Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2′-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley-Liss, Inc.     

Differential expression of cholinephosphotransferase in normal and cancerous human mammary epithelial cells

Membrane phospholipids as well as fatty acid profile of cell membrane phospholipids are altered in tumorigenicity and malignancy. Synthesis of total cellular phosphatidylcholine (PC) can be used as a marker for membrane proliferation in neoplastic mammary gland tissues. Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of PC, has an important role in regulating the acyl group of PC in mammalian cells. In this study, the effect of neoplasia on CPT was examined. The gene shows an elevated expression in cancerous (11-9-14) breast epithelial cell line when compared to that of normal non-tumorigenic (MCF-12A) breast epithelial cell line. Four nucleotide substitutions are observed in the cancer cell line. Of these, three are null mutations, but the third one shows an interesting serine to tyrosine substitution (at amino acid position 89 of our partial sequence which corresponds to position 323 of the CPT sequence reported as NM_020244 in GenBank) in 11-9-14 cells. The tyrosine is present in the right context of KSELYQDT, which directs tyrosine phosphorylation at the tyrosine site. Biochemical approach also reveals a 1.5-fold stimulation in CPT activity in 11-9-14 cells compared to that of the MCF-12A cells.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

Maintenance of normal rat mammary epithelial cells by insulin and insulin-like growth factor 1

Normal rat mammary epithelial cells were cultured within a rat tail collagen gel matrix formed under improved conditions for controlling pH and osmolarity. Under these conditions, growth can be maintained for up to 3 weeks with a 10- to 15-fold increase in cell number. The cells grow in response to prolactin, progesterone, epidermal growth factor, and cholera toxin, in a medium of DME: Ham's F12 supplemented with BSA and insulin at 10 micrograms/ml. When the insulin concentration was reduced to more physiological levels (10 ng/ml) the cells did not grow. However, at these more physiological concentrations it could be shown that insulin had a concentration-dependent effect on the maintenance of the cells with an optimum concentration around 25 ng/ml. The cells could be maintained in hormone-supplemented medium with low levels of insulin in a quiescent state for up to 14 days. The high levels of insulin needed for optimal growth could be replaced by insulin-like growth factor 1 (IGF-1) at much lower concentrations (25-50 ng/ml). The superphysiological level of insulin required for optimum growth is probably due to its acting weakly through an IGF-1-mediated growth-promoting mechanism. Insulin's effect on cell maintenance occurs at physiological levels and may better reflect its role in mammary cell growth.     

2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.     

Proliferative effects of insulin analogues on mammary epithelial cells

Structural modification of insulin results in the generation of insulin analogues that show altered binding affinities to the insulin receptor and/or IGF-I receptor. As a consequence these insulin analogues may have increased mitogenic potency. Nine benign or malignant human mammary epithelial cells, which show different insulin receptor and IGF-I receptor expression patterns, were studied regarding mitogenicity of insulin and insulin analogues. Only insulin glargine showed a significantly higher proliferative effect on MCF-7 breast cancer cells compared to regular insulin among a panel of short- or long-acting insulin analogues, that are in clinical use.     

Response of cultured normal human mammary epithelial cells to X rays

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique.     

Estrogen synthesis in human colon cancer epithelial cells

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [³H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis–Menten kinetic with apparent Km values of 20 nM and V_max values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 μM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 μM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.     

The effect of recombinant human tumour necrosis factor on growth and macromolecular synthesis of human epithelial cells

We have studied the effect of recombinant human tumour necrosis factor (rHuTNF) on growth and macromolecular synthesis in a range of normal and transformed epithelial cell types. Tumour necrosis factor did not affect the growth of normal human mammary epithelial cells, but its growth-inhibitory action on the SV40-transformed human mammary epithelial cell line HBL-100 increased with passage number in association with a progression of malignant phenotype. However, of two lines derived from nude mouse tumours of HBL-100 lines, one, HBLT-12, did not respond to rHuTNF, and the other, HBLT-11 showed some growth stimulation by high dose rHuTNF. Macromolecular synthesis in HBLT-11 was not affected by rHuTNF. The breast cancer cell lines MCF-7 and BT20 were sensitive to the cytotoxic effects of rHuTNF. In MCF-7 a gradual decrease in RNA and DNA synthesis occurred over 48 h, ending with an accumulation of cells in S and G2 phase of the cell cycle and cell death. The addition of alpha- or gamma-interferon increased, but did not accelerate the cytotoxicity of rHuTNF.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

The activation of phosphatidylinositol-specific phospholipase C by insulin in mammary epithelial cells of lactating mouse

We have previously demonstrated in vitro that, in the endoplasmic reticulum and Golgi apparatus of mammary epithelial cells of lactating and pregnant mice, inositol 1,4,5-trisphosphate releases Ca2+ that has been stored in these organelles. In this study, we examined whether insulin and prolactin, essential for the growth of mammary gland and for lactation, influenced the activity of phosphatidylinositol-specific phospholipase C in mammary cells. In the plasma membrane fraction of mammary epithelial cells of the DDY mouse strain 5 days after the start of lactation after the first pregnancy, and with phosphatidylinositol as substrate, it was shown that the activity of phospholipase C was enhanced by about four times in the presence of insulin compared with the control. Such enhancement was not found in the membrane fraction treated with prolactin.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

Interactions between normal mammary epithelial cells and mammary tumour cells in a model system

Abstract. Normal mammary epithelial (NME) cells and MCF-7 cells aggregate and grow as spheroids when cultured on extracellular matrix derived from Engelbreth/ Holmes/Swarth (EHS) tumour. NME cells stop dividing and differentiate but MCF-7 cells continue to proliferate, although growth is counterbalanced by cell death. In mixed cultures of NME cells and MCF-7 cells, the two cell types form mixed aggregates but then segregate to form well separated domains, often joined by only a narrow neck of cells. In these mixed cultures the growth of MCF-7 cells is inhibited by a factor secreted by NME cells into the medium.     

Serum-free primary culture of human normal mammary epithelial cells in collagen gel matrix

Collagen gel matrix has been used successfully to promote sustained growth of human normal mammary epithelial cells in primary culture using serum-containing medium supplemented with hormones and growth factors (Nandi et al., 1932). Sustained growth can now be accomplished in a serum-free medium consisting of a 1:1 mixture of Ham's F12 and DMEM supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, cortisol, and BSA. Human normal mammary epithelial cells derived from reduction mammoplasties can be routinely propagated in this serum-free medium. The extent of growth and the resulting three-dimensional outgrowths in this serum-free medium, using the collagen gel matrix system, are comparable to those seen in serum-containing medium. This is the first demonstration of sustained growth of human normal mammary epithelial cells in serum-free primary culture.     

Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines

Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off‐line thin layer chromatography (TLC)‐MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47‐D, and MDA‐MB‐231), using TLC‐MS, validated by hydrophilic interaction liquid chromatography‐MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non‐malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non‐malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy. J. Cell. Physiol. 228: 457–468, 2013. © 2012 Wiley Periodicals, Inc.