Milk Epithelial Cells Research Articles

Malignant transformation of simian virus 40–immortalized human milk epithelial cells by chemical carcinogenesis accompanied by loss of heterozygosity on chromosome 1 but not microsatellite instability

Simian virus 40–immortalized human milk epithelial cells (HuMI) are anchorage dependent and nontumorigenic but can spontaneously progress to anchorage-independent and tumorigenic cells. To see whether HuMI cells can be transformed into anchorage-independent cells by chemical carcinogens, we treated them with 3-methylcholanthrene (MCA, 10 μg/mL). After 7–8 wk of culture, none of the treated cells grew in soft agar. However, when HuMI cells treated with MCA were cultured with 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/mL), they grew in soft agar; cells treated with TPA alone did not. TPA at this dose was cytotoxic to HuMI cells but not to their tumorigenic subline HuMI-TTu2. The response of the anchorage-independent HuMI-T cells was intermediate. These results indicate that HuMI cells can be transformed by treatment with MCA plus TPA, possibly because TPA selects those cells that are progressing toward malignancy. All five clones from MCA plus TPA–induced transformed cells formed malignant carcinomas in nude mice. When microsatellite changes at 17 loci in HuMI, HuMI-T, HuMI-TTu2, and five MCA plus TPA–transformed cells were examined, none of these cell lines showed instability at any locus, and no change in microsatellite length was found. However, all five MCA plus TPA–transformed cell lines showed loss of heterozigosity at 1q21-23 and 1q42 loci. This region of chromosome 1 is known to contain at least one antiproliferative gene, and our results suggest that inactivation of such a gene may be essential for full transformation of HuMI cells by chemical carcinogens. Mol. Carcinog. 23:20–24, 1998. © 1998 Wiley-Liss, Inc.

Persistent HTLV-I Infection of Breast Luminal Epithelial Cells: A Role in HTLV Transmission?

Human T cell leukemia viruses are predominantly transmitted from mother to child by breastfeeding. Endemic levels of HTLV infection are associated with ethnic groups that have traditionally practised long-term breastfeeding. In the course of long-term lactation, we have found that human milk contains leukocytes and epithelial cells and that mixed primary cultures of these milk cells are susceptible to HTLV-I infectionin vitro.We have established and characterized an immortalized line of milk epithelial cells, HTLV-LEC, that are productively infected and transformed with HTLV-I. This is the first reported case of human cells, other than T cells, that are transformed with HTLV-I. Cultures of HTLV-LEC are distinctive because of the synthesis of an extensive extracellular matrix that appears to supportin vitromorphogenesis. HTLV-I infection can be transmitted from HTLV-LEC into normal epithelial cells and leukocytes. Our results suggest that infected epithelial cells could be involved in the persistence and transmission of virus infection in HTLV-I carriers.

Malignant progression of SV40-immortalised human milk epithelial cells

A human breast epithelial cell line (Hu-MI), established by microinjecting SV40 DNA into human milk epithelial cells, exhibits the phenotype of luminal epithelial cells and is neither clonogenic nor tumorigenic. From this cell line we have selected two sublines, HuMI-T and HuMI-TTul, reflecting different stages of spontaneous transformation. HuMI-T cells grow anchorage-independently, but do not induce tumours in nude mice. HuMI-TTul cells are clonogenic as well as tumorigenic. Cells from both lines exhibit polymorphic structural and numerical chromosome aberrations. Immortalisation of normal luminal epithelial cells from human mammary gland with SV40 DNA alone may thus cause random genetic changes eventually resulting in tumorigenic cell lines. Since Hu-MI, HuMI-T and HuMI-TTul represent some of the consecutive stages taking place during cellular transformation, they are particularly suited as a novel in vitro model system to study progression of human breast cancer.

Morphological differentiation of hybrids of human mammary epithelial cell lines is dominant and correlates with the pattern of expression of intermediate filaments

Hybrids have been developed from two cell lines originally derived from human mammary epithelial cells. MTSV1-7 (hygro), developed from cultured milk epithelial cells, shows a cuboidal morphology, expresses high levels of keratins (but no vimentin), and forms ball-like three-dimensional structures in collagen gels. 5.3.1E (neo), derived from a cell cultured from tissue taken from a primary breast cancer, shows an elongated morphology, expresses high levels of vimentin and low levels of keratins, and does not form structures on collagen gels. An examination of the hybrids formed by fusion of MTSV1-7 cells and 5.3.1E cells showed that while all could form three-dimensional structures in collagen gels, the type of structures formed resembled either ductal or alveolar-ball-like structures depending on the individual hybrid. Nine hybrids were examined and a clear correlation was observed between cell shape as seen on plastic, intermediate filament expression, and the form of the structures as seen in collagen gels. All the hybrids resembling the parent MTSV1-7 cells by showing a cuboidal morphology and high level of keratin expression formed ball-like structures; on the other hand, hybrids resembling 5.3.1E by showing an elongated morphology and a high level of vimentin expression formed duct-like structures in collagen gels. The results show that the ability to form structures in collagen gels was inherited in a dominant fashion from the MTSV1-7 parent. They also suggest that the profile of intermediate filament expression in the hybrids may influence both cell shape on plastic and the morphology of the three-dimensional structures formed in collagen gels.

Selective immortalization of a phenotypically distinct epithelial cell type by microinjection of SV40 DNA into cultured human milk cells

An immortal cell line, MMSV-1, has been developed which exhibits many features of the common luminal epithelial cell of the human mammary gland. The cell line was developed by microinjection of SV40 DNA into individual cells in selected colonies in cultures of human milk epithelial cells. Immunohistochemical staining shows that the MMSV-1 cells express keratins 7, 8, 18 and 19 homogeneously in organized filaments which lead into well-developed desmosomes. They do not express vimentin or keratins found in stratified epithelia or keratin 14 found in basal cells in the mammary gland. The PEM mucin, recognized by the antibody HMFG-I, is also expressed and appears to be processed normally. Fibronectin is detected but shows the punctate pattern typical of cultured normal milk epithelial cells. MMSV-1 cells show a reduced requirement for added growth factors, including cyclic AMP-elevating agents, but do not grow in agar or form tumours in nude mice. Since the transfected cells could be selected on the basis of an extended in vitro life span, antibiotic resistance markers were not introduced and the cells remain sensitive to hygromycin and neomycin.

Modulation of phenotype in cultures of human milk epithelial cells and its relation to the expression of a membrane antigen

Primary cultures of human milk epithelial cells, labelled with HMFG-1, a monoclonal antibody that recognises a differentiation antigen on the membranes of breast epithelia, were separated from unlabelled cells in a fluorescence-activated cell sorter (FACS). Cells not expressing the HMFG-1 antigen have a greater in vitro growth potential than the positive ones, and give rise to cultures containing both HMFG-1 antigen positive and negative cells. The cultures derived from negative cells usually contain colonies with ‘open’ cuboidal cells and these do not express the HMFG-1 antigen. However, when they differentiate into other phenotypes such as the ‘closed’ cuboidal cell type, expression of the differentiation antigen becomes evident and the cultures then show a reduced growth potential. When milk cells are subcultured several times, a new cell type emerges which does not express the HMFG-1 antigen or the intermediate filaments typical of epithelial cells but instead express high levels of fibronectin which is found in an extracellular matrix. Whether these cells emerge by selection of an existing phenotype or by phenotypic modulation is discussed.

Junctional communication pattern of cells cultured from human milk

Junctional communication between cultured human milk cells and fibroblasts derived from human foreskin or mammary gland has been investigated. The dividing cells in primary cultures from milk, which have been shown to be epithelial by immunological and morphological criteria, showed selectivity in communication and did not transfer 3H-labelled nucleotides to either type of fibroblast. With passage the communication phenotype changed, selectivity was lost and a morphologically homogeneous cell type emerged which communicated not only with both types of fibroblast, but also with the milk epithelial cells found in primary culture.

Types of Cells Present in Cow's Milk

Summary The various types of cells present in the milk are described. In both normal and abnormal milk epithelial cells form a large proportion of the cellular elements. Multi-nucleated epithelial cells (pseudopolymorphs) and micro-pseudocells originate from the epithelial lining of the mammary gland. Apart from lymphocytes of both large and small varieties, no other leucocytes are usually found in normal milk. Neutrophil polymorphonuclear leucocytes exhibiting atypical morphology, may be present in milk presumed normal in very small numbers, usually not exceeding 2-3 per cent. of the total number of cells. The appearance of typical neutrophils and eosinophils in milk deposits obtained from apparently healthy cows, should be looked upon as an early indication of a developing mastitis. Neutrophils are by far the most common abnormal cellular elements of milk, but in some cases the number of eosinophils may exceed that of the neutrophils.