Amplification of adenosine deaminase gene sequences in deoxycoformycin-resistant rat hepatoma cells.

Deoxycoformycin-resistant rat hepatoma cells exhibit up to a 2000-fold increase in adenosine deaminase activity compared to the sensitive parental cells.The increased enzyme activity in these cells is accompanied by similar increases in 1) the amount of adenosine deaminase protein, 2) the relative rate of adenosine deaminase synthesis in vivo, and 3) adenosine deaminase mRNA activity.To further investigate the mechanismfs) responsible for the overproduction of adenosine deaminase in these cells, we have isolated a recombinant plasmid containing a 1.4-kilobase insert complementary to at least part of the adenosine deaminase mRNA.Using this cDNA as a specific hybridization probe, all deoxycoformycin-resistant variants were shown to have increased amounts of adenosine deaminase mRNA and gene sequences.The relative increase in the level of mRNA and gene copy number was similar to the relative increase in enzyme activity for most resistant cell lines.However, the degree of adenosine deaminase gene amplification in one deoxycoformycinresistant cell line (6-10-200) was 3-4-fold less than the relative increase in adenosine deaminase mRNA.These results indicate that the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells is due in large part, but not exclusively, to gene amplification.The ability to produce increased amounts of a particular protein appears to be a common mechanism by which tissue culture cells may develop resistance to a specific inhibitor of that protein (1).The mechanism responsible for the increased production of the target protein has been investigated in several drug-resistant tissue culture systems.The most notable of these are the overproduction of dihydrofolate reductase, a multifunctional enzyme possessing carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activities, metallothionein I, and argininosuccinate synthetase in cells resistant to methotrexate, N-phosphonacety1-Laspartate, cadmium, and canavanine, respectively.Methotrexate-and canavanine-resistant cells produce increased amounts of a structurally normal enzyme (2-5).The increase in the amounts of enzyme synthesized by these cells has been shown to be the result of an increase in the rate of synthesis of these proteins (2,6).