Further characterization of the in vitro assay for inhibitors of metabolic cooperation in the Chinese hamster V79 cell line

12-O-Tetradecanoylphorbol-13-acetate (TPA) has been previouslyshown to inhibit metabolic cooperation in Chinese hamster V79cells. An in vitro assay, based on this phenomenon, has beendeveloped to study tumor promoters. Several parameters concerningthe metabolic cooperation assay using V79 Chinese hamster cellswere further investigated in this report. Pretreatment of thecells with TPA in situ for different periods of time did notresult in any detectable change in the inhibition of metaboliccooperation. If cells were replated after TPA treatment, a differentresult was obtained. There was an apparent decrease in the abilityof TPA to inhibit metabolic cooperation when TPA was added backto the TPA-pretreated cultures. However, when TPA was omittedfrom the TPA pretreated cultures after replating, the inhibitionof metabolic cooperation remained high. It was also found thatpretreatment of the cells with another chemical, aldrin, exhibitedthe same pattern as the in situ TPA pretreatment effect on inhibitionof metabolic cooperation. In order to obtain a high level ofinhibition of metabolic cooperation when using aldrin in thisassay, it was determined that the chemical needed to be presentfor more than one day. Our studies also showed that a 24 h treatmentwith 6-thioguanine did not kill 6-thioguanine-sensitive cellsquickly, nor did it prevent them from performing metabolic cooperation.The relationship of cell density and TPA concentration was alsostudied. It was observed that a higher cell density requiredhigher TPA concentration to inhibit, maximally, metabolic cooperation.A ‘down regulation’ type effect was noted when culturewas challenged with different concentrations of TPA. These resultswere interpreted to be consistent with the hypothesis that inhibitedgap-junctional intercellular communication is one of the componentsof tumor promotion.