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

Abstract

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.

Malignant progression of SV40-immortalised human milk epithelial cells

“…As for Hu-MI cells (Garcia et al, 1991) there was no immunostaining of HuMI-T and HuMI-TTul cells using an anti-carcinoembryonic antigen antibody (data not shown). (Garcia et al, 1991;Bartek et al, 1990;Chang et al, 1982). They retain a phenotype compatible with the putative role of breast cancer precursor cells and are neither clonogenic in soft agar nor tumorigenic in nude mice.…”

“…The expression of the viral large T-antigen induces continuous production of growth signals in several cell systems (Bartek et al, 1991;Lemoine et al, 1989;Poirier et al, 1988) via binding to a variety of proteins endowed with antiproliferative functions, such as the p53 protein (Lane & Crawford, 1979;Linzer & Levine, 1979) and the retinoblastoma gene product (Huang et al, 1990;Hu et al, 1990). SV40 large T-antigen is capable of transforming cells and causing tumours in the absence of any cooperating oncogene (Green, 1989;Choi et al, 1983) but SV40-infected normal human epithelial cells from various tissues have repeatedly been reported to be non-tumorigenic (Cussenot et al, 1991;Garcia et al, 1991;Bartek et al, 1990;Chang, 1986) or at best to grow anchorage-independently (Caron de Fromentel et al, 1985). Although generally considered to be a rather unlikely event, malignant transformation of normal cells from mammoplasty reduction surgical samples has been observed upon SV40-immortalisation (Berthon et al, 1992 Fromentel et al, 1985) or keratin 19-positive cell lines (Bartek et al, 1990), thus suggesting that different cell populations had been immortalised.…”

“…SV40 large T-antigen is capable of transforming cells and causing tumours in the absence of any cooperating oncogene (Green, 1989;Choi et al, 1983) but SV40-infected normal human epithelial cells from various tissues have repeatedly been reported to be non-tumorigenic (Cussenot et al, 1991;Garcia et al, 1991;Bartek et al, 1990;Chang, 1986) or at best to grow anchorage-independently (Caron de Fromentel et al, 1985). Although generally considered to be a rather unlikely event, malignant transformation of normal cells from mammoplasty reduction surgical samples has been observed upon SV40-immortalisation (Berthon et al, 1992 Fromentel et al, 1985) or keratin 19-positive cell lines (Bartek et al, 1990), thus suggesting that different cell populations had been immortalised. Indeed, the proportion of keratin 19-positive Hu-MI cells already decreases from 100% at early passages to 40% at passage 80 (Garcia et al, 1991).…”

“…Many human breast cancer cell lines have been established (O'Hare, 1991). In contrast, only few HMEC lines from normal breast cells are available (Paine et al, 1992;Soule et al, 1990;Briand et al, 1987), most of which, due to the difficulty of growing breast epithelial cells in culture, have been spontaneously (Caron de Fromentel et al, 1985) or selectively established by immortalisation through chemical agents, viral infection, transfection or microinjection of transforming genes (Berthon et al, 1992;Bartek et al, 1991;Garcia et al, 1991;Band et al, 1990;Bartek et al, 1990;Stampfer & Bartley, 1985;Chang et al, 1982). Invasive breast cancer probably originates from ductal luminal epithelial cells (Russo et al, 1987;Bartek et al, 1985a;Wellings et al, 1975).…”

“…Differentiated luminal epithelial cells not contaminated by fibroblasts can be obtained and cultured from early lactation milk samples (Taylor-Papadimitriou et al, 1977;Buehring, 1972). This also avoids the risk of coculturing contaminating myoepithelial cells (Bartek et al, 1991;Garcia et al, 1991;Bartek et al, 1990). Indeed, cell lines established from luminal cells are valuable in vitro tools to study carcinogenesis of the human mammary gland.…”