It has recently been found in this laboratory that the expression of the malignant phenotype in the C3H/10T1/2 CL8 assay system for malignant transformation can be repressed by high levels of serum. This repression can be reversed by exposure to sub-optimal concentrations of serum. This proposal is concerned with the mechanisms whereby the expression of the malignant phenotype can be modified. The proposal is divided into 2 stages: 1) an investigation of other selective media for the transformed phenotype; specifically it is proposed to investigate the effects of suboptimal concentrations of isoleucine and of calcium, 2) an investigation of the mechanisms whereby expression of the malignant phenotype can be inhibited or enhanced. In stage 1 concentrations of isoleucine and of calcium will be chosen which cause a restriction in growth rate of normal but not of transformed cells, carcinogen treated cultures will then be exposed to the selective procedure at various times after treatment, and the rate of development and number of transformed foci measured. In stage 2 of this proposal the mechanism of the effects of selective medium (serum, isoleucine, calcium) will be studied in carcinogen treated cultures containing latently transformed cells, and in reconstruction experiments utilizing mixed cultures of normal and transformed cells. Two questions will be asked: 1) is the observed effect due to cell/cell interaction i.e. can normal cells repress the expression of malignancy in transformed cells? This will be approached by adding cells and cell products to cultures in which expression is maximal. 2) Is the observed effect mediated via cyclic nucleotides? This will be approached by the addition of exogenous dibutryl cyclic AMP and dibutyryl and cyclic GMP, and by the use of drugs which stimulate the synthesis (prostaglandin E1), or retard the degradation of cyclic AMP (1-methyl-3-isobutly xanthine), or which elevate cyclic GMP levels and depress cyclic AMP levels(insulin).