Maintenance of milk protein gene expression in a subpopulation of 7,12-dimethylbenz (a) anthracene-induced rat mammary carcinoma cells grown on attached collagen gels

Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels. Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin (5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs. Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels. Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone) in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen gels. This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship, HD 06157.     

Prolactin-induced accumulation of casein mRNA in mouse mammary explants: A selective role of glucocorticoid

The role of glucocorticoid in the prolactin-induced accumulation of casein mRNA in mammary explants from midpregnant mice has been studied after an initial 4-day incubation to allow the level of messenger to decline to undetectable levels. Subsequent culture for 3 days: 1) with insulin and glucocorticoid did not result in detectable accumulation of messenger; 2) with insulin and prolactin resulted in a very small accumulation; 3) with insulin, glucocorticoid and prolactin elicited a 20-fold greater accumulation of casein mRNA than the system with only insulin and prolactin. Therefore, although glucocorticoids are not an absolute requirement for casein gene expression in mouse mammary tissue, they are necessary for massive accumulation of casein mRNA induced by prolactin. It appears that this dependence is not a result of either mRNA stabilization or alteration in prolactin receptors. By contrast, stimulation of total epithelial RNA synthesis by prolactin does not have this glucocorticoid dependency.     

Hormonal regulation of the synthesis of casein and alpha-lactalbumin in a primary mammary cell culture system

The addition of 5 micrograms/ml of both insulin and prolactin, 3 microM cortisol and 5% fetal bovine serum stimulated casein synthesis during a 5 day culture of mammary epithelium from lactating mice using a floating collagen gel as a culture substratum. Omission of any of the three hormones or serum decreased casein synthesis substantially. The use of 10% serum or the attached gel culture system also decreased casein synthesis. Cells cultured with the combination of the three hormones and 5% serum contained a low level of casein mRNA on day 2, but it increased to much higher levels on day 4 and 5, amounting to over 30% of total mRNA on day 5. In contrast to casein synthesis, the maximal increase in alpha-lactalbumin synthesis required the presence of 0.03 microM cortisol. The combination of insulin, prolactin and 3 microM cortisol or insulin and prolactin elicited smaller increases. The translatable mRNA for alpha-lactalbumin in cells cultured with insulin, cortisol and prolactin for 5 days was detected, but not in cells with insulin and cortisol. Both a high and low concentration of cortisol in combination with insulin increased prolactin binding capacity of cultured cells to the same extent, whereas cells cultured with insulin alone contained much lower levels of prolactin binding. The difference in the capacity of prolactin binding between cells cultured with insulin alone and those cultured with insulin and cortisol correlated well with their ability to synthesize casein in response to prolactin.     

Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland

The effect of progesterone on the synthesis of milk proteins, casein and alpha-lactalbumin was investigated by culturing mammary explants from mid-pregnant mice in serum-free medium. The addition of progesterone at concentrations above 10 ng/ml inhibited both the casein and alpha-lactalbumin accumulation that were induced by the synergistic actions of insulin, prolactin and cortisol. The maximal inhibition was attained at a progesterone concentration of 100 ng/ml. The maximal level of inhibition of the alpha-lactalbumin accumulation was about 90% in the presence of insulin and prolactin or insulin, prolactin and 0.01 microgram/ml of cortisol. The inhibition of the casein accumulation by progesterone was about 80% in the presence of insulin and prolactin, and about 40% in the presence of insulin, prolactin and 1 microgram/ml of cortisol, indicating that cortisol partially antagonized the action of progesterone on the casein synthesis. When the inhibitory effect of progesterone on the accumulation of both alpha-lactalbumin and casein was examined in cultured mammary tissues from virgin, early pregnant, mid-pregnant and late pregnant mice, the degree of inhibition was markedly reduced in tissue from late pregnant mice. This indicates that the susceptibility of mammary gland to the inhibitory action of progesterone varies with the developmental stage of the tissue.     

The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture.

Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.     

The differential actions of cortisol on the accumulation of α-lactalbumin and casein in midpregnant mouse mammary gland in culture

The dose-response relationship between cortisol and the accumulation of the two milk proteins, casein and α-lactalbumin, was studied in organ culture of mammary gland from midpregnant mice. The accumulation of casein was low in culture with insulin but was enhanced by the further addition of prolactin. Further increases in casein were effected by the addition of cortisol in increasing concentrations up to 3 × 10^?6 M, which was optimal for the accumulation of this protein. The content of α-lactalbumin in explants was similarly low in culture with insulin alone, but, in contrast, was increased to a maximal level by the addition of insulin and prolactin. The addition of cortisol up to 3 × 10^?8 M with insulin and prolactin did not further increase the level of α-lactalbumin; in fact, at concentrations above 3 × 10^?7 M the steroid caused progressive inhibition of the accumulation of this protein in cultured explants. Studies of the appearance of casein and α-lactalbumin in incubation medium during organ culture revealed the presence of substantial amounts of these milk proteins. During the first 2 days of culture with insulin, prolactin and 3 × 10^?6 M cortisol, the amount of α-lactalbumin in culture medium was almost equal to the level found in tissue, whereas in the presence of 3 × 10^?8 M cortisol, or in the absence of exogenous steroid, over 70% of total α-lactalbumin was retained in tissue. The observed difference in the amount of α-lactalbumin in culture medium can, however, only partially account for the inhibitory effect of high doses of cortisol on the accumulation of α-lactalbumin in cultured mammary explants. In contrast to α-lactalbumin, the relative amount of casein in culture medium containing insulin and prolactin was smaller—19% of total casein synthesized—and was further reduced to 16% and 11% of the total in the presence of 3 × 10^?8 M and 3 × 10^?6 M cortisol, respectively. The above results indicate that cortisol exerts dose-dependent differential actions on the accumulation of casein and α-lactalbumin in mouse mammary epithelium in vitro.     

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.     

The interaction of cortisol and prostaglandins on the phenotypic expression of the alpha-lactalbumin gene in the mouse mammary gland in culture

Addition of cortisol at concentrations above 300 nM selectively inhibited the synthesis of alpha-lactalbumin and the accumulation of its mRNA in the mouse mammary gland cultured in the presence of insulin and prolactin, whereas the same treatment augmented casein synthesis and the accumulation of casein mRNA. Prostaglandin E2 or F2 alpha reversed the inhibitory effects of cortisol in a dose-dependent manner, without affecting casein production. The levels of prostaglandin E2 or F2 alpha in tissue explants cultured with insulin and prolactin increased about 2.6-fold over those in uncultured tissue, and the addition of cortisol decreased these levels approximately 2-fold. These results indicate the ability of prostaglandins to counteract the inhibitory effect of cortisol on the alpha-lactalbumin gene expression in the mouse mammary gland.     

Hormonally induced elevations of alpha- and beta-casein mRNA levels are blocked by cyclic adenine nucleotide and prostaglandins

Normal mammary gland development during pregnancy follows a coordinated program of morphological development (formation of lobuloalveoli) and biochemical differentiation (casein production). In culture, whole mammary glands of Balb/c mice can be similarly induced by application of a mixture of insulin, prolactin, aldosterone and hydrocortisone (IPAH) for 7 days. Our previous reports have shown that lobuloalveolar development, induced by IPAH, is competitively inhibited by the simultaneous presence of dibutyryl cyclic AMP (Bt2cAMP), prostaglandins (PGs) E1, E2, and B1, and papaverine (pap). However, if this mixture is not added until day 4, lobuloalveolar development is relatively unaffected but casein synthesis is repressed. This report explores the mechanism by which cyclic adenine nucleotides and prostaglandins interfere with the normal developmental pathway. The accumulation of alpha- and beta-casein mRNAs induced by prolactin, hydrocortisone and aldosterone is blocked by the combination of Bt2cAMP, PGs E1, E2, and B1, and pap added to the medium for the final 3 days (days 4-7). Under these conditions the glands retain their lobuloalveoli, and little squamous metaplasia can be discerned. Furthermore, de novo synthesis of both caseins is selectively inhibited, despite the continued presence of casein mRNAs in the glands and normal protein synthesis. In contrast, the synthesis of keratin is stimulated. Incomplete mixtures of Bt2cAMP and pap or the combination of PGs E1, E2, and B1, are only partly effective in preventing the accumulation of casein mRNAs. All three mixtures bring about similar effects on both alpha- and beta-casein mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ouabain on the lactogenic action of prolactin and on the level of mammary prolactin receptors

Ouabain added to the culture medium of rabbit mammary gland inhibits prolactin action on the initiation of lactose and casein synthesis. The degree of inhibition is a function of the ouabain concentration in the medium. Likewise, ouabain blocks the accumulation of casein mRNA supported by prolactin. In addition, ouabain provokes a rapid disappearance of prolactin receptors. Conversely prolactin keeps its capacity to enhance the concentration of casein mRNA and the parallel casein synthesis when K+ ions are totally absent from the culture medium. These results suggest that although prolactin induces a modification of the K+/Na+ ratio in the mammary cell and ouabain prevents this effect of prolactin, the inhibitory action of ouabain on lactogenesis can be explained essentially by its effect on the hormone receptors.     

Correlation between nuclear histone acetylation and casein messenger RNA induction in the mammary gland

Sodium butyrate prevented the accumulation of casein mRNA induced by the combined action of prolactin and glucocorticoid in the presence of insulin in the cultured mammary gland. This inhibition was reversible and dose-dependent. In addition to the inhibition of the mRNA induction, both nuclear histone acetylase and deacetylase activities were inhibited by the incubation of the glands with butyrate, whereas these enzyme activities were stimulated by glucocorticoid and prolactin, or by glucocorticoid alone, in the presence of insulin. These data strongly suggest that the increased metabolism of histone acetyl groups is involved in the hormone-mediated casein mRNA induction and that glucocorticoid plays a preferential role on this increased metabolism.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

Response of end bud cells from immature rat mammary gland to hormones when cultured in collagen gel

End buds from 4- to 5-week-old rat mammary glands were isolated and cultured within a rat tail tendon collagen gel matrix. Media containing equine serum or porcine serum and cholera toxin promoted growth, but not the production of casein or thioesterase II, nor did they induce a state of differentiation as assessed by cell ultrastructure. Medium supplemented with only 5% porcine serum, insulin and cholera toxin did not support growth or differentiation. However, when prolactin, estradiol, progesterone and hydrocortisone were added to this medium, growth was stimulated greatly and a differentiated state was induced as assessed by the production of casein and thioesterase and by the appearance of a highly secretory ultrastructure.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Inhibition of casein synthesis by progestagens in vitro: modulation in relation to concentration of hormones that synergize with prolactin

We studied the effect of progesterone and its agonist, R 5020, on casein and transferrin production in pregnant rabbit mammary gland explant culture and its modulation by hormones that synergize with prolactin. The glands were obtained from rabbits on days 12-14 of gestation. The progestins had no effect alone, but significantly inhibited ovine and porcine prolactin stimulation of casein synthesis in a dose dependent manner. There were no effects on transferrin content of the tissue, demonstrating a specific effect of progesterone on casein synthesis. In approx 15% of the cultures, prolactin stimulated casein production to very high levels and the progestins lost their inhibitory action. Progestins were also ineffective when the tissue was cultured with prolactin and unphysiologically high levels of insulin (5 mg/l) or cortisol (280 nmol/l), which stimulated casein synthesis to higher levels than prolactin alone. The concentration of cortisol used was 10 times higher than the serum levels seen in rabbits at the stage of gestation studied (approx 10 ng/ml) and corresponded to levels seen at the end of gestation, a period when the glands are secreting milk and progesterone serum levels have commenced to decrease. Thus, when the prolactin effect upon casein synthesis had been potentiated, whether spontaneously or through synergism with insulin or corticoids, progestins were unable to inhibit it, as is the case in lactating tissues. The results show that utilization of unphysiological levels of hormones in culture may distort the response of the tissue, masking responses that are clearly seen in vivo.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and alpha-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. These results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

Summary The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

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.