Mammalian drug resistant mutants with multiple gene amplifications: Genes encoding the M1 component of ribonucleotide reductase, the M2 component of ribonucleotide reductase, ornithine decarboxylase, p5-8, the H-subunit of ferritin and the L-subunit of ferritin

Abstract

Hydroxyurea was used to select two very highly drug resistant cell lines, designated H R-15 and H R-30. Both drug resistant lines contained elevated levels of ribonucleotide reductase activity. Northern and Southern blot analysis indicated that the two drug resistant lines contained increased levels of mRNA for the two components, M1 and M2, of ribonucleotide reductase, and M1 and M2 gene amplifications. Alterations in M1 and M2 protein levels were also evident in Western blot analysis. Further studies of H R-15 and H R-30 cells by Northern and Southern blot analysis showed that the drug resistant cell lines had elevated levels of ornithine decarboxylase mRNA and p5-8 mRNA, as well as increased ornithine decarboxylase and p5-8 gene copy numbers, respectively. Furthermore, characterization of H R-15 and H R-30 drug-resistant cell lines revealed increased mRNA levels for both H- and L-ferritin. Both cell lines exhibited by Southern blot analysis, amplification of the H- and L-ferritin genes. Increases in the cellular levels of H- and L-ferritin subunit proteins were also observed in both H R-15 and H R-30 cells, by Western blot analysis. This is the first description of mutant cell lines containing this complex combination of modified gene expressions and gene amplifications. The alterations exhibited by these lines confirm and extend present models of hydroxyurea resistance, are in agreement with and help substantiate models of ribonucleotide reductase regulation and provide interesting links between the expressions of several cellular activities important in proliferation.