Inhibition of metabolic cooperation between mammalian cells in culture by tumour promoters

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Abstract
The influence of phorbol-related tumour promoters and non-promoters on metabolic cooperation between wildtype and mutant Chinese hamster cells has been studied. The recovery, in medium containing 8-azaguanine, of hypoxanthine phosphoribosyl transferase-deficient (HPRT−) V79 cells co-cultured with an excess (2 × 106 per 9 cm petri-dish) of wild-type cells was determined in the presence and absence of each compound. Under the latter conditions (solvent treatment only) metabolic cooperation consistently reduced the cloning efficiency of HPRT− cells to approximately 10% of that in cultures without wild-type cells. However, 12-O-tetra-decanoyl-phorbol-13-acetate (TPA), the most potent known tumour promoter, almost totally reversed the effect of wild-type cells on mutant recovery when included in the medium at concentrations as low as 1 nM. TPA also markedly enhanced the expression, in crowded cultures, of HPRT− mutants induced by the carcinogen N-methyl-N-nitrosourea. This property was not shared by other phorbol esters which are inactive as tumour promoters, or by polycyclic aromatic hydrocarbons, but was exhibited to a lesser degree by mezerein, a diterpene ester of significant but weaker promoting activity than TPA. Confirmation that the effect of TPA on mutant expression is the result of inhibition of metabolic cooperation was obtained in experiments using autoradiography, which showed that low doses are able to block the transfer of [3H]-uridine nucleotides from prelabelled V79 cells to unlabelled V79 cells in contact. These findings have prompted us to formulate a working hypothesis for the mode of action of TPA in vivo as a tumour promoter based on its interference with this type of intercellular communication.