Malignant Mammary Epithelial Cells Research Articles

ONCOSTATIN M-SPECIFIC RECEPTOR EXPRESSION AND FUNCTION IN REGULATING CELL PROLIFERATION OF NORMAL AND MALIGNANT MAMMARY EPITHELIAL CELLS

Oncostatin M (OSM) is a cytokine produced by activated T lymphocytes and macrophages. OSM is structurally and functionally related to leukaemia inhibitory factor (LIF), another cytokine in the interleukin 6 (IL-6) family. The biological activities of OSM are mediated through two types of receptor complexes, the LIF/OSM shared receptor (type I) and OSM-specific receptor (OSM-R type II), which is composed of gp130 as a binding subunit and a newly identified affinity conversion subunit, OSM-Rβ. Previous research conducted in the authors' laboratory has shown that OSM inhibits the growth of several breast cancer cell lines. To investigate whether OSM has a similar effect in primary normal human mammary epithelial (HME) cells, the activity of OSM in HME cells derived from four donors was examined. OSM produced a dose-dependent inhibition of DNA synthesis in these cells. In order to determine the receptor subtypes mediating OSM activity in HME and breast cancer cells, flow cytometry analysis using anti-gp130mAb and anti-OSM-RβmAb was performed. In these studies, the authors were able to examine expression of gp130 and OSM-Rβ. In addition, quantitative RT-PCR assays were conducted to measure expressions of the mRNAs of the subunits for type I and II OSM receptor. The results show that HME cells and most breast cancer cell lines express both the type I and the type II OSM receptors. However, type II, OSM-specific receptors are expressed at a higher level than type I, OSM/LIF shared receptors. Accordingly, we compared the growth regulatory activities of OSM with LIF in HME cells and in breast cancer cells. In contrast to the inhibitory activity of OSM, LIF stimulated the growth of breast cancer cells, whereas it had no effect on normal mammary epithelial cell growth. Together, these data suggest that OSM plays an inhibitory role in normal and malignant mammary epithelial cell growth in vitro. OSM activity is mediated by the OSM-specific receptor (type II), not by the OSM/LIF shared receptor.

Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: Correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.

Transcriptional Down-regulation of the Metastasis-inducing S100A4 (p9Ka) in Benign but Not in Malignant Rat Mammary Epithelial Cells by GC-factor

The S100-related calcium-binding protein S100A4 (p9Ka) is expressed at a low level in rat mammary epithelial cells from normal mammary gland and benign mammary tumors. In transgenic mice, expressed rat S100A4 transgenes co-operate with the activated c-erbB-2 oncogene, neu, to form metastatic mammary tumors. Elevated levels of S100A4 (p9Ka) in cultured benign rat or mouse mammary epithelial cells are associated with the induction of metastatic capability. A cis-acting sequence related to the consensus recognition sequence of GC-factor, 1,300 base pairs upstream of the start site of transcription of the rat S100A4 gene, acts as a cis-acting inhibitor of transcription of the S100A4 (p9Ka) gene in a low S100A4 (p9Ka)-expressing benign rat mammary epithelial cell line, but not in highly expressing rat mammary epithelial cell lines. There is an inverse relationship between the level of S100A4 (p9Ka) mRNA and the level of GC-factor mRNA in a range of rat mammary cell lines. The results suggest a novel mechanism for regulating the expression of the mRNA encoding an S100 protein.

EGF-related peptides in the pathophysiology of the mammary gland.

Normal mammary gland development is the result of complex interactions between a number of hormones and growth factors. Normal and malignant human mammary epithelial cells are able to synthesize and to respond to various different, locally acting growth factors and growth inhibitors. Among these, the EGF-related peptides play an important role in regulating the proliferation and differentiation of human mammary epithelial cells. EGF4 and TGF4 are able to stimulate the lobulo-alveolar development of the mammary gland in vivo as well they are involved in the pathogenesis of human breast cancer. Experimental evidence suggests that estrogen-induced proliferation of breast carcinoma cells is mediated in part by EGF-related growth factors. It has also been demonstrated that activation of certain cellular protooncogenes such as c-Ha-ras in human mammary epithelial cells results in cellular transformation and in an increased production of several EGF-related growth factors such as TGFalpha and amphiregulin. Coexpression of both EGF-related peptides and their own receptors frequently occurs in human breast carcinomas and in human breast cancer cell lines, suggesting that an autocrine pathway of uncontrolled cell growth sustains neoplastic transformation.

Growth factor and sex steroid interactions in breast cancer

Mitogenic and inhibitory growth factors and steroid ovarian hormones play important roles as selective modulators of normal mammary development and in the onset and the progression of human breast cancer. The focus of this article is to review past and current research on the interactions of these two classes of effectors in mammary gland development and neoplasia. Steroid hormones regulate synthesis of growth stimulatory and inhibitory growth factors, growth factor receptors, and growth factor binding proteins. In turn, growth factor pathways may modulate phosphorylation and function of steroid receptors and potentiate or inhibit the mitogenic actions of steroids. Ultimately, during the progression of the malignant mammary epithelial cell to hormonal autonomy, overexpression, mutation, or disregulation of key elements of growth factor signal transduction pathways all may play critical roles.

Expression and Regulation by Serum of Multiple FGF1 mRNA in Normal Transformed, and Malignant Human Mammary Epithelial Cells

In normal (NMEC), transformed (HBL-100) and malignant human mammary epithelial cells (MCF 7, BT-20, MDA-MB 231), we have examined the expression and the regulation by serum of FGF1 and FGF2 mRNA. FGF2 mRNA level was higher in NMEC and in a HBL-100 than in malignant cell lines (MDA-MB-231, BT-20). No FGF2 mRNA was detected in the malignant cell line, MCF-7. In contrast, the FGF1 mRNA was detected in all the mammary epithelial cells but at different levels. NMEC, HBL-100 and MDA-MB-231 cells expressed similar level of FGF1 and higher than that observed in BT-20 and MCF-7. In contrast to FGF2 which is only expressed in nonmalignant cells, no correlation between FGF1 mRNA expression and the phenotype of the cells was observed. We followed the expression of four FGF1 mRNA, heterogenous in their 5′ untranslated regions. This study demonstrated that (i) the FGF1 mRNA 1.A was not expressed by mammary epithelial cells, (ii) the FGF1 mRNA 1.B was only expressed in normal mammary epithelial cells and (iii) the transcripts 1.C and 1.D were expressed in normal and malignant cells with specific patterns. The expression of FGF1 mRNAs responded in a cell specific manner to serum starvation. The mRNA 1.A was only expressed in normal cells cultured in the absence of serum while 1.C was either up- or down-regulated by serum in transformed cells and the expression of 1.D was greater in presence of serum in all cell lines. These results show that the regulation of FGF1 mRNAs expression is cell specific and does not correlate with a tumorigenic or transformed cell phenotype.

Modulations of the H19 gene expression in normal or malignant mammary epithelial cells

Modulations of the H19 gene expression in normal or malignant mammary epithelial cells

Radiation-Induced Autophosphorylation of Epidermal Growth Factor Receptor in Human Malignant Mammary and Squamous Epithelial Cells

In an effort to identify events initiating up-regulation of epidermal growth factor receptor after single and repeated radiation exposures, we investigated the role of epidermal growth factor receptor, a receptor protein tyrosine kinase, in radiation-induced signal transduction. Human malignant mammary, MCF-7, and squamous, A431, cells showed low baseline phospho-tyrosine levels of epidermal growth factor receptor, permitting reproducible dose-dependent stimulation of epidermal growth factor receptor autophosphorylation after exposure to epidermal growth factor. MCF-7 cells exhibited a mean 2.3-fold increase (95% confidence interval: 1.91, 2.65; P < 0.0001) in levels of epidermal growth factor phosphorylation in response to exposures of 2 Gy, which was substantially less than the epidermal growth factor receptor Y phosphorylation induced by epidermal growth factor. A quantitatively similar radiation response was seen in A431 cells. In the dose range of 1 to 4 Gy, no clear dose response was seen. There was a rapid induction of radiation-induced epidermal growth factor receptor Y phosphorylation, starting within 2 min, with maximum values between 0.5 and 5 min after radiation exposure followed by a slower decline to baseline levels after 20 min. The data presented identify the epidermal growth factor receptor protein tyrosine kinase associated with the plasma membrane as one target for ionizing radiation in the dose range used in radiotherapy.

Production and localization of mammary-derived growth factor 1 in the normal and malignant mammary gland

Previously, we have detected a human milk and breast cancer-derived, acidic, 62 kDa, N-glycosylated growth factor named mammary-derived growth factor 1 (MDGF1). The N-terminal amino acid sequence of MDGF1 showed it to be a new growth factor. The factor stimulated collagen synthesis by fibroblasts and proliferation in normal and malignant mammary epithelial cells. Polyclonal antisera raised against the N-terminal synthetic peptide has been used to demonstrate that some human breast cancer and normal mammary epithelial cell strains and cell lines secrete the factor into their conditioned media. The antibody could adsorb the biological activity of the factor present in conditioned media. The objective of the present study was to utilize this high titre polyclonal anti-MDGF1 antisera to establish the presence and distribution of the factor in benign and malignant human breast tissues as determined by immunohistochemical analysis and enzymelinked immunosorbent assay (ELISA) assays. Frozen sections of normal and malignant breast tissue were used for immunostaining, while homogenized soluble fractions were used for ELISA testing. Antigen absorption confirmed the antibody staining to be specific. MDGF1 is localized in the duct and lobular epithelial cells of the mammary gland. Staining was nuclear and cytoplasmic in tumour and normal specimens. All of the breast tumour specimens demonstrated intense staining of the tumour cells. Localization of MDGF1 in benign and malignant human breast tissue suggests that it is universally present in breast epithelial derived cells. Study of MDGF1 could eventually provide new insights into breast development and/or tumour formation and progression.

Altered expression of epidermal growth factor receptor and estrogen receptor in MCF-7 cells after single and repeated radiation exposures

Purpose : Studies on radiation-induced changes in gene expression are likely to be very important in developing a better understanding of cellular responses to ionizing radiation. While there is some information on the activation of cellular signal transduction pathways after radiation, few late reacting target genes have been identified. This study focuses on the characterization of expression modulation of two critical growth regulatory genes, estrogen receptor and epidermal growth factor-receptor in malignant mammary epithelial cells in response to single and repeated ionizing radiation exposures. Methods and Materials : MCF-7 cells were used for single radiation exposure (2–50 Gy) experiments and MCFIR-3 cells, generated by exposure to cumulative doses of 60 Gy in 2 Gy fractions, respectively, were used to study the effects of repeated exposures. Steady-state messenger ribonucleic acid levels for estrogen receptor, epidermal growth factor-receptor, and transforming growth factor-α were determined by ribonucleic acid protection experiments. Estrogen receptor and epidermal growth factor-receptor protein expression was quantitated by competitive binding studies with 3H-estradiol and 125I-EGF. Results : MCF-IR-3 cells showed a permanent three-fold down-regulation of the estrogen receptor messenger ribonucleic acid and protein, while epidermal growth factor-receptor was upregulated about nine-fold. Epidermal growth factor-receptor was substantially up-regulated in MCF-7 cells, at both the mRNA and protein levels, within 24 h of a single 2 Gy exposures, while there was a two-fold concomitant increase in transforming growth factor-α messenger ribonucleic acid expression. A decrease in estrogen receptor messenger ribonucleic acid and protein was suggested only after higher doses of single radiation exposures. Conclusion : Single and repeated radiation exposures modulate the expression of two critical growth promoting genes, estrogen receptor and epidermal growth factor-receptor, in MCF-7 cells. The inverse expression of estrogen receptor and epidermal growth factor-receptor established for estrogen receptor-positive malignant mammary epithelial cells is maintained in MCF-7 cells after single and repeated exposures suggesting that radiation acts through common regulatory circuits and may modulate the cellular phenotype.

Generation of a murine monoclonal antibody to normal mammary epithelium using mice rendered immune-tolerant to malignant mammary epithelium.

A monoclonal antibody (MAb) that distinguishes normal from malignant mammary epithelia in tissue or cell lines was generated using a procedure that involved immune-tolerization before immunization. Immune-tolerance to two transformed mammary epithelial cell lines (MCF.7 and MDA.MB.231 cell lines combined) was induced in neonatal mice within 24 hr of birth. Successful induction of immune-tolerance was determined by an indirect immunohistological method, testing sera from mice against the tolerogen (i.e., the MCF.7 and MDA.MB.231 cell lines). Mice lacking antibodies in their sera against the immune-tolerogen were subsequently immunized with an extract of normal breast epithelium. One mouse was selected for hybridoma production based on evidence of serum antibody that showed reactivity with normal mammary epithelial cells (MEC) but not with invasive breast carcinoma cells, as determined by an indirect immunohistological method. Spleen cells from the selected mouse were fused with a mouse myeloma cell line to generate MAb. After extensive screening, one MAb was further studied on the basis of reactivity with normal MEC in tissue and absence of staining of malignant MEC in tissue or tumorigenic MEC lines. This test of specificity of reactivity revealed that the antigen detected by the specific antibody was expressed on the apical plasma membrane of normal glandular epithelia that included breast, cervix, colon, lung, pancreas, and stomach, but not on their malignant counterparts in tissue sections. The antigen recognized by the MAb was termed luminal epithelial antigen with an apparent MW of 92 KD (LEA.92). This study illustrates the practical usefulness of the immune-tolerization/immunization approach in the generation of antibodies with particular specificity requirements, as in the identification of an antigen that is differentially expressed in two tissues (e.g., normal and malignant) which otherwise have a multiplicity of antigens in common.

ALTERATIONS IN INTEGRIN AND BASEMENT-MEMBRANE PROTEIN EXPRESSION BY MALIGNANT BREAST CELLS

We investigated expression of eight integrin subunits and four basement membrane proteins by normal and malignant human mammary epithelial cells (HMEC) in culture and in tissue sections. Normal HMEC expressed the same six integrin subunits in culture and in vivo. Expression of some of these was altered in vivo in breast carcinomas: alpha1 was not observed and beta4 was missing from some cells-changes that were not genetic, as malignant cells in primary culture could express these subunits. Nor was expression of these subunits related to expression of their basement membrane protein ligands. Other integrin subunits were reduced in culture, and expression of basement membrane proteins differed between normal and malignant cells and cells in vivo and in culture. Our results suggest that breast carcinoma cells have altered regulation of expression of adhesion receptors and their ligands, and these alterations can be manipulated in culture for further study.

Identification of a cell-surface glycoprotein associated with normal mammary and extramammary epithelial cells.

The goal of the study was to identify any normal genes that may become inactivated in malignant cells, with associated modifications or loss of gene products. Consequently, attempts were made to identify such products by generating monoclonal antibodies using an immune tolerisation-immunisation procedure. Using such a technique, a plasma membrane-associated glycoprotein with an apparent molecular weight of 92 kDa was identified. The glycoprotein was termed luminal epithelial antigen (LEA.92). The pattern of expression of LEA.92 was demonstrated by an indirect immunostaining technique. Using an in vitro model system representing various stages of breast oncogenesis, LEA.92 was detected on normal or immortalised mammary epithelial cell (MEC) lines which were dependent on epidermal growth factor (EGF) and anchorage formation for growth and non-tumorigenic in nude mice. In contrast, LEA.92 was undetectable on oncogenically transformed or established lines of mammary carcinoma cell lines which were independent of EGF or anchorage formation for growth and were highly tumorigenic. The results appear to suggest a correlation between the down-regulation of LEA.92 and the development of tumorigenicity in malignant MEC lines. Furthermore, the patterns of expression of LEA.92 on breast cells in tissue mirrored those of breast epithelial cells in cell cultures. LEA.92 was detected on the surface of normal but not malignant epithelial cells, which included breast, cervix, colon, lung, pancreas and stomach. LEA.92 appeared to be distinct from receptor for epidermal growth factor, antigens associated with milk fat globule membrane and the family of epithelium-specific keratins.

Epithelial Cells Are an Important Source of Tenascin in Normal and Malignant Human Breast Tissue

The extracellular matrix glycoprotein tenascin is limited to the periductal matrix of normal breast tissue but is markedly increased in both malignant and fibroadenomatous proliferations. It has been hypothesized that the changes in tenascin expression in these tissues are the result of epithelial induction of tenascin expression by the underlying mesenchyme. We have used Western and Northern blotting techniques to examine tenascin expression by normal and malignant mammary epithelial cells in culture. Normal mammary epithelial cells express tenascin in culture and incorporate the protein into the underlying matrix. The SV40-transformed mammary epithelial cell line HBL100 and some established mammary carcinoma cell lines also express tenascin. In contrast to normal mammary epithelial cells, carcinoma cells incorporate very little tenascin into the underlying matrix. To examine the source of tenascin expression in vivo, we have used in situ hybridization to demonstrate that the epithelial cells are a significant source of tenascin in both normal and malignant breast tissues.

Epidermal growth factor-related peptides in the pathogenesis of human breast cancer

A number of different epidermal growth factor (EGF)-related peptides such as EGF, transforming growth factor alpha (TGF alpha), amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1), are coexpressed to varying degrees in both normal and malignant mammary epithelial cells. However, in general the frequency and level of expression of TGF alpha, AR, and CR-1 are higher in malignant breast epithelial cells than in normal mammary epithelium. In addition, several of these peptides such as TGF alpha and AR can function as autocrine and/or juxtacrine growth factors in mammary epithelial cells, and their expression is stringently regulated by mammotrophic hormones such as estrogens, activated proto-oncogenes that have been implicated in the pathogenesis of breast cancer, and other growth factors. The redundancy of expression that is observed for a number of these structurally related peptides in both normal and malignant mammary epithelial cells suggests that some of these peptides may be involved in regulating other aspects of cellular behavior such as differentiation in addition to proliferation.

Induction of the metastatic phenotype in rat mammary benign tumour cells by the gene for p9Ka a novel calcium-binding protein

Induction of the metastatic phenotype in rat mammary benign tumour cells by the gene for p9Ka a novel calcium-binding protein

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract Collagen-binding proteins ( CBPs) of rat mammary tumors are identical to Ca 2+-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I. In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12.) gene expression in two malignant human mammary epithelial cell lines: BT-20 and MCF-7. Regulation of gene expression by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the control of glycolysis. Its gene expression was analyzed in two breast cancer cell lines of human origin, BT-20 and MCF-7. We used a cDNA probe of 1.3 kb for Northern blot hybridization. It is found that GAPDH mRNA is overexpressed only in the poorly differentiated BT-20 cell line and that treatment of these cells by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) stimulates GAPDH mRNA expression in a dose- and time-dependent manner. The present investigation on the BT-20 cells indicates that the expression of GAPDH is sensitive to 1,25-(OH) 2D 3 and up-regulated by low doses of this steroid.

Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines

A mammary-derived growth factor, MDGF1, which stimulates collagen synthesis and proliferation in mammary epithelial cells was previously detected and purified from human milk and primary human breast tumors. MDGF1 binds to putative cell-surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. Partial protein sequence (N-terminal 18 amino acid sequence) shows that MDGF1 has no homology to any other known growth-promoting peptides. Polyclonal antiserum raised against this synthetic peptide recognizes native milk-derived MDGF1. We hypothesize that MDGF1 might be an autocrine or paracrine factor produced by and acting on normal and malignant human breast epithelial cells possessing MDGF1 receptors. As a first step in testing this possibility, we examined whether human breast epithelial cells in culture produce the growth factor. A protein with the size of MDGF1 was immunologically detected in the concentrated conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but nontumorigenic HBL-100 line, and the normal reduction mammoplasty-derived, nonimmortalized 184 cell strain. A competitive radioreceptor assay (RRA) was used to estimate the level of MDGF1 in the conditioned medium. MDGF1 was present in the nanogram range per 1 million cells. A 62-kDa protein was detected in the above cell lysates by Western immunoblotting or by immunoprecipitation of metabolically labeled cell-conditioned media. The polyclonal antisera directed against the 18 amino acid peptide sequence from milk-derived MDGF1 could adsorb MDGF1 biological activity from conditioned medium. In vitro translation of cell mRNA yielded a protein of 55 kDa which was immunoprecipitated by anti-MDGF1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of glycoproteins containing α- d-galactosyl groups on normal human breast epithelial cells and MCF-7 human breast carcinoma cells

Cell surface glycoproteins were isolated from the lysates of 125I-labeled normal human mammary epithelial cells (NHMEC) and from the human breast carcinoma cell line MCF-7, of blood-group O phenotype, by affinity chromatography on Griffonia simplicifolia I lectin-Sepharose. Specific elution of glycoproteins from the column with methyl α- d-galactoside suggests the presence of α- d-galactosyl groups on these moieties. SDS-PAGE analysis of isolated glycoproteins revealed both quantitative and qualitative differences between glycoproteins from normal and malignant cells. Three major glycoproteins of M r 180 kDa, 85 kDa and the 44 kDa were obtained from MCF-7 cells. The 180-kDa glycoprotein was absent in NHMEC and the 44-kDa glycoprotein was very weakly expressed in these cells. The only glycoprotein which was found in almost equal amount in the lysate from both normal and malignant cells was the 85-kDa glycoprotein. These results indicate differences between normal human mammary epithelial cells and one kind of malignant human mammary epithelial cells, in the expression of glycoproteins containing α- d-galactosyl groups, irrespective of blood-group phenotype; they also demonstrate that α- d-galactosyl group are expressed in a very restrictive manner on the surface of this tumor cell line.