Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S₁-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.     

Establishment of bovine mammary epithelial cells (MAC-T): An in vitro model for bovine lactation

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic “cobblestone” morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in β-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) α_s- and β-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

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.     

Cloning, expression, purification, distribution and kinetics characterization of the bacterial β-galactosidase fused to the cytoplasmic transduction peptide in vitro and in vivo

Cytoplasmic transduction peptide (CTP) offers exciting therapeutic opportunities for the treatment of many diseases caused by cytoplasmic functional molecules. It can transduce large, biologically active proteins into the cytoplasmic compartment of several mammalian cells. However, other intriguing features of CTP, including its activity in vitro, and distribution and tissue infiltration abilities in vivo, remain to be explored. The present study was initiated to (1) further confirm the cytoplasmic localization preference and the enzymatic activity of the transduced CTP-β-gal in vitro and (2) examine the kinetics and tissue distribution of the CTP-β-gal fusion protein in mice. A CTP-β-gal fusion protein was expressed in Escherichia coli and either transduced into BaF3-BCR/ABL cells or administered intravenously into female Balb/C mice at a dose of 100 μg per mouse. Its localization in BaF3-BCR/ABL cells was evaluated by immunocytochemistry and in situ X-gal staining, and its distribution in various tissues was analyzed both by in situ X-gal staining and quantitative enzymatic activity assay. β-Galactosidase enzyme activity was observed in BaF3-BCR/ABL cells and in all tissues tested, with peak activity occurring at 15 min in most tissues and at 24 h in brain. These data will not only allow rational selection of delivery schedules for therapeutic CTP, but will also aid the use of CTP fusion protein transduction in the development of protein therapeutics targeting the cytoplasmic compartment both in vitro and in vivo.     

Pten Regulates Development and Lactation in the Mammary Glands of Dairy Cows

Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of β-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for β-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPARγ, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of β-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased β-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of β-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.     

In vivo evaluation of epidermal growth factor and transforming growth factor β1 in mouse tumor models

The influence of modulating circulating levels of epidermal growth factor (EGF) and transforming growth factor β₁ (TGF-β₁) on tumorgrowth was examined in a variety of mouse models. Removal of the EGF-rich submandibular gland from host mice failed to alter the growth of a variety of human tumor xenografts or a C3H mouse tumor. Infusion of EGF from Alzet minipumps raised circulating EGF levels. However, only the A549 human tumor xenograft showed any significant increase in growth in the presence of EGF infusion and this response was marginal. The growth of Wehi 3BD+ and A549 tumor lines in culture was inhibited by TGF-β₁. The growth of these lines in vivo, however, was not significantly altered by the administration of TGF-β₁ via a variety of routes.     

The Association of the Nm23-M1 Protein and β-Tubulin Correlates with Cell Differentiation

The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with β-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/β-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with β-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with β-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/β-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with β-tubulin and that the number of complexes increases during the differentiation process of murine cells.     

In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

Background

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

Methodology

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

Principal Findings

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

Conclusions

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).     

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.     

常规培养与缺氧条件下BCG菌体内外sRNA的表达检测

近期发现细菌的sRNA在菌体内和菌体外均具有一定的生物学功能。为研究结核分枝杆菌菌体内外sRNA的表达情况,通过分析卡介苗(Bacillus Calmette-Guerin Vaccine, BCG)菌体和外泌体RNA测序结果,采用RT-qPCR法检测常规培养与缺氧条件下BCG菌体内外sRNA相对表达量,分析菌体内外sRNA谱的差异。结果显示,常规培养时,菌体内丰度较高的sRNA为MTS2823、MTS1338与ASdes,菌体外丰度较高的为Mcr3、MTS2823和AS1890。菌体内受缺氧诱导表达增加的是MTS2823、MTS1338、MTS0997和G2。其中MTS1338与G2的启动子区发现了DosR的结合基序。无论是否缺氧,MTS0997、G2、Mcr7和AS1890在菌体外的相对表达量均高于菌体内,而C8只在缺氧时菌体外表达增高。研究揭示了BCG菌体内外sRNA表达谱不同,且一部分sRNA在常规培养和缺氧应激时向菌体外释放,同时发现了细菌受缺氧诱导的sRNA种类。     

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.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Expression of β1 Integrins by Cultured Articular Chondrocytes and in Osteoarthritic Cartilage

Expression of β1 integrins was studied in vitro as articular chondrocytes reestablished a matrix in culture and in situ in a nonhuman primate model of osteoarthritis in order to investigate a potential role for integrins in mediating cell-extracellular matrix interactions in cartilage. Chondrocytes were found to express α1β1, α3β1, and α5β1 integrins both in vitro and in situ. Cell surface expression of β1 integrins increased as chondrocytes were maintained in culture from 3 to 7 days. Increased β1 integrin expression was also observed in osteoarthritic cartilage compared with normal cartilage. The greatest relative increase in both systems was noted for the α1β1 integrin. The increase in chondrocyte β1 integrin expression in vitro was noted in both monolayer and alginate cultures and occurred prior to detectable changes in the differentiated phenotype of the chondrocyte. Disruption of the cytoskeleton with the drug dihydrocytochalasin B inhibited the cell culture induced increase in integrin expression, while treatment of cultured cells with TGF-β resulted in increased expression of the α5β1 integrin. The modulation of β1 integrin expression noted in vitro and in situ indicates that chondrocytes are capable of regulated expression of β1 integrins and suggests that β1 integrins may play an important role in mediating chondrocyte-extracellular matrix interactions in cartilage.     

Sesquiterpenoids from Teucrium ramosissimum

An antiplasmodial bioguided investigation of the EtOAc extract of the aerial parts of Teucrium ramosissimum led to isolation and identification of three sesquiterpenoids, teucmosin, 4α-hydroxy-homalomenol C, 1β,4β,7α-trihydroxy-8,9-eudesmene and two trinorsesquiterpenoids, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione and 1β,4β-dihydroxy-11,12,13-trinor-8,9-eudesmen-7-one together with five known sesquiterpenoids, oplopanone, homalomenol C, oxo-T-cadinol, 1β,4β,6β-trihydroxyeudesmane, 1β,4β,7α-trihydroxyeudesmane and four flavonoids, 5-hydroxy-7,4′-dimethoxyflavone, salvigenin, genkwanin and cirsimaritin. The structures and the relative stereochemistry were elucidated by extensive spectroscopic studies including 1D and 2D NMR and mass spectrometry (MS). Homalomenol C, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione, oxo-T-cadinol and 1β,4β,6β-trihydroxyeudesmane displayed a significant in vitro antiplasmodial activity against Plasmodium falciparum with IC₅₀ values ranging from 1.2 to 5.0 μg/ml. Furthermore, no cytotoxicity was observed upon the human diploid lung cell line MRC-5 for these compounds.     

Early Stages of Notochord and Floor Plate Development in the Chick Embryo Defined by Normal and Induced Expression of HNF-3β

We have cloned a cDNA encoding the chick HNF-3β gene and have used RNA and antibody probes that detect HNF-3β to monitor the normal and induced expression of the gene in early embryos. HNF-3β is expressed in Koller's sickle, at the onset of primitive streak formation, and later in Hensen's node. At neural plate and neural tube stages, HNF-3 β is expressed transiently in the notochord and is then expressed by floor plate cells. Prospective floor plate cells that are located in the epiblast immediately anterior to Hensen's node prior to its regression do not express HNF-3β, providing evidence that floor plate fate is normally determined only after these cells populate the midline of the neural plate and overlie the notechord. Removal of the notochord in vivo prevents floor plate development and in this condition HNF-3β is not expressed by cells at the ventral midline of the neural tube. Notochord grafts induce ectopic floor plate development and ectopic neural expression of HNF-3 β. In vitro, neural plate explants are induced to express HNF-3β by notochord cells in a contact-dependent but cycloheximide-resistant manner, providing evidence that expression of HNF-3 β is a direct response of neural plate cells to notochord-derived inducing signals.     

Small Molecule Antagonists of the Wnt/Beta-Catenin Signaling Pathway Target Breast Tumor-Initiating Cells in a Her2/Neu Mouse Model of Breast Cancer

Background

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.

Methods/Findings

To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118–310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118–310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118–310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118–310 treated mice were unable to seed the growth of secondary tumors after transplant.

Conclusions

These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.