Inhibition of metabolic cooperation by phorbol esters in a cell culture system based on adenosine kinase deficient mutants of V79 cells

In Chinese hamster V79 cells, stable mutants which are greater than 1000-fold resistant to the adenosine analog, tubercidin (Tubr mutants), and which exhibit high degree of cross-resistance to various other adenosine analogs, viz. toyocamycin, formycin A, 6-methylmercaptopurine riboside and 8-azaadenosine, have been isolated. The inability of the mutant cells to phosphorylate [3H]tubercidin and lack of adenosine kinase activity (AK- phenotype) in their cell extracts provide evidence that the mutant cells are unable to convert adenosine analogs into their toxic phosphorylated derivatives. When AK- cells are co-cultured with increasing numbers of parental V79 (AK+) cells in medium containing tubercidin, then due to metabolic cooperation between AK+ and AK- cells, a cell density-dependent decline in recovery of the resistant cells is observed. However, diphtheria toxin resistant (Dipr) mutants of V79 cells, which are altered in elongation factor-2, showed no similar cell density effect. Addition of 0.01 microgram/ml of phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to growth medium in these experiments markedly enhanced the recovery of the Tubr mutants, but it had no effect on the recovery of Dipr mutants, which suggests that TPA was enhancing recovery of AK- mutants by inhibiting metabolic cooperation between AK- and AK+ cells. Maximum effect of TPA on the recovery of AK- mutants (3- to 5-fold enhancement) was observed at a density of 6 X 10(5) V79 cells/60 mm diameter dish and it was independent of the particular adenosine analog that was used as selective agent. Studies with a number of different phorbol derivatives show that only those phorbol esters which show tumor promoting activity in the mouse skin system inhibited metabolic cooperation between AK+/AK- cells in a dose-dependent manner. An excellent correlation was observed in these studies between the relative tumor promoting activity of various phorbol esters, their relative binding affinities to the cell surface receptors, and the concentrations at which they inhibited metabolic cooperation in the AK-/AK+ cell system. The AK-/AK+ cell system thus provides a new system for examining the effect of tumor promoters on metabolic cooperation between cells.