Effects of a trinucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, on mammalian cells in culture

Abstract

The nonionic 2'-O-methyribooligonucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, is complementary to the...ApCpC...sequence found in the amino acid accepting stem of most tRNAs and the anticodon region of tRNAgly and to the threonine codon of mRNA. Gmp(Et)Gmp(EtU forms hydrogen-bonded complexes with the amino acid accepting stem of tRNApheyeast and unfractionated tRNA Escherichia coli under physiological salt conditions at 37 degrees C as determined by equilibrium dialysis. The extent of phenylalanine aminoacylation of tRNApheE.coli is inhibited 39% by Gmp(Et)Gmp(Et)U at 37 degrees C in solution. The triester is resistant to hydrolysis by serum nucleases and cell lysates. The triester is readily taken up by transformed Syrian hamster fibroblasts growing in monolayer. Within the cell, the triester is deethylated to give the trinucleotide species Gmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU and is also hydrolyzed to dimeric and monomeric units. Treatment of transformed fibroblasts in monolayer with 25 micronM Gmp(Et)Gmp(Et)U results in a 40% inhibition of cellular protein synthesis with a concurrent slight increase in cellular RNA synthesis during the first 4 h. After 4 h, the rate of cellular protein synthesis begins to recover while RNA synthesis returns to that of the control. Our biochemical studies suggest that inhibition of cellular protein synthesis might be expected if Gmp(Et)Gmp(Et)UGmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU, which have been taken up by or formed within the cell, physically bind to tRNA and mRNA and inhibit the function of these nucleic acids. The reversible inhibition of protein synthesis may be a consequence of further degradation of the trinucleotide species within the cell as well as to an increase in supply of RNA molecules involved in protein synthesis. The growth of the transformed fibroblasts is inhibited during the first 24 h of incubation with 25 micronM Gmp(Et)Gmp(Et)U after which growth proceeds at a normal rate. In cloning experiments, the number and size of colonies formed by the transformed fibroblasts after 5 days exposure to 25 micronM triester is decreased by 50% relative to untreated controls. The temporary inhibition of cell growth may reflect the transitory inhibition of cellular protein synthesis caused by the triester.