Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines

Breast cancers require the presence of estrogens for the maintenance of growth at some time in the course of their development, as does normal breast tissue. Sulfation is an important process in the metabolism and inactivation of steroids, including estrogens, because the addition of the charged sulfonate group prevents the binding of the steroid to its receptor. Also, many of the therapeutic and chemopreventive agents used in the treatment of breast cancer are substrates for the sulfotransferases (STs). The activity and expression of four cytosolic STs, which are the human phenol-sulfating and monoamine-sulfating forms of phenol ST (PST), dehydroepiandrosterone ST, and estrogen ST (hEST), were assayed in normal breast cells and in breast cancer cell lines. ST activities and immunoreactivities were assayed in the estrogen receptor-positive human breast cancer cell lines ZR-75-1, T-47D and MCF-7; in the estrogen receptor-negative breast cancer cell lines BT-20, MDA-MB-468, and MDA-MB-231; and in normal human mammary epithelial cells. The PSTs were the most highly expressed ST activities in the breast cancer cell lines, although the levels of activity varied significantly. ZR-75-1 and BT-20 cells possessed the highest levels of activity of the human phenol-sulfating form of PST. The breast cancer cell lines showed only trace levels of dehydroepiandrosterone ST and hEST activities. In contrast, hEST was the only ST detectable in human mammary epithelial cells. Understanding the regulation of ST activity in these breast cancer and normal breast cell lines will improve our knowledge of the role of sulfation in breast cancer and provide a model with which to study the mechanism of action of estrogens in mammary cells.     

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.     

Cultures of normal human mammary cells

This paper describes a method of obtaining epithelial cells from large quantities of normal human breast tissue and the response of these cells in culture to lactogenic hormones. Suspensions of single cells and clusters of cells resembling normal ductal and alveolar structures were obtained by mechanical disaggregation and subsequent (3h) incubation of tissue fragments in 0.5 mg/ml collagenase. Cells rapidly attached to glass or plastic surfaces within 48 h and grew to form large colonies which maintained their epithelial appearance throughout 2 months of observation. Cell cycling as monitored by DNA synthesis was enhanced by insulin, hydrocortisone, or ovine prolactin (in concentrations of 5.0mug/ml each) at respectively 2,3 and 5 days of incubation. These results were observed in cultures derived from 3 premenopause samples of mammary tissue maintained in medium with 1% fetal calf serum. Prolactin at a concentration of 5 mug/ml induced phosphoprotein synthesis 8-fold over control values. In addition, prolactin induced morphological changes in cells including the development of distended endoplasmic reticulum, large microvilli, and the deposition of glycogen granules. These initial results led to the tentative conclusion that prolactin was sufficient to initiate some of the characteristics in cultured cells normally associated with lactating tissues.     

Overexpression of p27Kip1 inhibits the growth of both normal and transformed human mammary epithelial cells

We previously reported increased expression of p27Kip1 in a series of human breast cancer cell lines, as compared to cell lines established from normal mammary epithelial cells. These data were surprising because this protein exerts a growth-inhibitory function in normal cells, and p27Kip1 has been proposed as a candidate tumor suppressor gene. A possible explanation for the paradoxical increase in p27Kip1 in the breast cancer cell lines is that they had become refractory to the inhibitory effects of this protein. To address this question, here, we transfected the MCF7 breast cancer cell line and the MCF10F nontumorigenic mammary epithelial cell line with a vector containing the p27Kip1 cDNA to obtain derivatives that express increased levels of p27Kip1. The increased expression of p27Kip1 in both of these cell lines was associated with lengthening of the G1 phase, an increase in the doubling time, a decreased saturation density, and a decreased plating efficiency. In the MCF7 cells, anchorage-independent growth and in vivo tumorigenicity were also suppressed. These effects were associated with decreased cyclin E-associated in vitro kinase activity in both cell lines. The increased expression of p27Kip1 was associated with a decreased level of expression of cyclin D1 in the MCF10F cells but an increased level of the cyclin D1 protein in the MCF7 cell line. Both derivatives expressed slightly increased levels of the cyclin E protein. Thus, breast cancer cells are still responsive to p27Kip1-mediated inhibition of cell growth despite the high basal level of this protein. These results suggest that therapeutic strategies that further increase the level of expression of p27Kip1 or mimic its activity might be useful in cancer therapy.     

The epidermal growth factor receptor is not required for tumor necrosis factor-alpha action in normal mammary epithelial cells

Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.     

Estrogen receptor-negative epithelial cells in mouse mammary gland development and growth

Records of violent behaviour and its sequels gain increasing interest as a matter of quality assurance. The paper presents scales and instruments applied in psychiatric institutions until now. While problems of reliability are solved sufficiently, there are major problems of validity in the measurement of violence. To be usable for quality management, a documentation has to provide a clear cut-off for the severity of violent incidents that should be reported. Otherwise uncomparable data and underreporting will result.     

Promotion-sensitive epidermal and mammary epithelial cells maintained in suspension over agarose

The molecular basis of tumour promotion is still largely unknown. In in vitro model of tumour promotion, the promotion-sensitive cells are induced to grow under anchorage-independent conditions in the presence of promoting agent. The customary way of providing such conditions is to immobilize these cells in soft agar, but such cells cannot be readily recovered to study the induced biochemical and molecular events. In the present report, we analysed these events using JB6 mouse epidermal cells maintained in suspension in liquid medium over agarose. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent synthesis of DNA in promotion-sensitive P+ (but not in promotion-resistant P-) JB6 cells and this TPA-induced synthesis of DNA positively correlated with TPA-induced formation of colonies in soft agar. The TPA-induced synthesis of DNA began on or shortly before 24 h after the introduction of TPA, peaked at about 48 h and then declined to the control levels over the next several days. All trans-retinoic acid and dexamethasone inhibited and calcitriol (1 alpha,25-dihydroxy-vitamin D3) synergistically stimulated this TPA-induced DNA synthesis. Western immunoblot analysis of cyclins (A, B1, D1 and E) and p27Kip1, a cyclin-dependent kinase inhibitor, indicated that TPA induced cyclin A and cyclin B1 expression in P+ (but not in P-) JB6 cells and this induction coincided in time with TPA-induced synthesis of DNA. TPA also strongly induced cyclin D1 expression in P+ (but not in P-) JB6 cells, but this induction started prior to the expression of cyclin A and cyclin B1. TPA did not affect the expression of either cyclin E or p27Kip1 to any significant extent. We also found that NMU38 rat mammary epithelial cells were operationally equivalent to the promotion-sensitive P+ JB6 cells, but in these cells 17 beta-oestradiol exerted a strong synergistic effect on TPA-induced synthesis of DNA. Based on these observations, we tentatively propose a sequence of molecular events which possibly lead to the anchorage-independent synthesis of DNA in these cells.     

Surface proteins of normal and transformed epithelial liver cells

Surface protein patterns of 12 various tumorigenic and non-tumorigenic liver epithelial cell lines (IAR series) were studied by means of lactoperoxidase catalyzed iodination followed by the gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis. These patterns were found to be very similar except a protein with an apparent mol. wt. about 150 kilodaltons which appeared in cells of tumors derived from in vitro transformed epithelial liver cells. Surface fibronectin was present in all the three non-tumorigenic lines, and in 7 of 9 tumorigenic cell lines examined. The indirect immunofluorescence revealed that in non-tumorigenic lines this protein was localized predominantly in the regions of cell-to-cell contacts, while in tumorigenic cells fibronectin often formed fibrillar networks similar to those of fibroblasts.     

Autocrine regulation of protein secretion in mouse mammary epithelial cells

Milk secretion is under autocrine control by an inhibitory milk protein, named FIL (feedback inhibitor of lactation). Lactating mammary acini and epithelial cells cultured on reconstituted basement membrane (EHS matrix) with lactogenic hormones were used to study the characteristics of autocrine inhibition. FIL inhibited milk protein secretion in lactating acini, but not in epithelial cells on EHS matrix. The latter's insensitivity to FIL was due to formation of multicellular structures termed mammospheres, in which cell surrounded a central luminal space. Cell polarization, and the formation of tight intercellular junctions prevented FIL access to the apical cell surface, which faced the mammosphere lumina. When apical access was permitted either by incomplete mammosphere formation or EGTA treatment, FIL inhibited mammosphere protein secretion to the same extent as in lactating acini. The study shows that autocrine inhibition by FIL occurs specifically through interaction with the apical surface of the mammary epithelial cell, and suggests the presence of a FIL receptor on this, but not the basolateral cell membrane.     

Influence of Streptococcus dysgalactiae surface hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary macrophages

Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells. Streptococcus dysgalactiae (n = 60) isolated from bovine intramammary infections were examined for expression of SH after growth in Todd-Hewitt broth (THB) and THB supplemented with skim milk, whey, lactose, and casein. Strains were significantly more hydrophobic after growth in THB and THB plus whey and more hydrophilic after growth in THB plus skim milk. Both trypsin and proteinase K abolished SH in three strains tested. Mild pepsin treatment had little effect on SH, while heat treatment at 70 degrees or 80 degrees C abolished SH in two strains tested. A hydrophilic strain of S. dysgalactiae did not adhere as well to bovine mammary epithelial cells as a hydrophobic strain. Trypsin treatment significantly reduced adherence of a hydrophobic strain of S. dysgalactiae to epithelial cells while adherence of a hydrophilic strain remained unaltered. A hydrophilic strain of S. dysgalactiae was significantly more resistant to phagocytosis by bovine mammary gland macrophages than a hydrophobic strain. Differences in expression of SH may play an important role in determining the ability of S. dysgalactiae to establish successfully within the mammary gland.     

Growth factor dependency and gene expression in preneoplastic mouse mammary epithelial cells

The TM preneoplastic mammary outgrowth lines were established in vivo from mammary epithelial cell lines and have been characterized with respect to their tumorigenic, morphological, and functional properties. The TM outgrowth lines were then established as in vitro cell lines. A comparison of the growth factor dependencies of the TM preneoplastic lines and their progenitor cell lines grown in monolayer cell culture indicated that the TM preneoplastic cell lines exhibited a decreased dependence on epidermal growth factor for growth in vitro. The exception to this result was the TM3 cell line which still exhibited a marked dependence on epidermal growth factor for growth. An examination of several genes for mRNA levels indicated that the expression of c-neu, c-H-ras, c-myc, and retinoblastoma was not elevated in those TM preneoplasias which exhibited a decreased dependence on epidermal growth factor. Additionally, there was no evidence that c-H-ras was activated in the preneoplastic outgrowths or tumors. In contrast, mouse mammary tumor virus long terminal repeat mRNA was increased in preneoplasias and tumors, whereas gelsolin mRNA was decreased in tumors but not in preneoplasias. The down-regulation of gelsolin mRNA is consistent with recent reports in human breast cancers. These results together with those reported in another paper (D. Medina et al., Cancer Res., 53: 663-667, 1993) indicate that the TM3 outgrowth line is a minimally deviated preneoplasia which does not share many of the molecular alterations exhibited in tumorigenic TM preneoplastic outgrowth lines. These data, along with other recent data, are interpreted in a hypothesis which views the three essential characteristics of the mammary preneoplastic state as independent and dissociable genetic alterations. Importantly, each of the three characteristics is independently isolated in one or more of the in vivo outgrowth populations. These outgrowth lines should allow identification of the nature and function of the molecular alterations associated with each stage of mammary preneoplasia.     

Alteration of oestradiol metabolism in myc oncogene-transfected mouse mammary epithelial cells

Targeted overexpression of the c-myc oncogene induces neoplastic transformation in immortalized, non-tumorigenic mouse mammary epithelial cells (MMEC). Experiments in the present study were conducted to examine whether cellular transformation induced by c-myc oncogene is associated with altered metabolism of 17beta-oestradiol (E2). The parental, MMEC and the stable c-myc transfectant (MMEC/myc3) cell lines were compared for major oestrogen metabolic pathways, namely E2 and E1 interconversion, and C2- and C16alpha-hydroxylation by both high-pressure liquid chromatography (HPLC) analysis and the 3H release assay using specifically labelled [C2-3H]E2 or [C16alpha-3H]E2. The reductive conversion of E1 to E2 was about 14-fold and 12-fold higher than the oxidative conversion of E2 to E1 in MMEC and MMEC/myc3 cells respectively. However, in MMEC/myc3 cells, both reductive and oxidative reactions were decreased by about 32% and 12% relative to those seen in the parental MMEC cells (P = 0.0028). The extent of C16alpha-hydroxylation was increased by 164.3% (P < 0.001), with a concomitant 48.4% decrease (P < 0.001) in C2-hydroxylation in MMEC/myc3 cells; this resulted in a fourfold increase in the C16alpha/C2 hydroxylation ratio in this cell line. Thus, a persistent c-myc expression, leading to aberrant hyperproliferation in vitro and tumorigenesis in vivo, is associated with an altered oestrogen metabolism. However, it remains unclear whether this represents a result of oncogene expression/activation or is rather a consequence of phenotypic transformation of the cells.     

Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells

Binding data point to the coexistence of three endothelin receptors (ET-R) in rat heart myocytes. Induction of phosphoinositide hydrolysis in this preparation by endothelins (ET-1 and ET-3) or sarafotoxins (SRTX-b and SRTX-c) was demonstrated by measurement of labeled inositol phosphate generation. Pertussis toxin (PT) enhanced the induction of phosphoinositide hydrolysis by all four peptides. The process seems to be mediated by at least two heterotrimeric G-proteins, the one sensitive and the other insensitive to PT. Measurement of GTPase activity induced in rat myocytes clearly indicates for the first time the direct functional coupling between ET-R and a G-protein. These GTPase activity experiments provide evidence that phosphoinositide hydrolysis is stimulated via functional coupling between the endothelin receptor of the ETA-R subtype and a PT-insensitive G-protein, Gq/11. The involvement of PT-sensitive G-proteins, i.e. Gi-like and/or Go-like proteins, in the signal transduction pathways of ETs and SRTXs is discussed.     

New model of ErbB-2 over-expression in human mammary luminal epithelial cells

The ErbB-2 receptor has been strongly implicated in the development of breast cancer. To establish a new model system to investigate the role of erbB-2 in tumorigenesis of the breast, the conditionally immortalised human mammary luminal epithelial cell line HB4a was transfected with erbB-2 cDNA. Biological and biochemical characterisation of the resulting cell lines demonstrated that high levels of ErbB-2 expression were sufficient to cause transformation in vitro but did not cause tumours in vivo. Transformation by overexpression of ErbB-2 correlated with ligand-independent tyrosine phosphorylation of ErbB-2 and the adaptor protein Shc. Over-expression of ErbB-2 also resulted in the ligand-independent constitutive association between Shc and another adaptor protein, Grb2, indicating that receptor activation was sufficient to activate downstream signalling pathways. Using the model described, it was found that elevation of ErbB-2 expression levels caused marked quantitative and qualitative alterations in responses to the ligands epidermal growth factor and heregulin. Data indicate a central role for ErbB-2 in mediating the responses induced by these ligands and suggest that these altered ligand-dependent responses play an important role in tumorigenesis in vivo.     

Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro

Phenotypically stable cultures of untransformed mouse mammary epithelial cells (denoted 31EG4) were established and utilized to investigate the lactogenic hormone (glucocorticoids, insulin, and prolactin) regulation of tight junction formation. When 31EG4 cells were grown on permeable supports for 4 days in medium containing the synthetic glucocorticoid dexamethasone and insulin, confluent cell monolayers obtained a transepithelial electrical resistance (TER) of 1000-3000 omega.cm2. In contrast, over the same time period, confluent monolayers treated with insulin or insulin and prolactin maintained a low TER (35-150 omega.cm2). Consistent with the formation of tight junctions, apical to basolateral paracellular permeability was decreased from 12% to 1% for [14C]mannitol and 3.3% to 0.3% for [3H]inulin when cells were cultured in dexamethasone. This effect of dexamethasone on TER required extracellular calcium, de novo protein synthesis, dose-dependently correlated with glucocorticoid receptor occupancy, and was not due to an increase in cell density. As shown by direct and indirect immunofluorescence microscopy, dexamethasone treatment did not modulate the production or location of filamentous actin, the tight junction protein ZO-1, or the cell adhesion protein E-cadherin. Our results suggest that glucocorticoids play a fundamental role in the function and maintenance of cell-cell contact in the mammary epithelia by inducing the formation of tight junctions.     

Identification of an alpha2-macroglobulin receptor in human mammary epithelial cells

Several cases of zinc (Zn) deficiency in human infants caused by abnormally low concentrations of Zn in breast milk were recently reported, the underlying mechanism of which is not known. Alpha2-macroglobulin (alpha2-M), a major Zn-binding ligand in serum, presents a potential vehicle for mammary Zn uptake. This study was conducted to determine if an alpha2-M receptor is present in human mammary epithelial cells, where it may be involved in the endocytosis of alpha2-M into the mammary gland. Normal human mammary epithelial cells were grown to confluency in serum-free medium. For all binding and uptake studies, alpha2-M, preactivated with methylamine and labeled with 125I, was added to cells for varied lengths of time to determine saturation over time and at varied concentrations to determine saturation over increasing concentration of ligand. Nonspecific and competitive binding were measured by addition of a 100-fold molar excess of unlabeled alpha2-M and serum albumin or lactoferrin, respectively. Binding at 4 degreesC was specific for alpha2-M and approached saturation kinetics at 56 nmol/L. Scatchard plot analysis of the binding data demonstrated more than one binding site: a high affinity, saturable binding site and a low affinity, nonsaturable binding site. Uptake of alpha2-M at 37 degreesC was rapid and continuous over increasing concentrations of alpha2-M, and internalized alpha2-M was rapidly degraded. Results from this study present evidence for receptor-mediated uptake of alpha2-M in human mammary epithelial cells, which in turn, provides a potential mechanism for Zn acquisition by the cell.