for oPRL and hPL did not differ from each other on any day of pregnancy examined. A triphasic pattern of response was suggested when slope means of dose-response curves for both hormones were plotted as a function of day of gestation. Peak responses were observed on days 10, 13 and 17-18. Combinations of oPRL and hPL, in ratios of oPRL:hPL = 2:1 and oPRL:hPL = 1:2, also produced a triphasic pattern of sensitivity very similar to that produced by either hormone alone. These results suggest that mouse mammary explants may be more sensitive to oPRL and hPL on days 10, 13 and 17-18 of pregnancy.     

Casein turnover in rabbit mammary explants in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.     

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

Isolation and partial characterization of secreted mouse pituitary prolactin.

Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 +/- 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radio-receptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at --20 degrees C for 2 months.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice.

Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 . 10(-9) M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydrocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.     

Correlation between nuclear glucocorticoid receptor levels and casein gene expression in murine mammary gland in vitro

The relationship between nuclear binding of glucocorticoid-receptor complex and casein gene expression was studied in organ culture of the whole mammary gland of the mouse. Pyridoxal 5'-phosphate was used as a modulatory agent for measuring nuclear binding of the receptor complex. Addition of 2 mM and 5mM pyridoxal-5'-P in the medium (Waymouth's MB752/1) resulted in 4- and 12-fold increase of its concentration in the glands incubated with insulin, prolactin, and hydrocortisone. Pyridoxal-5'-P also caused a 52% and 92% inhibition of nuclear binding of [3H]dexamethasone in the glands at 2 mM and 5 mM concentration in the presence of the same hormones in the medium. Corresponding to the reduced nuclear binding of the receptor complex casein mRNA levels, measured by a specific cDNA probe was reduced 86% and over 90% in the glands exposed to 2 mM and 5 mM pyridoxal-5'-P, respectively, in presence of insulin, prolactin, and hydrocortisone in the medium. Withdrawal of pyridoxal-5'-P from the medium restored nuclear binding of the receptor complex near the level of control glands incubated only with the hormones. mRNA casein levels also increased in the gland in the pyridoxal-5'-P-free medium containing the same hormones. This indicates that pyridoxal-5'-P does not alter the specific hormone responsiveness of the mammary cells and its action mediated at the level of the glucocorticoid receptor can influence hormone-inducible expression of the casein genes. Thus, glucocorticoid plays a major role in the multiple hormone regulation of the milk protein gene(s). The findings also suggest that the breast tissue concentration of the vitamin B6 derivative may influence the physiology of lactation in nursing mothers.     

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.     

Prolactin induction of casein mRNA in organ culture. A model system for studying peptide hormone regulation of gene expression

The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth in vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogen- or prolactin-dependent growth. Primary tumors still displayed a constitutional expression of alpha-, beta-, and gamma-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in alpha- and beta-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of alpha-, beta-, and gamma-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10-14 days. The accumulation of beta-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance beta-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.