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Potential role of treatment artifact in the effect of cell density upon frequencies of C3H/10T1/2 cell transformation.

Reports of unusual increases in transformation frequency in low density cultures of C3H/10T1/2 cells suggest that transformation occurs via an indirect multistage mechanism. The effect of surviving cell density upon subsequent focus production was examined in C3H/10T1/2 cultures treated with acetone, 12-O-tetradecanoylphorbol-13-acetate (TPA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNNG plus TPA, or 3-methylcholanthrene (MCA). Foci developed independent of cell density in 0.5 and 5.9% of all cultures exposed to acetone and TPA (0.25 micrograms/ml), respectively. Transformation after treatment with MNNG (0.5 micrograms/ml) occurred with low frequency (less than or equal to 7 X 10(-4)/surviving cell) and was enhanced by TPA. In MNNG plus TPA treated cultures containing less than or equal to 140 viable cells the fraction of dishes with foci was dependent upon the number of cells present at the time of MNNG treatment. As a result, relatively constant frequencies of focus formation were obtained (less than or equal to 6 X 10(-3) after correction for focus formation in TPA treated solvent controls). Focus frequency declined at cell densities greater than or equal to 350 cells. In contrast, treatment with MCA (1.0 microgram/ml) produced transformed foci with frequencies that varied from 3.3 X 10(-2) at the lowest density (5.5 cells) to 5.4 X 10(-4) at the highest (4400 cells). In low density cultures (5.6-56 cells), the fraction of dishes with foci was independent of the number of cells treated. Thus cell density had differential effects upon the frequency of foci produced by MCA or MNNG plus TPA. However, binding studies demonstrated that 6-7% of the MCA added to cell culture dishes was retained after the termination of carcinogen treatment. This residual MCA possessed biological activity which may be sufficient to elevate transformation frequencies in low density cultures.