C3H/10T½ mouse embryo cells were exposed to 3-methylcholanthrene (MCA) for 24 hr. The daily addition of ascorbic acid at the noncytotoxic concentration of 1 µg/ml completely prevented the expression of transformed foci at 42 or 56 days after the experiment was begun if the ascorbate was added immediately after MCA exposure and daily thereafter for a total of 22 days. Ascorbic acid also could be added as late as 23 days after MCA treatment and still completely inhibit morphological transformation if maintained in the cultures until the dishes were stained.

The expression of the transformed phenotype in approximately 80% of the cultures containing transformed foci was also completely blocked following subculture at 6 to 8 weeks after MCA treatment if the transformed cells from these foci subsequently received ascorbic acid, 1 µg/ml, daily. Only 40% of cultures containing transformed foci returned to a normal morphological phenotype if the cultures were maintained for 10 weeks after MCA exposure before passage. The reversion of transformed cells to a normal morphological pattern often appeared to be irreversible since, after four subcultures in the presence of ascorbate, the ascorbic acid could subsequently be removed without reexpression of the morphological transformation. Transformed cells, however, became refractory to the effect of ascorbic acid if they were initially subcultured several times without ascorbate and then were exposed to the vitamin. This variation in the response to ascorbic acid when comparing early- and late-passaged transformed cells may indicate a fundamental biological difference between these transformed cells although they have a similar morphological pattern.

The exact mechanism(s) responsible for inhibition and reversion of the transformed phenotype is presently unknown. However, differential cytotoxicity between transformed and nontransformed cells, the production of an extracellular matrix in ascorbate-treated cultures, adipocyte formation, or increases in adenosine cyclic 2′:3′-monophosphoric acid were not found to be implicated in producing these effects.

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This work was supported by Grant CA-14226 from the National Cancer Institute.

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