No abstract

Impure and pure samples of saccharin (2 milligrams per milliliter) did not produce oncogenic transformation of C3H/10T1/2, clone 8, mouse embryo fibroblasts. However, after treatment of the cells with a nontransforming initiating dose (0.1 microgram per milliliter) of 3-methylcholanthrene, continuous treatment with either sample of saccharin (100 micrograms per milliliter) led to significant transformation. It is concluded that in this system saccharin is a cocarginogen, probably functioning as a promoting agent that is 1000-fold less active than the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate.

Abstract. Cell lines derived from C3H mouse ventral prostate grow to a monolayer, do not pile up, and do not give tumors on inoculation into isologous mice. When treated with carcinogenic hydrocarbons, these cells produce piledup colonies that give rise to fibrosarcomas in mice; these colonies are termed transformed. Individual single cells were treated with 0.5 per cent dimethyl sulfoxide (DMSO), and 2 per cent of them gave rise to clones containing transformed colonies. Under optimal conditions, treatment of individual single cells with 3-methylcholanthrene (MCA) led to the formation of transformed colonies in 100 per cent of the clones. From recloning experiments, it appears probable that all the progeny of the MCA-treated cells were potentially transformed, although the time required for the transformation to be scored was variable. It is concluded that in this system the carcinogen does not select for pre-existing malignant cells.

When C3H/10T1/2 cells are treated with a given concentration of a chemical carcinogen, the transformation frequency can vary over 4 orders of magnitude, depending primarily upon the number of cells plated. To explain this phenomenon, we have developed a probabilistic theory of the formation of transformed foci in this system. We define p1 as the probability that a cell will be activated by carcinogen treatment, p2 as the probability per cell generation that an activated cell will be transformed, and p3 as the probability per cell generation that an activated cell will be deactivated. The equation we have derived: log (F/N) = log [2p1p2(1 -- p3)/2(1 -- p3) -- 1]+ n log (1 -- p3) describes focus formation; F is mean number of loci per dish after carcinogen treatment, N is number of cells in a dish at confluence, and n is number of cell generations to confluence. This equation has been verified experimentally; p3 = 0.24 and p1p2 = 3.8 X 10(-6) at a single concentration of 3-methylcholanthrene. This relationship explains previously inexplicable effects of cell density on transformation frequency.

Starting with a thrice-cloned highly malignant fibroblastic line of cells derived from