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Glycosylation is a very important posttranslational modification of many biologically relevant molecules. A change in the structure of glycans added to glycoproteins and glycolipids is a common feature of the change to malignancy. With the cloning of many of the glycosyltransferases and the identification of specific target molecules, it is now possible to define these changes at the molecular level and to dissect the mechanisms involved. Within the mammary gland, mucin-type O-linked glycosylation has been studied most extensively. In normal resting, pregnant and lactating breast, mucin O-glycans are largely extended (core 2 type) structures. In contrast, mucin O-glycans found in breast carcinomas are often truncated (core 1 type). One mechanism that is responsible for this increase in core 1 structures is a change in the expression of glycosyltransferases, particularly an increase in the expression of the sialyltransferase, ST3Gal-1. The loss, at least to some degree, of core 2 based glycans is a consistent feature of MUC1 mucin when it is expressed by mammary tumours as demonstrated by the unmasking of the SM3 epitope in greater than 90% of breast carcinomas.

The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 --> Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibited when cotransfected with a c-ErbB2 or a V12-H-ras expressing vector. Transient transfections using mutant forms of the ras oncogene, and the inhibitor chemical wortmannin, indicated that the pathway activated by c-ErbB2 proceeds via activation of Ras and that the Raf and phosphoinositide 3-kinase pathways are involved. Finally, cotransfection assays using a reporter gene driven by the MUC1 promoter carrying abolishing mutations in some of the cis-acting elements showed that a GC box at -99/-91 is crucial for responsiveness to c-ErbB2 inhibition of transcription.

Constitutive overexpression of c-erbB2 in the mammary epithelial cell line MTSV1-7 has been shown to result in epithelial-mesenchymal conversion, anchorage-independent growth and loss of organized morphogenesis in collagen. To elucidate the events leading to this drastic change, MTSV1-7 cells and its subclone HB2 (which shows a more strictly epithelial phenotype) were transfected with the hybrid trk-neu receptor consisting of the extracellular domain of the trkA nerve growth factor (NGF) receptor and the transmembrane and cytoplasmic domains of c-erbB2 (neu). In cells expressing this construct, c-erbB2 homodimerization can be mimicked by addition of NGF. In trk-neu transfectants of HB2 cells, modest expression led to increased cell proliferation upon NGF treatment. When clones with higher expression levels were grown in collagen, NGF instead induced cell scattering, diminished viability and dramatically increased apoptosis. Interestingly, both the dissociation of colonies and loss of cell viability could be completely reversed by treatment of the cells with antibodies that activate the adhesive capacity of the alpha2beta1 integrin. Long-term NGF treatment of high-expressing transfectants generated fibroblastic clones displaying a reduced expression of integrin alpha2 and E-cadherin, and extensive apoptosis in collagen. These results, which indicate that strong c-erbB2 signalling may lead to downregulation and/or inactivation of the alpha2beta1 integrin, promoting apoptosis in collagen, provide one possible explanation to the increased apoptosis frequently seen in early tumour development.

The effects of heregulin on the cell line HB2, derived from immortalised human luminal mammary epithelial cells, have been examined. HB2 cells, which normally form smooth spherical colonies in collagen gels, exhibited a striking heregulin-induced morphological change to colonies projecting a large number of spiky branches. A mitogenic effect of heregulin on HB2 cells was also seen, which was more pronounced on collagen than on plastic, whereas cell motility was unaffected. HB2 cells were found to express the heregulin receptor subunits HER2 and HER3, but not HER4. Treatment of HB2 cells with heregulin also induced tyrosine phosphorylation of a band shown by immunoprecipitation to contain HER3. Using specific receptor-blocking antibodies, it was found that both the morphogenetic and proliferative responses of heregulin in HB2 cells were mediated by HER2 and HER3. To compare the effects of HER2 in heregulin signaling to heregulin-independent HER2 homodimerisation (thought to be a carcinoma-associated event), HB2 cells were transfected with the trk-neu hybrid receptor which could be induced to form homodimers by NGF. Although activated HER2 homodimers induced proliferation in the HB2 transfectants in collagen, a morphological response in collagen was not seen, suggesting that HER3 signaling is important for morphogenesis in this cell type.

The HB2 cell line, developed from luminal epithelial cells cultured from milk, forms ball-like structures in collagen gels which show a uniform branching response to hepatocyte growth factor. The alpha2beta1 integrin is the major integrin expressed by luminal epithelial cells, and the role of this integrin in mammary morphogenesis has been analysed using HB2 cells cultured in collagen gels and antibodies which affect integrin function. Selectivity of response was followed by comparing effects on morphogenesis in fibrin, where the alphavbeta1 integrin interacts with the matrix. In the presence of hepatocyte growth factor, using alpha2 and beta1 antibodies in collagen and alphav and beta1 antibodies in fibrin, complete blocking of the cell-matrix interaction inhibits cell survival. With partial blocking of the integrin-ligand interaction, the cells proliferate but form dissociated colonies. Activating antibodies to the beta1 integrin subunit which enhance the matrix interaction dramatically inhibit the branching and motility responses to hepatocyte growth factor. A series of non-blocking alpha2 reactive antibodies also inhibit these responses specifically in or on collagen. Studies with ras-transfected HB2 cells emphasise the importance of the alpha2beta1 collagen interaction in the development of form since HB2ras cells, which express reduced levels of the alpha2beta1 integrin, form dissociated colonies in collagen but not in fibrin. Treatment of HB2ras cells with a beta1 activating antibody, however, induces the formation of compact colonies. Even though the ras-transformants form colonies in agar, complete blocking of the alpha2beta1/collagen interaction does not allow survival in collagen. The results indicate that in mammary morphogenesis, the strength of the interaction of integrins with the extracellular matrix modulates the response to motogenic factors and contributes to the definition of form.

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAcalpha2-3Galbeta1-3GalNAc-ol, Galbeta1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

Loss of normal tissue morphology is one of the first changes seen in the development of solid malignant tumours, including breast cancers. Since cell adhesion molecules play a crucial role in maintaining normal tissue architecture, investigations into the molecular mechanisms involved in the morphological changes occurring in malignancy that focus on the expression and function of cell adhesion molecules in malignant progression are extremely relevant. This review discusses the cadherin and integrin families of adhesion molecules which have been shown to be extremely important in the cell-cell and cell-matrix interactions of epithelial cells. Immunohistochemical studies using tissue and tumour sections indicate that a reduction in levels of expression or function of both types of cell adhesion molecules is indeed observed in many breast cancers. A specific and crucial role for these molecules in the maintenance of normal morphological differentiation has been demonstrated in vitro, where the noninvasive differentiated phenotype correlates with the normal functioning of E-Cadherin and alpha 2 beta 1 integrin. Further in vitro evidence suggests that in mammary epithelial cells, oncogenes may be upstream regulators of both the expression and function of E-Cadherin, the alpha 2 beta 1 integrin, and other epithelial specific molecules important for maintaining epithelial differentiation. In this way they could manifest their effects on the tumorigeneic potential of epithelial cells.

Overexpression of the B cell leukemia/lymphoma-2 (bcl-2) gene has been shown to confer a survival advantage on cells by inhibiting apoptosis. In epithelia, the bcl-2 gene is also related to development and differentiation, and the protein is strongly expressed in the embryo in the epithelial cells of the developing mammary gland. To investigate directly the effect of bcl-2 on human epithelial cells, we used an amphotropic recombinant retrovirus to introduce the gene into nontumorigenic cell lines developed from luminal epithelial cells cultured from milk. Here we demonstrate that while bcl-2 overexpression does not directly induce the tumorigenic phenotype, it provides a survival advantage to the mammary epithelial cells by inhibiting cell death at confluence or under conditions of serum starvation, bcl-2 can also affect the phenotype of the original epithelial cells, and promote epithelial-mesenchymal conversion, accompanied by loss of the cell adhesion molecules E-cadherin and alpha 2 beta 1 integrin. The extent of the epithelial-mesenchymal conversion varies with small differences in the phenotype of the parental line and with the level of expression of Bcl-2 and in some cases cell lines emerge with a mixed phenotype. The increased survival of Bcl-2-expressing cells at confluence results in multilayering, and the development of three- dimensional structures. Where a mixed phenotype is observed these structures consist of an outer layer of polarized epithelial cells separated by a basement membrane-like layer from an inner mass of fibroblastoid cells. Branching morphogenesis of bcl-2 transfectants is also observed in collagen gels (in the absence of fibroblast growth factors). The results strongly indicate that by increasing their survival under restrictive growth conditions, and by modifying the epithelial phenotype, bcl-2 can influence the specific morphogenetic behavior of mammary epithelial cells.

To study the morphogenesis of human epithelial cells in vitro we have used a three-dimensional collagen matrix and a newly developed mammary epithelial cell line, 1-7 HB2. In standard medium 1-7 HB2 cells formed compact balls/spheres inside collagen type I gels, while cocultivation with various fibroblast cell lines or growth in fibroblast-conditioned media resulted in the appearance of branching structures. At least two different soluble factors secreted by fibroblasts were found to be implicated in the branching morphogenesis. Firstly, hepatocyte growth factor/scatter factor could induce branching in a concentration-dependent manner. Moreover, a polyclonal serum against hepatocyte growth factor/scatter factor completely inhibited the branching morphogenesis induced by medium conditioned by MRC-5 fibroblast cells. In contrast, a morphogenetic activity secreted by human foreskin fibroblasts was identified that appears to be different from hepatocyte growth factor/scatter factor and from a number of other well-characterized growth factors or cytokines. This model system has been used to examine the role of integrins in mammary morphogenesis. The expression of the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins was decreased when cells were plated on collagen gels. The addition of specific blocking monoclonal antibodies directed to the alpha 2- and beta 1-integrin subunits to growth media impaired cell-cell interactions and interfered with the formation of compact structures inside collagen gels, suggesting that the alpha 2 beta 1 integrin can control intercellular adhesion in mammary morphogenesis. In contrast one of the blocking monoclonal antibodies against the alpha 3-integrin subunit (P1B5) mimicked the effect of soluble 'morphogens'. Our results suggest that the modulation of alpha 3 beta 1 activity may represent an important event in the induction of branching morphogenesis of human mammary epithelial cells.

Overexpression of the ERBB2 receptor in transfectants of a human mammary epithelial cell line (MTSV1-7) is associated with a reduced ability to undergo morphogenesis in vitro and with a decreased level of expression of the E-cadherin and alpha 2 integrin genes. The inhibition of expression of the adhesion molecules has been shown to be at the level of transcription by using nuclear run-on assays and by following transcription of a reporter gene fused to 5' sequences of the E-cadherin gene. To relate the effects on gene transcription to a functional ERBB2 protein, signaling from the receptor was inhibited by the antibody 4D5, which blocks phosphorylation of ERBB2 on tyrosine residues and association of the protein with the GRB2/Sem5 protein. After treatment with the antibody 4D5, the ERBB2 transfectants regain the ability to form three-dimensional structures in collagen gels and the rates of transcription of the genes encoding the E-cadherin and the alpha 2 integrin subunit are restored to the levels seen in MTSV1-7neo cells. These results demonstrate that the inhibition of morphogenesis and transcription of specific adhesion molecules in human mammary epithelial cells can be affected by signals generated by the ERBB2 receptor and suggest a role for ERBB2 overexpression in tumor progression and metastasis.

The c-erbB2 (or Her2) oncogene is amplified and/or overexpressed in a significant proportion of breast cancers. To assess the role of the c-erbB2 oncogene in mammary tumorigenesis, we have transfected the corresponding human c-erbB2 cDNA into an immortalized human mammary epithelial cell line, MTSV1-7, that was derived from luminal epithelial cells cultured from milk. Three transfectants expressing different levels of the c-erbB2 gene product have been isolated which form colonies in agar and produce tumours in nude mice with high efficiency. We have observed that MTSV1-7 cells form three-dimensional structures in collagen gels and that alpha 2 beta 1-integrin plays a crucial role in the process of morphogenesis. We now find that the c-erbB2 transfectants exhibit an impaired ability to undergo morphogenesis in collagen gels as compared with the parental cell line or the control neomycin transfectant, and that the degree of impairment is related to the level of c-erbB2 expression. Moreover, overexpression of the c-erbB2 product was found to be correlated with a specific decrease in the expression of alpha 2-integrin subunit and in the alpha 2-mRNA. The breast cancer cell line SKBr3, which carries multiple copies of the c-erbB2 gene and overexpresses the 185-kDa product, was also found to express very low levels of the alpha 2-integrin protein and mRNA. Our results confirm the involvement of the alpha 2 beta 1-integrin in collagen-induced morphogenesis of mammary epithelial cells and suggest that the c-erbB2 gene product may inhibit this morphogenesis by inhibiting the expression of the alpha 2-integrin subunit.

The cell line MTSV1-7, originally derived by immortalizing mammary epithelial cells cultured from human milk was able to form three-dimensional structures in collagen gel. We have now found that these cells, cultured as a monolayer, are able to undergo rapid morphogenesis forming ridges and balls around collagen fibres, when soluble collagen type I is added to the medium. Monoclonal antibodies to the alpha 2 (P1E6) and beta 1- (mAB13) subunits of VLA-2, but not to the alpha 3-subunit (P1B5) of VLA-3, could block this collagen-induced rapid morphogenesis (CIRM). The effect of the antibodies on cell attachment, spreading, and migration on collagen gels was analyzed to identify alpha 2 beta 1 dependent steps which might be involved in CIRM. The results suggest that while other proteins, besides alpha 2 beta 1, are also involved in cell attachment and migration, cell spreading was specifically blocked by antibodies to the VLA-2, but not to the VLA-3 integrin. The results demonstrate that the alpha 2 beta 1 integrin plays a crucial role in the collagen-induced morphogenesis of human mammary epithelial cells and implicate the process of VLA-2-dependent cell spreading as an important step in this morphogenesis.

When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line.

In the adult human mammary gland, most of the luminal epithelial cells express keratin 19 (K19+). However, in some small ducts and terminal ductal lobular units where branching would be expected to occur during pregnancy, the pattern of expression of this keratin is heterogeneous. While the keratin 19 negative cells (K19-) appear to have a high proliferative potential in vitro and in vivo, they have a lower secretory activity than the K19+ cells as monitored by expression of secretory component in the resting breast or casein in the pregnant gland. That the K19- cells form a separate proliferative compartment in the luminal cell lineage is suggested by the fact that they are absent in the prepubertal breast and only appear at puberty associated with branching ducts, and newly formed lobules. Our observations are consistent with the hypothesis that the K19- luminal cell is less differentiated than and may be precursor to the K19+ luminal cell, which represents the fully differentiated phenotype able to produce milk in response to a hormonal stimulus.

Human breast cancer cell lines, as well as transformed mammary epithelial cells (HBL-100) and growth-stimulated normal breast epithelial cells showed positive cytochemical reaction with the proteinase substrate 2-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methoxynapht halene, in the presence of 5-nitrosalicylaldehyde. The reaction product, small fluorescent granules, was distributed throughout the cytoplasm, in the perinuclear zone, in some cytoplasmic projections, and at the cell surface. Using a panel of various proteinase inhibitors, we found that the formation of the reaction product was an enzymic function of a cysteine proteinase. Using the substrate 7-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methylcoumarin, we evaluated some biochemical properties of the cysteine proteinase, including pH-activity profile, pH stability, apparent relative molecular mass and sensitivity toward various proteinase inhibitors. We found that the proteinase from the studied breast epithelial cells exhibited characteristics of a mature form of cathepsin B. Taken together, the cytochemical and biochemical data provide evidence that human breast epithelial cells of cancer origin, as well as in the transformed or growth-stimulated state express active cathepsin B and compartmentalize it into specific subcellular sites.

The growth of human epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.

The monoclonal antibodies HMFG-1 and HMFG-2 are directed against different epitopes on a large molecule in the human milk fat globule and are also expressed on breast tumors. Here we examine the relative intensity of staining of the two antibodies in formalin fixed paraffin embedded tissue sections from malignant and benign mammary lesions. Most ductal carcinomas and their lymph node metastases stained more strongly with HMFG-2 whereas primary lobular carcinomas are more variable but the majority of their lymph node metastases stained more strongly with HMFG-1. Western blots of gel separated extracts of formalin fixed, paraffin embedded tumor tissue, stained with the antibodies in an ELISA assay, show that the molecular weight of components expressing the antigenic determinants varies. Smaller molecular weight components recognised by HMFG-2 and present in metastatic tumors may be lost during tissue processing. To assess the importance of the molecular weight parameter in tumor classification and prognosis it will be necessary to use frozen tissue.

Human breast epithelial cells cultured from milk have been transformed with SV40. Indirect immunofluorescence tests using monoclonal antibodies show that cells from clones grown in soft agar have SV40 large T-antigen in their nuclei and epithelia-specific tonofilament antigens on their intermediate filaments. In primary cultures of milk epithelial cells, the tonofilaments form a characteristic delicate basketwork throughout the cytoplasm, but in the SV40-transformed epithelial cell strains, the filament network is grossly distorted. Insulin, hydrocortisone, and serum stimulate the growth of the cell strains. At passages 8 to 11, the cell strains become quiescent and usually die. One cell strain survived this crisis period and gave rise to the fR series of cell lines. Most cell lines have a cuboidal morphology and react with a monoclonal antibody that recognizes a differentiation antigen on the membranes of breast epithelia. Line fR2 expressed the highest level of this antigen whereas fR5, the only fR line isolated with fusiform morphology, had relatively little. The in vitro-transformed lines may be related to the two dominant epithelial cell types seen in primary milk cultures and could be useful for studying the relationship between transformation and differentiation in human mammary epithelial cells.

Human lymphoblastoid interferon inhibited the growth in vitro of fibroblasts and epithelial cells from normal, hyperplastic and neoplastic human breast tissue. At an interferon concentration of 10(3) inter-national units (IU) per ml, the inhibitory effects on monolayer growth were completely reversible but the growth potential of cells at lower density in colony-forming cultures was not completely recovered. Studies on the cell cycle distribution of interferon-treated cells demonstrated that the growth-inhibitory effect was not due to an effect on one specific phase of the cell cycle, but to a lengthening of all phases.

The authors have used the indirect immunoperoxidase technique to examine the presence and distribution of milk fat globule membrane antigens in 12 cases of mammary Paget's disease using two monoclonal antibodies, HMFG-1 and HMFG-2. These stain breast epithelial cells but do not stain normal epidermis. The Paget's cells showed a similar pattern of cytoplasmic staining to that seen in the underlying intraduct or invasive carcinoma, therefore confirming them to be malignant ductal cells. To support this one of the cases was stained with the antibody LE 61 which is specific for nonepidermal epithelial cytokeratins. The result was strongly positive in Paget's cells in the epidermis but not in squamous cells.

The luminal and basal epithelial cells in the human mammary gland can be distinguished in tissue sections on the basis of the pattern of keratins they express. Moreover, the invasive cells in primary carcinomas show a keratin profile that corresponds to that of the dominant luminal cell (7, 8, 18, 19). When homogeneous populations of luminal epithelial cells from milk or from breast cancer metastases are cultured the profile of keratin expression seen in vivo is maintained. We have therefore used monospecific antibodies reactive with individual keratins to examine the phenotype of cells cultured in three different media from reduction mammoplasty tissue that contains both luminal and basal cells. The phenotype of cells cultured from primary breast cancers in one of these media (MCDB170) has also been examined. In characterizing cell phenotypes, antibodies to a polymorphic epithelial mucin (PEM) expressed in vivo by luminal cells, and to smooth muscle (a) actin, expressed in vivo by basal cells, have also been used. Our results show that proliferation of different cell phenotypes is selected for in different media. In milk mix (MX) developed for growth of luminal cells from milk, only the luminal cell phenotype proliferates (for only 1 or 2 passages). In medium MCDB 170, which was developed for long-term growth of human mammary epithelial cells from reduction mammoplasty organoids, cells from the basal layer proliferate, while in MM medium the basal phenotype dominates, but a few cells with the luminal phenotype are found. Around passage 3, in medium MCDB 170, most cells senesce and a subpopulation of cells proliferates on further passage. These cells retain expression of the basal epithelial keratins but also express some features characteristic of luminal epithelial cells, suggesting that the basal layer may contain a stem cell that can develop along the luminal lineage. In culture, however, they do not express keratin 19, which in vivo is a feature of the fully differentiated luminal cell. The cells cultured from primary breast cancer in medium MCDB 170 have a similar keratin profile to that of the normal cells cultured in this medium. They do not express keratin 19, even though the invasive cells in primary cancers homogeneously express this keratin in vivo. The invasive phenotype, which in its keratin profile corresponds to the differentiated luminal cell and that of the metastatic cancer lines, cannot be cultured from primary breast cancers using MX, which supports proliferation of the corresponding normal cell.

The monoclonal antibodies BA16 and BA17 directed to different epitopes on human keratin 19 have been tested for their reaction with normal breast and with benign and malignant breast lesions and associated tissue. In Western blots of gel-separated extracts of fibroadenomas, malignant tumours or normal mammary epithelial cells, the antibodies reacted with only one component of 40 kd molecular weight. Immunoperoxidase staining of sections of normal breast tissues showed all basal cells and a few luminal cells to be unstained by the antibodies. The distribution of the unstained (keratin 19-) luminal cells in the mammary tree is consistent with that of cells with the proliferative potential to give rise to the growth of terminal ductal lobular units (TDLU) seen at pregnancy. A total of 42 benign and 141 malignant lesions were stained with the antibodies, and a clear difference in staining pattern was seen between the benign and malignant tumours. All but 3 of the benign lesions showed a heterogeneous staining pattern with 5-50% unstained cells. In contrast, the cancer cells in 106/116 invasive primary tumours and in all 21 metastatic lesions examined showed a homogeneously positive reaction with antibodies BA16 and BA17: the malignant cells in 4 cases of Paget's disease also showed homogeneously positive staining with the antibody. In the malignant tumours, the observed homogeneity in expression of keratin 19 was confined to the malignant cells; tumour-associated normal tissue and benign proliferative lesions contained keratin 19-cells. Seven pure in situ tumours were examined and 5 showed the homogeneous pattern of staining characteristic of invasive tumours while 2 contained a high number of keratin 19-cells. A general model is presented to explain the presence of keratin 19-cells in benign proliferation and the dominance of keratin 19-cells in invasive carcinoma.

Many existing MAbs raised against the human milk fat globule or against carcinoma cells recognise epitopes on high-molecular-weight glycoproteins. In a comparative ELISA assay a number of these antibodies have been shown to react with an extract of the human milk fat globule. In comparative immunoblots of cultured normal milk cells and breast cell lines, all were found to bind to large molecules which show some variation in molecular weight depending on the cell source. The HMFG-2 antibody, which is widely used in cancer diagnosis, also recognises epitopes on lower-molecular-weight components. In T47D cells these may be as small as 80,000 Mr, and with electron microscopy this cell line can be shown to accumulate HMFG-2-reactive components in the Golgi apparatus. Using an HMFG-2 affinity column we have immunopurified HMFG-2-reactive material from the 2 breast cancer cell lines MCF-7 and T47D and shown that all of the above antibodies react in a solid-phase ELISA with the purified material. In addition to high-molecular-weight components, the immunopurified material was found to contain lower-molecular-weight components including a glycoprotein of 68,000 Mr that was not, however, recognised by the HMFG-2 antibody on a Western blot. We have used this immunopurified material to generate new MAbs. All of these recognise the high-molecular-weight bands seen with the other antibodies, but 2 of them also recognise a band at 68,000 Mr in blots of MCF-7 and T47D. The second-generation antibodies show a spectrum of reactivity on tissues similar to HMFG-2 and one reacts at least as strongly as HMFG-2 with methanol-acetone-fixed sections of breast cancers.

Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins (Mr greater than 250,000) that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purified and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a lambda gt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven cross-reacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI.

The expression of human mammary epithelial (HME) antigens was studied in primary cell cultures of normal and abnormal breast tissue and of tissues from other organs. By indirect immunofluorescence it was shown that heterologous anti-HME bound to the cell surface of both suspension and monolayer cultures of human breast epithelial cells from normal breast tissue, normal breast fluid, non-malignant atypical lesions of the female breast, gynecomastia in the male breast, breast carcinomas, and peritoneal and pleural effusions of patients with breast carcinomas. The fibroblast-like cells cultured from either normal, non-malignant atypical, or malignant breast tissue never bound anti-HME, nor did histiobyte-like cells from breast fluid or a sarcoid granuloma of the breast. From a Paget tumor of the breast, two distinguishable epithelial-like colonies appeared in the cultures, only one of which expressed HME-antigens. Anti-HME did not bind to cells cultured from tissues of other organs, whether epithelial-like or fibroblast-like; these organs included thyroid, ovary, endometrium, and skin.

Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.