Mammary Epithelial Cell Line HC11 Research Articles

Estrogen receptors alfa (ERα) and beta (ERβ) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11

The mitogenic effect of 17beta-estradiol (E2) on the breast is mediated by estrogen receptor alfa (ERalpha), hence ERalpha antagonists are effective in the treatment of breast cancer. The possible use of estrogen receptor beta (ERbeta) as a target in treatment of breast cancer is under investigation. The mouse mammary cell line HC11 expresses both ERs and was used to study the role of the two receptors in proliferation. E2 had no effect on proliferation. The ERalpha-selective agonist 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) stimulated proliferation. The ERbeta-selective agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) inhibited cell growth and induced apoptosis. PPT upregulated while DPN downregulated cyclin D1 and proliferating cell nuclear antigen (PCNA). Upon inhibition of ERalpha expression with RNA interference, E2 caused a decrease in cyclin D1 and PCNA, and increased apoptosis. When ERbeta expression was blocked, E2 induced proliferation and cells gained the capacity to grow in soft agar. In summary, in HC11 mammary epithelial cells, ERalpha drives proliferation in response to E2 while ERbeta is growth inhibitory. The lack of effect of E2 on HC11 cell growth is the result of the combined actions of ERalpha (proliferation) and ERbeta (apoptosis). We suggest that use of ERbeta agonists will be a useful addition in treatment of breast cancer, which, at present, is only aimed at inhibition of ERalpha.

Molecular recognition characteristics in the insulin-like growth factor (IGF)-insulin-like growth factor binding protein -3/5 (IGFBP-3/5) heparin axis

Insulin-like growth factor binding proteins (IGFBPs) -3 and -5 are known to interact with various components of the extracellular matrix (ECM; e.g. heparin and heparan sulphate) and this interaction is believed to affect the affinity of both IGFBP species for their cognate ligands--IGF-I and -II. There is little detail on the nature of the molecular complex formed between ECM components, IGFBPs and IGFs although the glycosaminoglycan (GAG) heparin has been reported to reduce the affinity of IGFBP-5 for IGF-I. In order to investigate this phenomenon further, we have undertaken an extensive surface plasmon resonance based biosensor study to report the affinity of IGFBP-3 and -5 for binding heparin (22 and 7 nM respectively). We have also shown that pre-complexation of IGFBP with IGF-I and -II inhibits the subsequent association of IGFBP with heparin and conversely that heparin complexation of IGFBP-3 and -5 inhibits IGFBP binding to biosensor surfaces containing immobilised IGF-I. In addition we have used both IGF-I and heparin coated biosensor surfaces in an attempt to build ternary IGF-IGFBP-heparin complexes in order to gain some insight into the nature of inhibition by heparin of IGFI-IGFBP complex formation. Our data lead us to conclude that the inhibition by heparin is partly competitive in nature, and that ternary complexes of IGF-IGFBP-heparin are either unable to form, or only form unstable transient complexes. The potential biological significance of our data is highlighted by the demonstration that IGF-I and IGF-II can displace endogenous IGFBP-5 from monolayer cultures of the mouse mammary epithelial cell line HC11.

Leptin and prolactin modulate the expression of SOCS-1 in association with interleukin-6 and tumor necrosis factor-α in mammary cells: a role in differentiated secretory epithelium

Leptin and its receptors have been shown to be expressed in several tissues, suggesting that this protein might be effective not only at the CNS level but also peripherally. We have previously reported that leptin and its long form receptor are expressed in the mouse mammary epithelial cell line HC11. In this study, we report a specific relationship among leptin, prolactin (PRL), interleukin-6 (IL-6), and tumor necrosis-α (TNF-α) in the modulation of the suppressor of cytokine signaling 1 (SOCS-1). Furthermore, we show that leptin and PRL are able to effectively enhance SOCS-1 gene expression in the HC11 cell line. Finally, high concentrations of leptin (100 nM) and/or PRL significantly ( p<0.05) reduce the inhibitory effect of IL-6 (10 and 100 ng/ml) and TNF-α (10 and 100 ng/ml) on β-casein gene expression in HC11 cells transfected with pβcCAT, a chimeric rat-β casein gene promoter-cloramphenicol acetyl transferase (CAT) gene construct. These results provide evidence that leptin may be an important mediator in regulating mammary gland growth and development and that this role may be related to the immune factors that are involved in inflammation.

Epithelial mesenchymal transition is a characteristic of hyperplasias and tumors in mammary gland from MMTV-Cripto-1 transgenic mice.

Epithelial-mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto-1 (CR-1), a member of the epidermal growth factor-CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR-1 transgene by the MMTV promoter (MMTV-CR-1) and in mouse mammary epithelial cell line HC-11 overexpressing CR-1 (HC-11/CR-1). Western blot analysis showed decreased expression of E-cadherin in MMTV-CR-1 tumors and in HC-11/CR-1 cells. The expression of N-cadherin, vimentin, cyclin-D1, and of the zinc-finger transcription factor, snail, was increased in MMTV-CR-1 tumors. Increased snail mRNA was also found in HC-11/CR-1 cells. Expression of phosphorylated (P)-c-Src, P-focal adhesion kinase (FAK), P-Akt, P-glycogen synthease kinase 3beta (GSK-3beta), dephosphorylated (DP)-beta-catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV-CR-1 tumors. Immunohistochemistry showed positive staining for vimentin, N-cadherin, cyclin-D1, smooth muscle actin, fibronectin, snail, and beta-catenin in MMTV-CR-1 tumor sections. HC-11/CR-1 cells treated with the c-Src inhibitor PP2 reduced the expression of P-c-Src and of P-FAK, P-Akt, P-GSK-3beta, DP-beta-catenin all known to be activated by c-Src. Migration of HC-11/CR-1 cells was also reduced by PP2 treatment. These results suggest that CR-1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT.

Estrogen receptor functional activity changes during differentiation of mammary epithelial cells.

Mammary gland development involves complex cycles of proliferation, differentiation, and morphogenesis, regulated by hormones including estrogens, prolactin (PRL), and epidermal growth factor (EGF). The mouse mammary epithelial cell line HC11 has been shown to be valuable for investigations of differentiation of mammary gland. In this study, we show that HC11 cells express estrogen receptor (ER)alpha and ER beta proteins at all developmental stages. We have established two different stable HC11 cell lines; H-estrogen response element (ERE) containing an ERE-reporter and H-Bc containing a beta-casein reporter. Transcription of the ERE-reporter was activated only in proliferating cells in the presence of EGF. When the cells entered the differentiation program, in the absence of EGF, estradiol-induced transcription of the ERE reporter was repressed, and similar results were obtained when MAPK signaling was inhibited in proliferating cells. We propose that these findings are related to changes in ER corepressor levels, regulated by EGF. We also report that the beta-casein reporter was activated in terminally differentiated cells and that this induction was effectively repressed by estradiol treatment. Finally, we show a physical interaction between endogenous ER alpha and signal transducer and activator of transcription 5 in differentiated HC11 cells. In summary, our results show that ER functional activity changes during differentiation of HC11 cells.

Hormonal control of IGF-binding protein (IGFBP)-5 and IGFBP-2 secretion during differentiation of the HC11 mouse mammary epithelial cell line.

The mouse mammary epithelial cell line HC11 upregulates the synthesis of beta-casein (a differentiation marker) following treatment with the lactogenic hormone mix dexamethasone, insulin and prolactin (DIP). We demonstrate that the basal levels of IGF-binding protein (IGFBP)-5 secreted by undifferentiated HC11 cells are upregulated 10-fold during DIP-induced cellular differentiation whereas the level of the other IGFBP species secreted by HC11 cells (IGFBP-2) is downregulated during this process. As previously reported, the combination of all three of these hormones is required for synthesis of the differentiation marker beta-casein, whereas basal IGFBP-5 secretion is evident in the absence of any hormonal treatment and, unlike beta-casein, secretion of this protein can be stimulated by binary combinations of the hormones (although maximal levels of IGFBP-5 are achieved in the presence of all three lactogenic hormones). Additionally, levels of IGFBP-5 can be increased by DIP treatment under conditions (non-competency of HC11 cultures or presence of epidermal growth factor) where DIP treatment does not increase synthesis of beta-casein. For IGFBP-2, dexamethasone is a potent inhibitor of secretion whilst prolactin stimulated the secretion of this binding protein into the medium. For the IGFBP axis in HC11 cells we conclude that, although the levels of IGFBP-5 and -2 are influenced by the state of cellular differentiation, the hormonal regulation of the levels of these IGFBP species can be dissociated from the regulation of beta-casein synthesis. In a further series of experiments we demonstrate that IGF-I is able to replace insulin in the DIP lactogenic hormone mix and by the use of a specific IGF-I receptor blocking antibody indicate that the action of IGF-I is mediated through the cell surface IGF-I receptor and not by cross-reaction of IGF-I ligand at the insulin receptor. We discuss our data in the context of the potential role of the IGF axis in the process of cell differentiation and illustrate the significance of our findings in the context of the physiology and life cycle of the mammary epithelial cell.

Effect of leptin in proliferating and differentiated HC11 mouse mammary cells

Leptin and its receptors have been shown to be expressed in several tissues thus suggesting that this protein might be effective not only at the CNS level, but also peripherically. We demonstrated by RT-PCR analysis that leptin and its long isoform receptor are expressed in the mouse mammary epithelial cell line HC11, an in vitro cell model considered suitable to study the regulation of the functional development of the mammary epithelium. Furthermore, leptin secretion by HC11 cells was demonstrated by heterologous ELISA. Neither mRNA expression nor protein secretion changed throughout the different phases of differentiation of the cell line. Receptor mRNA was not modified when cells were induced to express β-casein. High concentrations of leptin (between 1.5 and 15 μM) significantly ( p<0.05) reduced cell growth as measured by MTT test. HC11 cells were transfected with pβcCAT, a chimeric rat-β casein gene promoter-CAT gene construct and CAT ELISA was used to determine gene expression. Leptin, from 1.5 nM to 15 μM, was shown to positively ( p<0.05) influence β-casein expression both in the presence or in the absence of prolactin. These data provide evidence that leptin, through its receptor, may be an important mediator in regulating mammary gland growth and development.

Effects of hyperactive Janus kinase 2 signaling in mammary epithelial cells

Prolactin, the Janus kinase 2 (JAK2) and the signal transducer and activator of transcription 5 (STAT5) are important for mammary gland development and have also been implicated in development and growth of breast tumors. In the present study we have investigated the role for JAK2 in proliferation, differentiation, and apoptosis of the mammary epithelial cell line HC11 by stably overexpressing two hyperactive JAK2 mutants. Cells expressing a JAK2 mutant consisting of only the kinase domain had high amount of nuclear STAT5 protein with low DNA-binding activity, which was rapidly induced to a DNA-binding state by prolactin treatment. Cells expressing JAK2 deleted of the kinase-like domain showed increased sensitivity to prolactin treatment compared to wild type cells. Proliferation was not affected by any of the mutants whereas the ability to undergo apoptosis was decreased implicating a transforming potential of the JAK2 mutants.

Tumor suppression by a proapoptotic calcium-activated chloride channel in mammary epithelium.

Little is known of the roles played by ion channels in cancer. Here we describe a pair of closely related calcium-activated chloride channels whose differential regulation in normal, apoptotic, and transformed mouse cells suggests that channel function is proapoptotic and antineoplastic. While mCLCA1 predominates over mCLCA2 under normal physiological conditions, this relationship is reversed by apoptotic stress both in developing mammary gland and in cultured HC11 mammary epithelial cells. Consistent with an apoptosis-promoting role, splicing of mCLCA2 is disrupted in apoptosis-resistant tumor cell lines and in HC11 cells selected for resistance to detachment-induced apoptosis (anoikis). Unexpectedly, mCLCA1 message is also down-regulated in these cells by at least 30-fold. These results suggest that both genes antagonize survival of mammary tumor cells by sensitizing them to anoikis. When MCF7 or HEK293 tumor cells were transfected with plasmids encoding either mCLCA1 or mCLCA2, colony formation was greatly reduced relative to a vector-transfected control, demonstrating that calcium-sensitive chloride channel (CLCA) expression is deleterious to tumor cell survival. Furthermore, mammary epithelial cells overexpressing mCLCA2 had twice the rate of apoptosis of normal cells when subjected to serum starvation and formed multinuclear giants at a high frequency in normal culture, suggesting that mCLCA2 can promote either apoptosis or senescence.

Recruitment of the Protein-tyrosine Phosphatase SHP-2 to the C-terminal Tyrosine of the Prolactin Receptor and to the Adaptor Protein Gab2

The protein-tyrosine phosphatase SHP-2 modulates signaling events through receptor tyrosine kinases and cytokine receptors including the receptor for prolactin (PRLR). Here we investigated mechanisms of SHP-2 recruitment within the PRLR signaling complex. Using SHP-2 and PRLR immunoprecipitation studies in 293 cells and in the mouse mammary epithelial cell line HC11, we found that SHP-2 co-immunoprecipitates with the PRLR and that the C-terminal tyrosine of the PRLR plays a regulatory role in both the tyrosine phosphorylation and the recruitment of SHP-2. Our results further indicate that SHP-2 association to the PRLR occurs via the C-terminal SH2 domain of the phosphatase. In addition, we determined that the newly identified adaptor protein Gab2, but not Gab1, is specifically tyrosine phosphorylated and is able to recruit SHP-2 and phosphatidyinositol 3-kinase in response to PRLR activation. Together, these studies suggest the presence of dual recruitment sites for SHP-2; the first is to the C-terminal tyrosine of the PRLR and the second is to the adaptor protein Gab2.

Role of androgens in proliferation and differentiation of mouse mammary epithelial cell line HC11.

Androgens have been found in mammary epithelium and in milk throughout the cycle of the mammary gland in vivo. The aim of this study was to investigate the possible role of these substances in mammary epithelial growth and differentiation in the mouse HC11 cell line. Cells were stimulated with testosterone, dihydrotestosterone, androstenedione and 5alpha-androstane-3alpha,17beta-diol at concentrations ranging between 0.3 nM and 30 nM. Cyproterone acetate or flutamide, androgen receptor antagonists, (3 microM) were used to block specific androgen effects. Proliferative effects were measured by an MTT (tetrazolium blue) conversion test and [(3)H]thymidine uptake. HC11 cells were transfected with pbetacCAT, a chimeric rat beta-casein gene promoter-chloramphenicol acetyl transferase (CAT) gene construct and CAT ELISA was used to determine gene expression. RT-PCR was performed to detect androgen receptor expression. After 24, 48 and 72 h androgens significantly (P<0.05) increased proliferation. Androgen antagonists significantly (P<0.05) reduced the proliferative effects. Furthermore androgens potentiated the lactogenic effect of prolactin, insulin and dexamethasone (P<0.05). Finally, the androgen receptor gene was expressed in both proliferating and differentiated HC11 cells. These observations lead us to hypothesize an activity of this class of steroids in mammary physiology. In particular, androgens stimulate cell proliferation and beta-casein gene expression; this influence appears to be mediated by androgen receptors.

Signal transduction pathways regulated by CSF-1 receptors modulate the in vitro radiosensitivity of mammary epithelial cells

Purpose: CSF-1 and its receptor have both been previously implicated in the basic biology and clinical course of mammary and female reproductive tract neoplasms. A recent study (1) demonstrated that expression of this receptor correlated with local relapse in early-stage breast cancer patients. In this communication, we investigated the role that this receptor/ligand pair plays in modulating cellular responses to ionizing radiation in a mammary epithelial cell line HC11. Methods and Materials: The radiosensitivity of HC11 clonal cells transfected to overexpress either the wild-type CSF-1 receptor or CSF-1 receptor mutated at one of the two major autophosphorylation sites (TYR → PHE 807 or TYR → PHE 721) was quantitated by standard in vitro clonogenic assays. Results: We demonstrated that a signal transduction pathway regulated by the phosphorylation of TYR-807 of CSF-1 receptor appears to play a major role in controlling the radiosensitivity of murine mammary epithelial cells. Conclusions: Our observations offer insights into potential pharmacologic and gene-therapeutic approaches for the modification of radiation response of mammary neoplasms.

Development of a constitutively active mutant form of the prolactin receptor, a member of the cytokine receptor family.

The extracellular domain of the PRL receptor (PRL-R) is composed of two subdomains of approximately 100 amino acids, S1 and S2. To explore the functional significance of these subdomains in PRL binding and signal transduction, deletion mutants of S1 or/and S2 subdomains were constructed. We report here the inability of each of these mutant receptor forms to bind PRL after expression in COS-7 cells. We also studied the abilities of these different mutant receptors to respond to hormonal stimulation after transfection of each mutant complementary DNA into CHO-K1 cells along with a chimeric gene containing the promoter of a milk protein gene (beta-lactoglobulin) fused to chloramphenicol acetyltransferase coding sequence. Somewhat unexpectedly, a constitutively (PRL-independent) mutant form of the PRL-R was obtained after deletion of the S2 subdomain. Moreover, we analyzed, in CHO-K1 cells, the biological activity of chimeric receptors constructs in which each subdomain sequence was replaced by an unrelated, but coding, sequence of foreign protein, and we confirmed a specific requirement for the S1 sequence in the constitutive activity. In contrast, the S2 subdomain produced an inhibitory effect on S1 constitutive activity. Cotransfection experiments with the wild-type receptor and the constitutive mutant receptor provided evidence that the wild-type receptor was able to inhibit the constitutive activity of the deleted mutant. Furthermore, in the mouse mammary epithelial cell line HC11, the constitutive PRL-R form was able to induce transcription of the beta-casein gene in the absence of PRL. These results suggest a complex signal transduction process that implicates each extracellular PRL-R subdomain. Possible mechanisms for the constitutive effect are discussed.

The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction

Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -80 and -100 is indispensable for hormonal induction of transcription. Binding of MGF activity to DNA is greatly enhanced by the action of the lactogenic hormones. Repression of transcription in the uninduced state is mediated by a promoter element located adjacent to the MGF binding site at positions -110 to -150. This repressor element consists of two interacting protein binding sites. A nuclear factor that binds specifically to the proximal site between positions -110 and -120 has been characterized and found to be identical with the nuclear factor YY1 (delta, NF-E1). YY1 does not bind to the distal site. The simultaneous mutation in the proximal and the distal sites results in high, hormone-independent transcription. This finding suggests that YY1 plays a functional role in the repression and acts in conjunction with a second DNA binding protein. Comparison of YY1 DNA binding activity in uninduced and hormone-induced cells showed that relief of repression is not mediated by changes in the concentration or binding affinity of YY1. Infection of HC11 cells with a YY1-expressing recombinant retrovirus resulted in overexpression of YY1 but did not suppress hormonal induction. The addition of purified MGF decreased YY1 binding to its DNA recognition site in vitro. This finding indicates that MGF regulates the DNA binding activity of YY1 and thereby may cause the relief of transcriptional repression.

The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction.

Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -80 and -100 is indispensable for hormonal induction of transcription. Binding of MGF activity to DNA is greatly enhanced by the action of the lactogenic hormones. Repression of transcription in the uninduced state is mediated by a promoter element located adjacent to the MGF binding site at positions -110 to -150. This repressor element consists of two interacting protein binding sites. A nuclear factor that binds specifically to the proximal site between positions -110 and -120 has been characterized and found to be identical with the nuclear factor YY1 (delta, NF-E1). YY1 does not bind to the distal site. The simultaneous mutation in the proximal and the distal sites results in high, hormone-independent transcription. This finding suggests that YY1 plays a functional role in the repression and acts in conjunction with a second DNA binding protein. Comparison of YY1 DNA binding activity in uninduced and hormone-induced cells showed that relief of repression is not mediated by changes in the concentration or binding affinity of YY1. Infection of HC11 cells with a YY1-expressing recombinant retrovirus resulted in overexpression of YY1 but did not suppress hormonal induction. The addition of purified MGF decreased YY1 binding to its DNA recognition site in vitro. This finding indicates that MGF regulates the DNA binding activity of YY1 and thereby may cause the relief of transcriptional repression.

Interaction of Two Sequence-Specific Single-Stranded DNA-Binding Proteins with an Essential Region of the β-Casein Gene Promoter is Regulated by Lactogenic Hormones

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones.

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat β-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.

Multiple forms of prolactin receptor messenger ribonucleic acid are specifically expressed and regulated in murine tissues and the mammary cell line HC11.

The expression of PRL receptor mRNA in mouse tissues was studied. Seven PRL receptor transcripts of different sizes were found. The pattern of expression was tissue specific. The PRL receptor protein exists in isoforms of approximately 300 and 600 amino acids, which differ in the sequence and length of the cytoplasmic domain. Probes specific for the lower mol wt forms of the receptor hybridized to transcripts of 1.4, 2.4, 3.5, and 4.2 kilobases (kb), which were predominantly expressed in the liver and kidney. Among the three isoforms of the small form of the receptor, PR-3 was highly expressed, PR-2 was weakly expressed, and PR-1 was undetectable. Probes specific for the higher mol wt receptor form detected transcripts of 9 and 10 kb, expressed most strongly in the ovary, mammary gland, and kidney. An additional 8.3-kb transcript was expressed in the kidney. The PRL-responsive mouse mammary epithelial cell line HC11 expressed only the 9- and 10-kb receptor mRNAs, as did the mammary gland. The transcripts for the two PRL receptor forms were independently regulated during pregnancy and lactation in a tissue-specific manner. The expression of the small receptor form in the liver increased 7-fold during pregnancy and decreased during lactation. Its expression in the kidney remained constant. Expression of the larger receptor form increased 2.5-fold in the kidney during lactation, but remained constant in the mammary gland. In the ovary the expression of the large receptor form could be elevated 4-fold after induction of hyperovulation with FSH and hCG. Thus, in the mouse there are at least two forms of the PRL receptor regulated independently in different tissues.

Lactogenic Hormone and Cell Type-Specific Control of the Whey Acidic Protein Gene Promoter in Transfected Mouse Cells

The whey acidic protein (WAP) is a major milk protein. It is abundantly expressed in mammary epithelial cells, and its gene is controlled by lactogenic hormones. The identification of regulatory cis-acting sequences of the mouse WAP gene was so far dependent on the analysis of transgenic animals. We report here the possibility of analyzing regulatory sequences by gene transfer experiments using the lactogenic hormone-dependent mammary epithelial cell line HC11. A WAP-chloramphenicol acetyltransferase construct containing 2.5 kilobases of the 5'-flanking sequence of the WAP gene was stably transfected into HC11 cells. High chloramphenicol acetyltransferase activity was induced in pools of transfected cells cultured in the presence of the lactogenic hormones glucocorticoid, PRL, and insulin. A lower induction was observed by glucocorticoid hormone alone. PRL by itself was not able to induce the WAP gene promoter above the level observed in the absence of lactogenic hormones. A time course of hormone induction showed a weak initial response with a steady increase over at least 4 days of hormone treatment. Induction was not observable in the mammary epithelial cell line NOG-8 and NIH3T3 fibroblasts, despite the presence of functional glucocorticoid receptor in these cells. This indicates the requirement for a cell type-specific transcription factor present in the mammary epithelial cell line HC11, but not in NOG-8 epithelial cells or NIH3T3 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)