The ability of eukaryotic cells in culture to proliferate in calciumpoor medium has been found to characterize populations of transformed cells, but the relationship between this phenotypic property and tumorigenicity at the cellular level is unclear. Thus, we have isolated 14 clonal subpopulations, based on their ability to colonize in calcium-poor medium, from a parental tumorigenic rat hepatic epithelial cell line which was transformed by multiple exposures to N-methyl-N′-nitro-N-nitrosoguanidine. These clonal subpopulations of cells were tested for their ability to grow in soft agar, to express γ-glutamyl transpeptidase activity, and to form tumors upon back-transplantation into isogeneic newborn rats. The results indicated that clonal subpopulations of cells selected by their ability to grow in calcium-poor medium were phenotypically heterogeneous for γ-glutamyl transpeptidase activity and anchorage-independent growth, and, more importantly, they were not more tumorigenic than the phenotypically heterogeneous parental cell line. This observation suggests that the capability of cultured hepatic epithelial cells to grow in calciumpoor medium is not tightly coupled to the tumorigenic phenotype.

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Supported by NIH Grant CA29323.

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