Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

Abstract

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of -30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice.The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T

“…Nevertheless, the effects of SV40 transformation in, for example, rodent hepatocytes (Isom et al, 1980(Isom et al, , 1981 and mammary epithelia (Garcia et al, 1986), and in human keratinocytes (Chang, 1986) have been broadly similar to our observations, i.e. acquisition of anchorage independence (with or without tumorigenicity) but retention of differentiated characteristics.…”

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras.

“…Under these conditions the transformants tended to form multilayers ranging from small clusters of cells in 17,2 to five layers of cells in R5. It has been reported that transformed mammary epithelial cells form bilayers when grown on collagen (Garcia et al, 1986). Why these differences in substratum have such an effect on the organization of transformed epithelial cells is not apparent.…”

Alterations in the establishment and maintenance of epithelial cell polarity as a basis for disease processes.

“…On the other hand, transformed cells that maintained epithelial characteristics and a high surface membrane uvomorulin content were noninvasive (59). Reductions in cell-cell contacts had been previously reported when pp60 v-src was expressed in MDCK cells (60), and when SV-40-DNA and ras-DNA were used to transform primary rabbit mammary epithelial cells (61).…”