Temperature-sensitive mutants of chemically transformed epithelial cells.

Abstract

The first temperature-sensitive mutants of epithelial cells transformed with chemical carcinogens have been isolated. Like the wild-type transformed parental cells, the mutants readily grow in agar suspension at 360, but in contrast to the wild type, they do not do so at 400. Detailed studies of one of these mutants, TS-223, indicate that at high temperature it also has reduced cloning efficiency in monolayer culture and a lower saturation density. Scanning electron microscopy revealed that at 40°confluent cultures of TS-223 consist of a monolayer of generally ilat polygonal cells, whereas 360 cultures contain many patches of piled-up cells that are spherical and have rougher surface membranes. All of these cellular changes are reversible with upward or downward temperature shifts. The temperature-sensitive lesion appears to reside in a host cell gene which modulates expression of the transformed cell phenotype. These mutants may provide a useful system for elucidating the minimal biochemicai changes required for expression of the transformed phenotype in epithelial cells.Mutants that are temperature sensitive (TS) in the expression of cell transformation afford a powerful tool for analyzing mechanisms of transformation since one can, at will, turn off or on the tumor phenotype. Several TS mutants of RNA and DNA tumor viruses have been isolated (1-10). The cells transformed by these viruses have characteristics of normal cells when grown at high temperature and of tumor cells when grown at lower temperature. Cell mutants of this type have also been isolated from 3T3 mouse fibroblasts transformed by simian virus 40 (11).It is important to develop similar mutants of cells transformed by chemical carcinogens, since the phenotype of chemically transformed cells may differ from that of virally transformed cells. Aside from the cold-sensitive chemically transformed BHK cells described by DiMayorca et al. (12), there are no published reports of TS mutants in cells transformed by chemical carcinogens.In the present study we report the isolation of five mutants of epithelial cells transformed by the carcinogen, N-acetoxyacetylaminofluorene, which are temperature sensitive in the maintenance of several properties associated with transformation. Our laboratory has emphasized the use of epithelial cells because about 80% of human tumors arise from epithelial tissues (13), and the growth properties of tumorigenic epithelial cultures (14-16) differ in several respects from those of transformed fibroblast cultures (17). Because of their epithelial origin, W-8 cultures do not display the criss-cross growth pattern seen with transformed fibroblasts (17) and they show "piling up" only when kept as confluent cultures for several days and only when the growth medium is changed repeatedly.The growth medium used throughout these experiments, unless stated otherwise, was Dulbecco's modification of Eagle's medium (GIBCO) supplemented with 10% fetal calf serum. This medium was also used when cells were grown in 0.4% agar suspens...

Glycopeptides from epithelial cell mutants: Temperature sensitive for the transformation phenotype

“…Their properties have been previously described . Clonal line W8 (Weinstein et al, 19756;Yamaguchi and Weinstein, 1975) underwent transformation in vitro 10-12 weeks after exposure of the cloned K16 line to a single exposure of 0.5 ,ug/ml of N-acetoxy-2-acetyl-amino-fluorene. The temperature-sensitive mutant, ts 223, was obtained by mutagenizing a cloned strain of W8 with N-methyl-N-nitro-N-nitrosoguanidine and selecting clones with decreased growth at high density by a slight modification of the procedure of Renger and Basilico (1972).…”

“…Recently Yamaguchi and Weinstein (1975) have isolated mutants of epithelial cells that are ts with respect to maintenance of the transformed phenotype. The source of these ts mutants was an in vitro transformed rat liver epithelial cell line (W8) which is highly tumorigenic.…”

“…Nor does it appear to involve the production of plasminogen activator since the mutant has negligible levels of plasminogen activator at either 36" C or 40" C (Yamaguchi and Weinstein, 1975). Because of the striking changes in the cell surface properties of ts 223 associated with temperature shifts, it is possible that the mutation affects a biochemical property of the cell surface.…”

Density-dependent regulation of growth of BSC-1 cells in cell culture: Control of growth by low molecular weight nutrients

“…There is considerable evidence (1)(2)(3)(4) that the growth controls of epithelial cells differ from those of fibroblasts in cell culture. Because of this and because of the importance of epithelial cells in the origin of tumors, we have made a thorough study of the factors that control the growth of BSC-1 cells, an epithelial cell line of African green monkey kidney origin (5).…”

Density-dependent regulation of growth of BSC-1 cells in cell culture: control of growth by serum factors.

“…It is suggested that a decrease in the number of available EGF receptor sites per cell, and the accompanying decrease in sensitivity of the cells to EGF, contributes to density-dependent regulation of growth of these cells. Information in the literature suggests that growth controls of epithelial cells differ from those of fibroblasts in cell culture (1)(2)(3). Epithelial cells often have a lower serum requirement, and the cell density to which they grow appears to be less dependent on the serum concentration and more dependent on the surface area of the culture dish (2).…”