Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. We have tested whether MAR sequences from the chicken lysozyme locus, the so-called A-elements, can confer position-independent regulation to a whey acidic protein (WAP) transgene in mammary tissue of mice. In the absence of MARs, expression of WAP transgenes was observed in 50% of the lines, and regulation during pregnancy, during lactation, and upon hormonal induction did not mimic that of the endogenous WAP gene and varied with the integration site. In contrast, all 11 lines in which WAP transgenes were juxtaposed to MAR elements showed expression. Accurate position-independent hormonal and developmental regulation was seen in four out of the five lines analyzed. These results indicate that MARs can establish independent genetic domains in transgenic mice.

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation.

Expression of the mouse whey acidic protein (WAP) gene is specific to the mammary gland, is induced several thousand-fold during pregnancy, and is under the control of steroid and peptide hormones. To study developmental regulation of the mouse WAP gene, a 7.2-kilobase (kb) WAP transgene, including 2.6 kb of 5'- and 1.6 kb of 3'-flanking sequences, was introduced into mice. Of the 13 lines of mice examined, 6 expressed the transgenes during lactation at levels between 3 and 54% of the endogenous gene. Although expression was dependent on the site of integration, the transgenes within a given locus were expressed in a copy number-dependent manner and were coordinately regulated. The WAP transgenes were expressed specifically in the mammary gland, but showed a deregulated pattern of expression during mammary development. In all six lines of mice, induction of the WAP transgenes during pregnancy preceded that of the endogenous gene. During lactation, expression in two lines increased coordinately with the endogenous gene, and in three other lines of mice, transgene expression decreased to a basal level. These data indicate that the 7.2-kb gene contains some but not all of the elements necessary for correct developmental regulation. At a functional level it appears as if a repressor element, which inactivates the endogenous gene until late pregnancy, and an element necessary for induction during lactation are absent from the transgene. Complementary results from developmental and hormone induction studies suggest that WAP gene expression during pregnancy and lactation is mediated by different mechanisms.

Regulation of expression of genes for milk proteins.

Our results demonstrate that mRNAs that encode two major milk proteins in the mouse, namely, WAP and beta-casein, are found predominantly in the mammary glands of lactating animals. During the course of mammary development from the virgin to the fully lactating animal the steady-state levels of the two RNAs increase about 10(4)-fold, with the most pronounced increase occurring around midpregnancy. Low levels of WAP and beta-casein RNAs are also found in some nonmammary tissues such as tongue, pancreas, and pituitary gland, but not in others, for example, heart and brain. This variation suggests that the transcriptional machinery in some nonmammary cells is capable of recognizing milk-protein genes. The analysis of transgenic animals that carry a hybrid gene composed of the WAP promoter plus the tPA gene strongly suggests that mammary specificity of gene expression is encoded in the promoter/upstream region of the WAP gene. Induction of the genes for WAP and beta-casein during development of mammary glands requires the synergistic action of insulin, hydrocortisone, and prolactin. The increase in levels of the two mRNAs in mammary tissue from midpregnant animals was about 50-fold upon incubation with all three of these hormones, suggesting that no additional hormones may be required in the intact animal. The characteristics of the hormonal induction of the WAP-tPA hybrid gene in transgenic animals differed from those of the induction of the endogenous milk-protein genes in that the presence of all three hormones was not sufficient to increase the levels of the hybrid RNA. This difference suggests that some elements involved in the hormone-dependent accumulation of WAP RNA are located outside the promoter/upstream region, (Pittius et al. 1988a and 1988b). Similar conclusions have been drawn by Groner and coworkers, who analyzed expression of the WAP promoter in two additional hybrid genes (Andres et al. 1988). In addition to the WAP gene, the beta-casein gene also appears to contain hormone-sensitive regulatory elements within the transcribed region. Hybrid genes containing the rat beta-casein promoter (Lee et al. 1989a and 1989b) are expressed in a mammary-specific fashion in transgenic animals but have lost their ability to be regulated by steroid and peptide hormones, a property of their endogenous counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone-responsive survival of mammary gland explants from the pregnant tammar wallaby (Macropus eugenii) in the absence of exogenous hormones and growth factors.

1. The level of beta-lactoglobulin mRNA increased maximally in mammary explants from late pregnant tammars cultured for 3 days in media containing either prolactin or insulin, cortisol and prolactin. 2. The same level of accumulation occurred when explants were first cultured for 4 days in a chemically defined medium with no exogenous hormones, serum or growth factors, suggesting that the tissue remains viable and hormone-responsive during the initial incubation. 3. Mammary explants cultured for 4 days in medium with no hormones demonstrated a progressive increase in the rate of RNA and DNA synthesis suggesting that the tissue is under a positive autocrine/paracrine stimulus.

Comparison of the regulation of the whey acidic protein gene with that of a hybrid gene containing the whey acidic protein gene promoter in transgenic mice.

The developmentally and hormonally regulated expression of the mouse whey acidic protein (WAP) gene and a hybrid gene containing the WAP gene promoter and a cDNA for human tissue plasminogen activator (tPA) were studied in a line of transgenic mice. During mammary gland development from the mature virgin state to the seventh day of lactation, the relative concentration of WAP mRNA increased about 10(4)-fold, the increase being most pronounced between days 14 and 16 of gestation. In mammary gland organ culture from virgin and midpregnant animals, the concerted actions of insulin, hydrocortisone, and PRL were required to increase WAP mRNA levels. Steady state levels of transcripts from the WAP-tPA hybrid gene increased about 100-fold during pregnancy; this occurred mainly around day 10 of gestation. Insulin, hydrocortisone, and PRL were necessary to maintain the levels of WAP-tPA RNA in explants from virgin and pregnant animals, but could not further elevate it. The results suggest that the WAP gene promoter and upstream region contains some, but perhaps not all elements conferring developmental and hormone regulated expression of the mouse WAP gene.

Comparison of some biological effects of epidermal growth factor and commercial serum albumin on the induction of alpha-lactalbumin in rat and rabbit mammary explants.

Commercial preparations of serum albumin from six species can markedly enhance the prolactin-independent induction of alpha-lactalbumin in mammary explants from pregnant rats, and evoke such induction in the tissue from virgin rats. These effects are similar to those of epidermal growth factor (EGF) reported previously. The stimulatory activity of bovine serum albumin (BSA) resides in a putative impurity in the albumin. Charcoal extraction and gel filtration of the BSA results in complete loss of activity. Of the five milk protein mRNAs studied, only alpha-lactalbumin mRNA is induced by insulin, glucocorticoid and serum albumin in the absence of prolactin. Despite these similarities, the biological effects of serum albumin and EGF on mammary tissue diverge in some respects. They appear to operate by different mechanisms since their effects on the rat system are additive. Furthermore, while both inhibit prolactin-mediated induction of alpha-lactalbumin in rabbit mammary explants, cortisol converts EGF into a stimulatory agent, but merely blocks the inhibitory effect of serum albumin. The results emphasize that commercial serum albumin is not to be regarded simply as an inert protein additive to culture media.

Progesterone and prolactin are both required for suppression of the induction of rat alpha-lactalbumin activity.

Progesterone prevents lactation during pregnancy. This anti-lactogenic effect includes suppression of the advent of alpha-lactalbumin activity, an effect which prevents the formation of lactose. Alpha lactalbumin activity can be induced to some extent in pregnant rat mammary explants by insulin and hydrocortisone alone, and to a greater extent with prolactin in addition, or with EGF in addition. Physiological levels of progesterone markedly inhibit the induction in the presence of prolactin plus insulin and hydrocortisone, only weakly inhibit in the presence of insulin and hydrocortisone alone, and have no inhibitory effect in the presence of EGF plus insulin and hydrocortisone. Prolactin permits some inhibition in the presence of EGF. The results suggest that progesterone does not subvert the essential insulin or glucocorticoid signals. It also appears that transduction of the prolactin signal is required in order that progesterone effectively block induction of alpha-lactalbumin activity.

Is EGF a physiological inhibitor of mouse mammary casein synthesis? Unphysiological responses to pharmacological levels of hormones.

It has been observed that EGF inhibits the induction of casein synthesis by mouse mammary tissue in vitro in addition to acting as a promoter of mammary epithelial proliferation. However, since the circulating level of EGF increases during lactation, and since functional EGF receptors are retained by the lactating cells, it seemed unlikely that EGF is an inhibitor of mammary differentiation in vivo. The current studies demonstrate, in fact, that EGF inhibits the induction of casein synthesis in vitro only when insulin is present in the culture medium at unphysiologically high concentrations. Other artifactual responses to high levels of hormones are described.

Comparison of the roles of insulin and insulin-like growth factor I in casein gene expression and in the development of alpha-lactalbumin and glucose transport activities in the mouse mammary epithelial cell.

The concentration-activity profiles for insulin and insulin-like growth factor I (IGF-I; in the presence of and insulin-like growth factor I (IGF-I; in the presence of hydrocortisone and PRL) have been compared in terms of the accumulation of beta-casein mRNA, total casein synthesis, and alpha-lactalbumin and basal carrier-mediated glucose transport activities in mammary epithelial cells from midpregnant mice. For the accumulation of the casein mRNA and the induction of casein synthesis and alpha-lactalbumin activity, the insulin ED50 is 1-2 ng/ml, while that for IGF-I is 10- to 20-fold greater. The effects of insulin and IGF-I are not additive in these instances. For the induction of basal carrier-mediated glucose transport, the insulin ED50 is 8 ng/ml, and that for IGF-I is 16 ng/ml. Either factor can induce transport activity up to the level present in the cells from 2-day lactating mice. In this instance the effects are additive; insulin and IGF-I together can induce the transport up to the 10-day lactating level.

Effects of estrogen-depletion on rat casein gene expression.

Mammary tissue from rats that had been ovariectomized and adrenalectomized 4 weeks previously was compared to that from intact rats in terms of epithelial content and hormone-responsiveness in vitro. The endocrinectomy resulted in about a 30% enlargement of the gland, but led to a loss of only about 12% of the epithelium. This estrogen-depleted epithelium was able to acquire full responsiveness in vitro to insulin in terms of the accumulation of alpha-aminoisobutyric acid, and induction of glucose-6-phosphate and gluconate-6-phosphate dehydrogenases. It was also fully responsive to cortisol in relation to the induction of NADH-cytochrome C reductase, and to prolactin in terms of total RNA synthesis. However, estrogen-depletion led to an 82% loss in the ability of a unit amount of the epithelium to synthesize casein in response to these 3 hormones, and to a similar loss in relation to the accumulation of 25K casein mRNA. Estrogen administration in vivo could prevent and reverse the casein lesion. The disparity between constitutive and casein hormone-responsiveness in the absence of estrogen is discussed in relation to cell commitment.

Prolactin-induced alpha-lactalbumin activity in mammary explants from pregnant rabbits. A role for epidermal growth factor and glucocorticoids.

Exogenous prolactin alone can induce alpha-lactalbumin activity in rabbit mammary explants. Under these conditions, exogenous corticosol has no effect. However, low levels of epidermal growth factor (EGF) can markedly inhibit the induction by prolactin, and this inhibitory effect, in turn, can be prevented by cortisol. The steroid can, in fact, convert EGF from a potent inhibitor into an agent which enhances the induction. None of the other growth factors tested inhibits induction of alpha-lactalbumin activity by prolactin, and no other type of steroid tested overcomes the effect of EGF. The results suggest that, under physiological conditions, glucocorticoid may be essential for induction of alpha-lactalbumin activity in the rabbit in order to override the effect of serum EGF.

Selective enhancement of the induction of alpha-lactalbumin activity in rat mammary explants by epidermal growth factor.

Epidermal growth factor (EGF) enhances the induction of alpha-lactalbumin in mammary explants from pregnant and virgin rats in the presence of insulin (I), hydrocortisone (F) and prolactin (P). EGF also enhances the prolactin-independent induction of alpha-lactalbumin in tissue from pregnant rats and evokes prolactin-independent induction of alpha-lactalbumin in mammary tissue from virgin rats in the presence of I and F. Casein synthesis and galactosyltransferase activity are unaffected by EGF in the IFP-system, and are not induced in the IF-EGF-system. Multiplication stimulating activity, nerve growth factor, fibroblast growth factor and platelet-derived growth factor do not mimic the selective effects of EGF on rat alpha-lactalbumin. These influences of EGF on the differentiation of isolated rat mammary tissue are compared with those on mouse and rabbit tissue studied previously.

A unique and essential role for insulin in the phenotypic expression of rat mammary epithelial cells unrelated to its function in cell maintenance.

Mammary explants from pregnant rats can be induced in regard to casein synthesis and alpha-lactalbumin activity when cultured in the presence of hydrocortisone, prolactin and levels of insulin approaching physiological concentrations. No detectable induction occurs in the absence of insulin. Although epidermal growth factor and multiplication stimulating activity, in the presence of hydrocortisone, can maintain the initial level of NADH-cytochrome c reductase as well as insulin, neither can substitute effectively for insulin in the induction of the milk proteins. Proinsulin, nerve growth factor, platelet-derived growth factor and fibroblast growth factor are also ineffective substitutes for insulin in this regard. Whereas prolonged tissue exposure to multiplication stimulating activity, hydrocortisone and prolactin does not result in induction of alpha-lactalbumin activity, subsequent addition of insulin leads to prompt response. The results suggest that the ability of insulin to function as a unique, essential factor in the induction of rat milk proteins is independent of its cell-maintenance activity. Thus, in addition to its well established functions in metabolic processes, insulin appears to play a vital role in certain developmental processes.

Deoxyribonucleic acid synthesis-dependent casein gene expression: species differences.

Mammary gland explants from mature virgin mice, rats, and rabbits exhibit an increased rate of both DNA and casein syntheses when cultured in the presence of specific combinations of insulin, hydrocortisone, and PRL. If cytosine arabinoside, a potent inhibitor of DNA synthesis, is added to the culture medium, casein synthesis is inhibited in explants from mice but not in those from rats or rabbits. This inhibition is at the level of accumulation of casein mRNA; an 89% reduction of stimulated levels was observed. The nature of this block was investigated further by examining the general response of mature virgin mouse mammary explants to hydrocortisone and PRL, hormones considered essential for casein gene expression in this species. Cytosine arabinoside did not prevent an increment in either hydrocortisone-induced NADH-cytochrome c-reductase or PRL-induced total RNA synthesis. Previous work has shown that certain insulin-induced responses are also unaffected. Taken collectively, these results suggest that the lesion induced by cytosine arabinoside inhibition of DNA synthesis is distal to the receptor for one or more of these hormones. The necessity for coupling DNA synthesis with overt differentiation in the mature virgin mouse, but not in the rat or rabbit, is one of numerous examples of species variation in regard to the interaction of hormones with the mammary gland.

Effect of vitamin A deprivation on maintenance of rat mammary tissue and on the potential of the epithelium for hormone-dependent milk protein synthesis.

Mammary tissue from rats severely deficient in vitamin A was compared with that from control animals. Glands from the deficient animals weighed only about 60% as much as those from control rats, a reflection largely of loss of material from the mammary fat pad. By contrast, vitamin A-deficient glands had as much epithelium as control glands. Furthermore, explants from the deficient glands were as responsive to insulin, glucocorticoid, and PRL as explants from control glands, in terms of the induction of casein synthesis and the alpha-lactalbumin and galactosyltransferase activities. It appears that vitamin A does not have a physiological role either in the maintenance of rat mammary epithelium or in its potential for hormone-dependent phenotypic expression. The results suggest that the anticarcinogenic activity of retinoids on rat mammary gland reported previously may lie outside the purview of these aspects of the mammary cell.

The induction of alpha-lactalbumin in rat mammary explants in the absence of exogenous prolactin: effects of progesterone and estrogen.

Mammary explants from both pregnant and virgin rats cultured in medium containing insulin, hydrocortisone and prolactin show a progressive increase in alpha-lactalbumin activity. However, when cultured with insulin and hydrocortisone only, explants from pregnant, but not those from virgin rats show an induction of alpha-lactalbumin-like activity similar to that seen when prolactin is present. The activity induced in the absence of exogenous prolactin corresponds to newly synthesized alpha-lactalbumin molecules, since 1) the activity is suppressed completely by rabbit anti-rat alpha-lactalbumin serum and 2) culture in the presence of [3H]-amino acids generates [3H]-alpha-lactalbumin, identified by SDS-polyacrylamide gel electrophoresis. Mammary tissue from virgin rats can be rendered responsive to insulin and hydrocortisone in this regard by administration in vivo of either progesterone or estrogen, but not by desoxycorticosterone acetate. This ability to convert virgin rat mammary cells to a state in which they are independent of exogenous prolactin in terms of alpha-lactalbumin synthesis represents a heretofore unreported effect of the female sex steroids.