Jeffrey D. Turner

Inhibition of cellular proliferation and modulation of insulin-like growth factor binding proteins by retinoids in a bovine mammary epithelial cell line.

Retinoids are potent inhibitors of growth and tumor progression in many mammary carcinoma cell lines, though regulation of growth in nontumorigenic mammary epithelial cells by retinoids is less clear. Here, we have characterized the inhibition of MAC‐T (a nontransformed bovine mammary epithelial cell line) cellular proliferation by retinoids and their role in regulating insulin‐like growth factor binding proteins (IGFBPs). Retinoic acid (RA) (100 nM) was a potent inhibitor of MAC‐T cell proliferation. Retinol was 10–100 times less effective. Neither retinoid could completely arrest growth at noncytotoxic concentrations. Retinoic acid inhibited cellular proliferation by 1 h (P < .05), but inhibition was fivefold greater by 24 h (P < .01). This second stage of growth inhibition (after 12 h) was dependent upon protein synthesis. However, RA‐induced inhibition of cellular proliferation did not persist, with thymidine incorporation increasing toward control levels by 4 days in culture. Retinoic acid was less effective in inhibiting thymidine incorporation when cells were stimulated with insulin, des(1–3) IGF‐I, or Long(R3) IGF‐I when compared to cells stimulated with native IGF‐I or serum. Inhibition of proliferation by RA was associated with increased levels of IGFBP‐2 in conditioned media and in plasma membrane preparations. Treatment with insulin or des(1–3) IGF‐I resulted in the appearance of IGFBP‐3 in conditioned media and on the cell surface. However, RA significantly reduced IGFBP‐3 levels in conditioned media and eliminated IGFBP‐3 associated with the plasma membrane. Thus, RA is a potent but transient inhibitor of bovine mammary epithelial cell proliferation, and this growth inhibition is correlated with increased IGFBP‐2 accumulation and inhibition of IGF‐I stimulated IGFBP‐3 protein secretion.

IGF-I-Induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29-32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditions by MAC-T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

CHARACTERIZATION OF LACTOFERRIN BINDING TO THE MAC-T BOVINE MAMMARY EPITHELIAL CELL LINE USING A BIOTIN-AVIDIN TECHNIQUE

1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line. 2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable. 3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete. 4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (Ka) of 2.36 x 10(7) and 3.36 x 10(6) M-1. 5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins

Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.

Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2).

A spontaneously established porcine granulosa cell line (PGC‐2) was cloned through the continuous culturing of primary granulosa cells collected from equine chorionic gonadotropin (eCG)‐treated prepubertal gilts. This established cell line has undergone ∼︁ 100 passages and shows contact‐inhibition of growth. PGC‐2 stained with a monoclonal antibody (mAb) directed against cytokeratin, indicating its epithelial nature, but not with a mAb directed against vimentin, suggesting that it is not fibroblast derived. Immunoblotting revealed that PGC‐2 expresses cadherin, an epithelial Ca??? dependent cell adhesion molecule. The cells were dependent on serum for growth and had a doubling time of ∼︁ 20 hr when cultured with 10% fetal bovine serum. The cell line was examined for the presence of FSH receptors, cAMP responses, and steroidogenic capabilities. The cell line lacks FSH receptors as assessed by radiolabelled‐ligand binding, and no transcripts for FSH receptor were detected by Northern blotting of total cellular RNA. Neither FSH nor cholera toxin (0.5 ng/mL) stimulated increases in cAMP levels in these cells, whereas forskolin 10 μM) induced a fivefold increase in cAMP production. When a higher concentration of cholera toxin (300 ng/mL) was used, however, cAMP levels doubled by 2 hr. Despite a lack of responsiveness to purified oFSH or oLH, the cells were capable of progesterone and estradiol production when provided with the appropriate substrates. We conclude that PGC‐2 display properties that are similar to immature granulosa cells and may provide a suitable in vitro model for the study of granulosa cell function.

Lactogenic hormones and extracellular matrix regulate expression of IGF-1 linked to MMTV-LTR in mammary epithelial cells

The cell line MD-IGF-1, containing an ovine IGF-1 cDNA driven by the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter, was used to study expression of IGF-1 linked to the MMTV-LTR in bovine mammary epithelial cells in response to various hormonal and substratum stimuli. Acute sensitivity of the MMTV-LTR promoter to glucocorticoids and sex steroids was ascertained by transient transfection of parental MAC-T cells with an MMTV-CAT construct. Specifically, CAT activity was induced by glucocorticoids, but not by 17 beta-estradiol or progesterone. Induction of MD-IGF-1 cells with dexamethasone (DEX) alone triggered a 29.5-fold increase in secretion of recombinant IGF-1 (348.9 vs 11.8 pg/micrograms DNA), and stimulated a 1.7-fold increase in total DNA within 72 h. Growth of MD-IGF-1 cells was enhanced by exogenous IGF-1, insulin, and TGF-alpha. In contrast, TGF-beta inhibited cell proliferation, while epidermal growth factor, estrogen, progesterone, and testosterone had no effect. Extracellular matrix from the Engelbreth-Holm-Swarm (EHS) tumor, in the presence of DEX, prolactin (PRL), and insulin stimulated a 29.4-fold increase in secretion of IGF-1 (591.9 pg/microgram DNA), compared with cells in absence of hormones (20.1 pg/micrograms DNA). EHS and DEX plus PRL triggered a 63.2-fold increase in IGF-1 secretion (689.1 pg/micrograms DNA), compared with MD-IGF-1 cells cultured on plastic (10.9 pg/micrograms DNA), in the absence of hormones. These data indicate that the MMTV-LTR is regulated by both lactogenic hormones and extracellular matrix in MD-IGF-1 cells and that the MMTV-LTR may be a useful regulatory element for targeting expression of foreign proteins in bovine mammary epithelial cells.

Advanced Spider Silk Fibers by Biomimicry

Spider silk is an ancient biomaterial that is useful for modern medicine and industry. This chapter explains the relevant spider biology, outlines the technical limitations of producing recombinant silk from published literature and describes how biomimicry works. The process begins with spider silk genes, production of recombinant dragline silk proteins in vitro and within in vivo lactation systems, and ends with the purification of silk proteins and their conversion into continuous silk fibers. Finally, this chapter offers a view to the future of the potential for nature inspired performance biomaterials

Chapter 13 - Culture of Mammary Tissue: Glucose Transport Processes

This chapter discusses the glucose transport processes. The chapter discusses the epithelial cells of the kidneys and the small intestine that transport glucose against a concentration gradient via a Na + -dependent transporter. In addition, in the same epithelial cells, Na + -independent glucose transporters are also identified. Mammary epithelial cells utilize large quantities of glucose because the monosaccharide is the precursor of lactose, the major carbohydrate found in milk of most mammalian species. The types of glucose transporters present in mammary epithelial cells have never been determined. The chapter outlines the experiments with an objective to determine: (1) if glucose transport by mammary epithelial cells was Na + -dependent or Na + -independent; (2) changes in glucose uptake by mammary epithelial cells at different stages of growth; and (3) the effect of glucose concentration on its accumulation by mammary epithelial cells at different stages of growth. The experiments demonstrated that monolayers of bovine mammary epithelial cells do not accumulate glucose in a Na + -dependent manner. The uptake of glucose reaches a maximum during the growth phase of the cells and diminishes as the cells reach confluency. The transport system(s) is regulated by the glucose concentration in the culture medium.

The Expression of Protooncogenes in Skeletal Muscle

Oncogenes are DNA sequences that when expressed lead to rapid cellular proliferation characteristic of malignant transformation. The nontransforming normal cellular cognates of the oncogenes are protooncogenes. These sequences are highly conserved and are thought to be involved in the process of normal cell replication (Bishop, 1983).