Normal Mammary Epithelial Cell Line Research Articles

Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

Purpose : Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes, Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4A overexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizing radiation. Methods and Materials : The normal mammary epithelial cell line MCF10A was stably transfected with full-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selected and treated with either 8 Gy of ionizing radiation or 7 J/M 2 of UV radiation. The profile of cell cycle progression and the accumulation of several cell cycle proteins were analyzed. Results : We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint in response to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizing radiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in the mock-transfected MCF10A cells, but not in the Cul-4A transfectants. Conclusion : Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly through disruption of cell cycle control.

Identification and Functional Characterization of a Human GalNAc α2,6-Sialyltransferase with Altered Expression in Breast Cancer

We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.

Changes in the cytologic distribution of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) during in vivo and in vitro mouse mammary epithelial cell expression and differentiation.

HIP/RPL29 is a small, highly basic, heparin/heparan sulfate interacting protein identical to ribosomal protein L29 and present in most adult epithelia. In the present study, we show that mouse HIP/RPL29 is ubiquitously present in adult mammary epithelia and is significantly increased during pregnancy and lactation. We observed for the first time that HIP/RPL29 intracellular expression and distribution varies, depending on the growth/differentiation state of the luminal epithelium. HIP/RPL29 was detected at low levels in mammary glands of virgin animals, increased markedly during lactation, and was lost again during involution. HIP/RPL29, preferentially found in the expanded cytoplasm of mature epithelial cells secreting milk, is present also in the nucleus of proliferating and differentiating ductal and alveolar elements. We used COMMA-D cells as an in vitro model for mammary-specific differentiation and examined similar intracellular redistribution of HIP/RPL29 associated with functional differentiation. However, no changes in HIP/RPL29 expression levels were detected in response to lactogenic hormones. Finally, the cellular distribution of HIP/RPL29 in both nuclear and cytoplasmic compartments was confirmed by transfecting a normal mammary epithelial cell line, NMuMG, with a fusion protein of HIP/RPL29 and EGFP. Collectively, these data support the idea that HIP/RPL29 plays more than one role during adult mammary gland development.

Characterization of C/EBPbeta isoforms in normal versus neoplastic mammary epithelial cells.

A member of the CCAAT Enhancer Binding Proteins (C/EBPs) family of transcription factors, C/EBPbeta, has recently proven to be an important player in both growth and differentiation of the epithelial cells in the mammary gland. When the gene for C/EBPbeta is disrupted in mice, these mice fail to either develop normal mammary ducts during puberty or pregnancy, or to lactate upon parturition. C/EBPbeta can be present in cells in three isoforms: C/EBPbeta-1, -2, and -3. These isoforms have the same carboxy terminus but different N-termini due to alternative translational initiation at three different initiator codons within the C/EBPbeta mRNA. Using a commercially available antibody specific to the C-terminus of C/EBPbeta and a novel antibody specific to the N-terminus of C/EBPbeta-1, we have uncovered a striking difference in the forms of C/EBPbeta present in normal mammary epithelial cells versus breast cancer cell lines. C/EBPbeta- 1 is found exclusively in normal mammary epithelial cells, whereas C/EBPbeta- 2 is found only in dividing cells, both normal and neoplastic. Our preliminary data suggest that the prevalent form of C/EBPbeta in cancer cells, C/EBPbeta- 2, can activate genes which push the cell to divide, such as cyclin D1.

Characterization of Cyp2d22, a Novel Cytochrome P450 Expressed in Mouse Mammary Cells

Endogenous steroids and numerous environmental agents have potent effects on mammary development and carcinogenesis. Locally produced cytochrome P450 enzymes that modify such molecules are therefore likely to be important regulators of these processes. Here we describe the characterization of a novel mouse gene, termed Cyp2d22, that is highly expressed in the mammary tumor derived cell line RIII/Pr1. Cyp2d22 is expressed at intermediate levels in the weakly tumorigenic cell line RIII/MG, whereas expression is low or absent in all normal mouse mammary epithelial cell lines tested and three C3H mammary tumor derived cell lines. Immunoblot analysis of mouse tissues with highly specific antisera indicates that 2D22 protein levels are most abundant in liver, while intermediate levels of expression are seen in adrenal, ovary, and mammary gland. Immunohistochemical staining of liver sections with these antisera demonstrates that 2D22 is most abundant in the first layer or two of parenchymal cells surrounding the central vein, with virtually no expression detected in periportal cells. Interestingly, sequence similarity and functional data suggest that Cyp2d22 may be the mouse ortholog of human CYP2D6. These observations support the hypothesis that 2D22 mediates a distinct, biologically significant activity in relation to other mouse 2D family members.

Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells.

Breast cancer is a heterogeneous disorder in which most tumors display some degree of aneuploidy, especially those at later stages of the disease. Aneuploidy and associated chromosome instability may be important in the progression of mammary tumorigenesis. Aneuploidy is prevented during normal cell division in part through regulation of a mitotic spindle checkpoint where mitotic arrest prevents segregation of misaligned chromosomes into daughter cells at anaphase. Mitotic arrest genes, including the MAD family, which was originally characterized in yeast, help regulate normal function of the mitotic spindle checkpoint. Decreased expression of the human gene MAD2L1 was previously reported in a breast cancer cell line exhibiting chromosome instability and aneuploidy. To explore further the potential role of MAD2L1 in breast cancer, we analyzed MAD2L1 gene expression in 13 minimally to grossly aneuploid human breast cancer cell lines and found significant differences of expression in three lines. Sequence analysis of MAD2L1 cDNA in these as well as nine additional aneuploid breast cancer and five immortalized normal human mammary epithelial cell lines revealed one heterozygous frameshift (572 del A) mutation in a cancer cell line that demonstrated a high level of transcript expression. In addition, two 3'UTR sequence variants were noted in breast cancer cell lines. The 572 del A mutation creates a truncated MAD2 protein product. Further functional studies in primary breast tumors are therefore warranted to determine the potential role MAD2L1 may play in breast cancer.

Prolactin-Dependent Up-Regulation ofBRCA1Expression in Human Breast Cancer Cell Lines

We report experimental evidence thatBRCA1,a breast and ovarian cancer susceptibility gene, is up-regulated in response to prolactin (PRL) stimulation. Expression of theBRCA1gene was monitored in 2 human breast cancer cell lines (MCF-7 and T-47D) and in the normal mammary epithelial cell line MCF10a. Using competitive RT-PCR, we have shown that PRL induced an increase inBRCA1mRNA level in MCF-7 and T-47D cell lines at a dose resulting in the maximal enhancement of cell proliferation. The up-regulation was 12-fold in MCF-7 cells and 2-fold in T-47D cells. No increase inBRCA1mRNA level was observed in the MCF10a cell line. The level of BRCA1 protein was quantified using an affinity chromatography strategy. At the protein level, PRL treatment induced a 4-fold increase of BRCA1 protein expression in MCF-7 and a 6-fold increase in T-47D cells, whereas BRCA1 protein expression was not affected by PRL in MCF10a.

Cell matrix adhesions and localization of the vitronectin receptor in MCF-7 human mammary carcinoma cells.

Interference reflection microscopy (IRM) was used to evaluate the status of cell matrix adhesions in the MCF-7 human mammary carcinoma cell line. Focal contacts were concentrated at the periphery of individual cells or small cell clusters. Close contact was detected as a band at cell peripheries and as localized patches throughout the ventral face of cells. The MCF-7 cells also exhibited a distinctive reflection pattern of an intensity midway between that of either focal or close contact. This novel reflection pattern was located primarily at the periphery of cells and often obscured visualization of focal contacts in live cells. A similar distinctive pattern was absent from the normal tissue-derived MCF-10A mammary epithelial cell line. Immunofluorescence staining using an antiserum that cross-reacts with both alpha(v)beta3 and alpha(v)beta5 integrins revealed a distribution of the vitronectin receptor similar to that of the novel adhesion pattern as well as to that of focal contacts. In addition, IRM demonstrated the presence of "tracks" associated with cells, which were also stained with the vitronectin receptor antiserum. The tracks are apparently residual material left behind as a result of cell migration. When MCF-7 cells were cultured in the absence of estradiol, the tracks were greatly diminished when visualized with either IRM or staining for the vitronectin receptor. In contrast, the addition of 17-beta-estradiol to the medium resulted in an increased presence of the tracks as well as the development of extensive close contacts throughout the ventral surface of cells and cell clusters. Cells treated with the estrogen antagonist ICI 182,720 in the presence of estradiol had few associated tracks, indicating that the process leading to the formation of these structures is dependent on an estrogen receptor-activated pathway. However, the antagonist did not prevent the estradiol-induced formation of extensive close contacts. The extensive close contact as well as the increase in trailing material suggests that estradiol may promote breast tumor cell motility. However, this migratory activity may be mediated by both estrogen receptor-dependent and -independent pathways.

Murine HIP/L29 Is a Heparin-binding Protein with a Restricted Pattern of Expression in Adult Tissues

Heparin/heparan sulfate (Hp/HS)-binding proteins are implicated in a variety of cell biological processes including cell adhesion, modulation of blood coagulation, and cytokine/growth factor action. Hp/HS-interacting protein (HIP) has been identified in various adult tissues in humans. HIP supports high affinity, selective binding to Hp/HS, promotes cell adhesion, and modulates blood coagulation activities via Hp/HS-dependent mechanisms. Herein, a murine ortholog of human HIP is described that is 78.8% identical to human HIP and 99.8% identical at the cDNA level and identical at the amino acid level to a previously described murine ribosomal protein, L29. Western blot analyses and immunohistological staining with affinity-purified antibodies generated against two distinct peptide sequences of murine HIP/L29 indicate that HIP/L29 is differentially expressed in adult murine tissues and cell types. In the normal murine mammary epithelial cell line, NMuMG, HIP/L29 is enriched in the 100,000 x g particulate fraction. HIP/L29 can be solubilized from the 100,000 x g particulate fraction with 0.8 M NaCl, suggesting that it is a peripheral membrane protein. HIP/L29 directly binds 125I-Hp in gel overlay assays and requires 0.75 M NaCl for elution from Hp-agarose. In addition, recombinant murine HIP expressed in Escherichia coli binds Hp in a saturable and highly selective manner, compared with other glycosaminoglycans including dermatan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid. Collectively, these data indicate that murine HIP/L29, like its human ortholog, is a Hp-binding protein expressed in a restricted manner in adult tissues.

Expression of CSF-I and CSF-I receptor by normal lactating mammary epithelial cells.

Previous studies suggested a potential role for macrophage colony stimulating factor (CSF-1) in lactogenic differentiation of the breast. The aim of this study was to define the regulation of CSF-1 and its receptor (CSF-1R, the product of c-fms proto-oncogene) by lactogenic hormones in the breast in vivo during pregnancy and lactation and in vitro in organ culture and mammary epithelial cell lines. Immunohistochemical staining assays for the expression of CSF-1 and CSF-1R antigens were performed on sections of breast biopsies from nonpregnant (n = 10), prepartum (n = 4), and postpartum lactating patients (n = 7) and on sections of human mammary glands cultured in the presence of the lactogenic hormones insulin, prolactin, and glucocorticoids. Northern blot analyses were used to study the regulation of CSF-1 and CSF-1R by these same lactogenic hormones in normal and neoplastic mammary epithelial cell lines in cell culture. Normal, nonlactating mammary epithelium did not express CSF-1R and synthesized only low levels of CSF-1. During lactation, significant levels of both proteins could be observed in the epithelial cells that line actively lactating ducts and alveoli. Very similar increases in epithelial cell expression of CSF-1 and CSF-1R were observed in organ cultures of normal mammary gland biopsies exposed to prolactin, insulin, and glucocorticoids. Colony stimulating factor mRNA levels were increased by prolactin and/or insulin in a normal mammary epithelial cell line, while glucocorticoids had no apparent effect on CSF-1 mRNA levels. In contrast, we found that the levels of CSF-1R transcript are regulated primarily by glucocorticoids in breast carcinoma cells, while prolactin merely modulates the glucocorticoid effect. The observed increases in the expression of CSF-1 and its receptor during lactogenesis and the regulation of CSF-1/CSF-1R by lactogenic hormones suggests that this cytokine/receptor pair might play a regulatory role in the cellular events leading to the lactogenic differentiation of mammary epithelial cells.

Novel alphaGalNAc containing glycans on cytokeratins are recognized invitro by galectins with type II carbohydrate recognition domains.

We report on a novel posttranslational modification of cytoplasmic proteins. Presented evidences suggest that cytokeratins are bound in vitro by mammalian galectin-3 and the galectins from the sponge Geodia cydonium via their type II carbohydrate recognition domains, whose highest binding affinity is directed towards terminal alpha-N-acetylgalactosamine-bearing glycans with the general sequence GalNAcalpha1-3Gal(NAc)beta. Specificity analyses and the characterization of the critical sugar residue on cytokeratins for galectin binding were done with cytochemical and biochemical methods using various plant and animal lectins. Binding of GalNAc-specific lectins was saturable, sensitive to mild periodate oxidation, inhibitable by glycoconjugates carrying terminal GalNAc, and abolished after treatment of the cytokeratins with alpha-N-acetylgalactosaminidase. Binding to bacterially expressed recombinant cytokeratins did not exceed background binding. The presence of GalNAc residues on highly purified cytokeratins from MCF-7 and HeLa SS6 cells was confirmed by sugar composition analyses using gas chromatography/mass spectrometry. This novel posttranslational modification was not restricted to cytokeratins of MCF-7 cells, but did also occur in all of 9 other examined human carcinoma cell lines and in a normal human mammary epithelial cell line. From these cytochemical and biochemical in vitro studies we hypothesize that this glycan with its terminal alpha1-3 linked GalNAc determinant might represent the first natural cytoplasmic ligand for endogenous galectins-3 detected so far.

Differential regulation of p34cdc2 and p33cdk2 by transforming growth factor-beta 1 in murine mammary epithelial cells.

Cyclin-dependent kinases (cdks) are a family of proteins whose function plays a critical role in cell cycle traverse. Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor of epithelial cells. Since cdks have been suggested as possible biochemical markers for TGF-beta growth inhibition, we investigated the effect of TGF-beta 1 on cdc2 and cdk2 in a normal mouse mammary epithelial cell line (MME) and a TGF-beta-resistant MME cell line (BG18.2). TGF-beta 1 decreases newly synthesized cdc2 protein levels within 6 h after addition. Coincident with this decrease in newly synthesized cdc2 protein was a marked reduction in its ability to phosphorylate histone H1. This decrease in kinase activity is not due to a change in steady-state levels of cdc2 protein, since mRNA and total protein levels of cdc2 are not reduced until 12 h after TGF-beta 1 addition. This suggests that the kinase activity of cdc2 is dependent on newly synthesized cdc2 protein. Moreover, the protein synthesis of another cyclin-dependent kinase, cdk2, is not effected by TGF-beta 1 addition, but its kinase activity is substantially reduced. Thus, it appears that TGF-beta decreases the kinase activity of both cdc2 and cdk2 by distinct mechanisms.

Carotenoids induce morphological changes in human mammary epithelial cell cultures

Epidemiological studies suggest that carotenoids may play a role in human breast carcinogenesis. To identify an anticarcinogenic mechanism, a laboratory model for examination of biologic effects is required. Efficacy of tetrahydrofuran (THF) for delivery of beta-carotene to a human mammary epithelial cell line has not been reported, and biologic effects of carotenoids on normal mammary epithelial cells or mammary epithelial cell lines have not been described. In these studies, we examined MCF-10A cells treated with 0.04%, 0.10%, and 0.35% THF (vol/vol) for morphological signs of toxicity and determined effects of THF on cell proliferation over a seven-day period. Cells treated with THF demonstrated a reduction in mean number of cells per dish (p < 0.05) but still underwent a 3.2- to 4.0-fold increase in cell number over the seven days. MCF-10A cells were also treated with a 7 mumol/l solution of beta-carotene and examined for morphological changes and effects on cell growth. Exposure to this concentration of carotenoid did not significantly affect proliferation but did induce the formation of cytoplasmic vacuoles similar to those seen in differentiating mammary epithelial cells. High-performance liquid chromatography analysis revealed a beta-carotene concentration of 0.004 nmol/10(6) cells in the treatment group. The effects of beta-carotene and the non-provitamin A carotenoid canthaxanthin were also examined in the in vitro cultures of primary human mammary epithelial cells obtained from reduction mammoplasties of two individuals. Exposure to these carotenoids induced morphological changes consistent with cellular differentiation and had a dramatic effect on the proliferative life span of these cells. Thus carotenoids may directly affect the proliferative capacity and differentiation of mammary epithelial cells, which may be among the chemoprotective activities of these compounds.

Quantitative Analysis of the Expression of Whey Acidic Protein (WAP) mRNA in the Prolactin-Responsive Mouse Mammary Gland Epithelial Cell Line, HC11.

The prolactin (PRL)-responsive cell line, HC11, derived from normal mammary epithelial cell line COMMA-1D, expressed the long form of prolactin receptor (L-PRLR) mRNA, though the level was considerably lower than that in the mammary gland of the normal lactating mouse. HC11 cells expressed whey acidic protein (WAP) mRNA, when they were cultured to confluence and 'in vitro lactogenesis' was induced by the addition of PRL, insulin and dexamethasone. The levels of PRL-induced WAP expression, as measured by reverse transcriptase mediated polymerase chain reaction (RT-PCR), increased in a dose-dependent manner, as PRL concentrations were raised from 0.05 μg/ml to 50 μg/ml. Treatment of HC11 cells with human growth hormone (hGH) caused increase in levels of WAP mRNA. In contrast to PRL, hGH showed its maximum effect at 0.5 μg/ml. Beyond this concentration, the effect of hGH was greatly reduced. These results suggest that hGH has a narrow optimal concentration range with regard to the stimulation of WAP expression in HC11 cells.

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.

Laminin and tenascin assembly and expression regulate HC11 mouse mammary cell differentiation.

HC11 is a normal mouse mammary epithelial cell line that requires certain growth factors, such as EGF or bFGF, to respond optimally to lactogenic hormones and produce the differentiation marker beta-casein. Growth in insulin (Ins) or PDGF does not produce cells competent to respond to lactogenic hormones. Here we show that competency for differentiation is due at least in part to the modulation of extracellular matrix components. In particular we have studied laminin and tenascin. EGF alters endogenous laminin assembly. In addition, promotion of competency can be partially mimicked by plating HC11 cells on the E8 laminin fragment, which is able to induce lactogenic responsiveness in cells grown in the absence of EGF or bFGF. The production and assembly of tenascin is also dependent upon the growth conditions of the HC11 cells. EGF- or bFGF-grown competent cells produce tenascin but do not assemble it at the extracellular matrix as efficiently as Ins- or PDGF-grown, non-competent cells. This alteration apparently leads to a change in the cellular microenvironment that supports beta-casein production. In addition, when competent cells are plated on dishes coated with tenascin, lactogenic hormone induction of beta-casein is inhibited. The data suggest that tenascin assembly and beta-casein production are opposing features of a coordinated differentiation program of HC11 cells.

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.

Control of Mammary Epithelial Cell DNA Synthesis by Epidermal Growth Factor, Cholera Toxin, and IGF-1: Specific Inhibitory Effect of Prolactin on EGF-Stimulated Cell Growth

Epidermal growth factor (EGF) stimulates mouse mammary cell proliferation in vivo , but its maximal level in the mammary gland is at midlactation, when little mammary growth takes place. The present studies use a normal murine mammary gland epithelial cell line (NMuMG) to determine the effects of the mammary mitogens EGF, cholera toxin (CT), and insulin-like growth factor-1 (IGF-1) on cell proliferation and how cell growth is altered by addition of the lactogenic hormone prolactin (PRL). EGF and CT stimulated over a fourfold increase of DNA synthesis in NMuMG cells when compared to basal levels. Only a twofold stimulation of DNA synthesis was observed when cells were treated with IGF-1. There was a slight increase in the percentage of cells in S-phase when these agents were added in combination with each other. Physiological levels of PRL had no significant effect on CT- or IGF-1-induced DNA synthesis but reduced EGF-stimulated cell proliferation to basal levels. Furthermore, PRL reduced the percentage of cells in S-phase to IGF-1- and CT-induced levels in the presence of EGF. Interestingly, PRL increased cellular levels of EGF mRNA after 2 h of treatment, which is similar to the response of mouse mammary glands cultured in lactogenic hormones. We conclude that even though PRL can increase the amount of EGF mRNA in mammary epithelial cells, it also eliminates EGF-induced mitogenesis.

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected’ cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le y, Le x, sialosyl-Le a, precursor of Thomsen Friedenreich antigen (T n), but not Thomsen-Friedenreich antigen and sialosyl-T n). Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2 + cells develop from the basal cell layer of the mammary gland.

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the ‘selected’ cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le y, Le x, sialosyl-Le a, precursor of Thomsen Friedenreich antigen (T n), but not Thomsen-Friedenreich antigen and sialosyl-T n]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2 + cells develop from the basal cell layer of the mammary gland.