Chinese hamster ovary cells resistant to beta-aspartylhydroxamate contain increased levels of asparagine synthetase

Abstract

The growth of Chinese hamster ovary cells in a complete medium lacking asparagine is inhibited by beta-aspartylhydroxamate. The inhibition is overcome by the presence of asparagine in the growth medium. beta-Aspartylhydroxamate inhibits the activity of both asparagine synthetase and asparaginyl-tRNA synthetase in vitro. beta-Aspartylhydroxamate-resistant clones of Chinese hamster ovary cells have been isolated and three of these have been characterized. One clone, AH12, is 3-fold more resistant to beta-aspartylhydroxamate than the parental line and has 2 times higher levels of asparagine synthetase activity. Strains AH2 and AH5 are 6- to 7-fold more resistant to beta-aspartylhydroxamate and have 5 times higher levels of asparagine synthetase. The regulation of the expression of asparagine synthetase is altered in all three resistant cell lines. Whereas asparagine synthetase activity varies 2- to 3-fold in response to the asparagine content of the medium or to the extent of aminoacylation of tRNALeu in the parental cells, the activity of asparagine synthetase in the resistant cells is elevated under all growth conditions. No significant changes in the Km for substrates, Ki for beta-aspartylhydroxamate, or thermal stability were found for the asparagine synthetase of the resistant cells. These variants should prove useful in understanding the mechanisms involved in regulating the levels of asparagine synthetase in mammalian cells.