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Various natural and synthetic retinoids have been studied for their activity in two biological systems: (a) their activity as inhibitors of methylcholanthrene-induced neoplastic transformation in the C3H/10T1/2 clone 8 mouse fibroblast line (System 1); and (b) their ability to increase the degree of adhesion of C3H/10T1/2 clone 8 cells to a plastic substrate (System 2). These activities were then compared with their known activity in maintaining epithelial differentiation (System 3). With the notable exception of retinoic acid and 13-cis-retinoic acid, which were inactive in Systems 1 and 2, an excellent correlation was observed between activities in Systems 1 and 3 for retinyl acetate, N-(4-hydroxyphenyl)retinamide, retinylidene dimedone, N-ethylretinamide, and N-benzoylretinylamine. Compounds shown to be inactive in System 1 had little or no activity in System 2. However, the ability of retinoids to cause increased adhesion could not be correlated with Systems 1 or 3 in all cases. For instance, retinyl acetate was highly active in Systems 1, 2, and 3, whereas retinylidene dimedone was highly active in Systems 1 and 3 but weakly active in System 2. Conversely, N-(4-hydroxyphenyl)retinylamide was highly active in Systems 1 and 3 but caused a decrease in System 2. The lack of activity of 3 but caused a decrease in System 2. The lack of activity of retinoic acid isomers in the C3H/10T1/2 clone 8 system is paradoxical and may provide important information on requirements for their activation and/or transport.

It has been demonstrated previously that nontransformed C3H/10T1/2CL8 mouse embryo fibroblasts (10T1/2) can induce a state of reversible growth inhibition in cocultured malignantly transformed mouse fibroblasts and that this inhibition is modulated by serum concentration. The present study suggests that cyclic nucleotides may be implicated in this intercellular communication. The phosphodiesterase inhibitors theophylline, caffeine, and 3-isobutyl-1-methylxanthine (IBX) at concentrations of 10(-3) M, maintained continuously, were all found to inhibit the expression of 3-methylcholanthrene-induced malignant transformation when added 7 days after removal of carcinogen. IBX was the most potent, causing 100% inhibition at 10(-4) M and 70% inhibition at 10(-5) M. This inhibition was partially reversible in the former case and completely reversible in the latter case by removal of drug. Complete inhibition by 10(-4) M IBX was still observed when treatment was delayed 21 days postcarcinogen. In reconstruction experiments, utilizing confluent monolayers of 10T1/2 cells overlaid with transformed cells, IBX caused a dose-dependent inhibition of colony size of the transformed cells. Adenosine cyclic 2':3'-monophosphoric acid (cAMP) and N6,O2'-dibutyryladenosine cyclic 3':5'-monophophoric acid potentiated this response. The presence of non-transformed 10T1/2 cells was required for this effect, since a concentration of IBX (10(-4) M) inhibitory for the growth of transformed cells in mixed cultures was without effect on the growth rate, plating efficiency, or saturation density of pure cultures of 10T1/2 cells or of their transformed counterparts. Conditioned medium removed from IBX-treated 10T1/2 cells was not growth inhibitory for transformed cells, indicating a requirement for cell-cell contact. IBX caused a dose-dependent increase in intracellular cAMP in confluent 10T1/2 cells and a more pronounced increase in cAMP concentration in the culture medium of these cells. The dose-response effects of IBX on growth inhibition of malignant cells in mixed cultures appear to correlate well with its ability to elevate cAMP levels. Thus, IBX increased the capacity of 10T1/2 cells to cause reversible growth arrest of transformed cells and appears to act in a manner analogous to the previously reported effects of serum.

Chronic p.o. feeding of small amounts of ellagic acid, a naturally occurring dietary plant phenol, to BALB/c mice in drinking water afforded significant protection against skin tumorigenesis induced by 3-methylcholanthrene, a polycyclic aromatic hydrocarbon carcinogen. A significant increase in the latent period for the development of skin tumors by 3-methylcholanthrene was observed in the ellagic acid-fed group of mice (9 wk on test) as compared to the control group of animals (6 wk on test). The observed protection against tumor induction in the ellagic acid-fed group of animals may be due to the inhibition of the metabolic activation of the polycyclic aromatic hydrocarbon since epidermal aryl hydrocarbon hydroxylase activity was found to be significantly inhibited. Our results suggest that dietary supplementation with small amounts of ellagic acid may prove useful in reducing the risk of skin carcinogenesis induced by environmental chemicals.