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. 1982 Sep 1;94(3):586–591. doi: 10.1083/jcb.94.3.586

Selection of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosaminyltransferase activity

PMCID: PMC2112219  PMID: 6215412

Abstract

Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) have been isolated by a stepwise selection procedure with progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the lipid carrier. The TM-resistant cells exhibited a 200-fold increase in their LD50 for TM and were morphologically distinct from the parental cells. The rate of asparagine-linked glycoprotein biosynthesis was the same for wild-type and TM-resistant cells. Membrane preparations from TM-resistant cells cultured for 16 d in the absence of TM had a 15-fold increase in the specific activity of the UDP-N- acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase as compared to membranes of wild-type cells. The products of the in vitro assay were N-acetylglucosaminylpyrophosphoryl-lipid and N,N'-diacetylchitobiosylpyrophosphoryl-lipid for membranes from both TM- resistant and wild-type cells. The transferase activity present in membrane preparations from wild-type of TM-resistant cells was inhibited by comparable levels of TM. The data presented are consistent with overproduction of enzyme as the mechanism of resistance in these variant CHO cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
  2. Alt F. W., Kellems R. E., Schimke R. T. Synthesis and degradation of folate reductase in sensitive and methotrexate-resistant lines of S-180 cells. J Biol Chem. 1976 May 25;251(10):3063–3074. [PubMed] [Google Scholar]
  3. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  4. Behrens N. H., Tábora E. Dolichol intermediates in the glycosylation of proteins. Methods Enzymol. 1978;50:402–435. doi: 10.1016/0076-6879(78)50047-5. [DOI] [PubMed] [Google Scholar]
  5. Gantt J. S., Bennett C. A., Arfin S. M. Increased levels of threonyl-tRNA synthetase in a borrelidin-resistant Chinese hamster ovary cell line. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5367–5370. doi: 10.1073/pnas.78.9.5367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gantt J. S., Chiang C. S., Hatfield G. W., Arfin S. M. Chinese hamster ovary cells resistant to beta-aspartylhydroxamate contain increased levels of asparagine synthetase. J Biol Chem. 1980 May 25;255(10):4808–4813. [PubMed] [Google Scholar]
  7. Keller R. K., Boon D. Y., Crum F. C. N-Acetylglucosamine- 1 -phosphate transferase from hen oviduct: solubilization, characterization, and inhibition by tunicamycin. Biochemistry. 1979 Sep 4;18(18):3946–3952. doi: 10.1021/bi00585a016. [DOI] [PubMed] [Google Scholar]
  8. Kempe T. D., Swyryd E. A., Bruist M., Stark G. R. Stable mutants of mammalian cells that overproduce the first three enzymes of pyrimidine nucleotide biosynthesis. Cell. 1976 Dec;9(4 Pt 1):541–550. doi: 10.1016/0092-8674(76)90036-2. [DOI] [PubMed] [Google Scholar]
  9. Krag S. S. A concanavalin A-resistant Chinese hamster ovary cell line is deficient in the synthesis of [3H]glucosyl oligosaccharide-lipid. J Biol Chem. 1979 Sep 25;254(18):9167–9177. [PubMed] [Google Scholar]
  10. Krag S. S., Robbins P. W. Sindbis envelope proteins as endogenous acceptors in reactions of guanosine diphosphate-[14C]Mannose with preparations of infected chicken embryo fibroblasts. J Biol Chem. 1977 Apr 25;252(8):2621–2629. [PubMed] [Google Scholar]
  11. Kuwano M., Tabuki T., Akiyama S., Mifune K., Takatsuki A., Tamura G., Ikehara Y. Isolation and characterization of Chinese hamster ovary cell mutants with altered sensitivity to high doses of tunicamycin. Somatic Cell Genet. 1981 Sep;7(5):507–521. doi: 10.1007/BF01549655. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Lehle L., Tanner W. The specific site of tunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett. 1976 Nov 15;72(1):167–170. doi: 10.1016/0014-5793(76)80922-2. [DOI] [PubMed] [Google Scholar]
  14. Lewis W. H., Kuzik B. A., Wright J. A. Assay of ribonucleotide reduction in nucleotide-permeable hamster cells. J Cell Physiol. 1978 Mar;94(3):287–298. doi: 10.1002/jcp.1040940306. [DOI] [PubMed] [Google Scholar]
  15. Lewis W. H., Wright J. A. Isolation of hydroxyurea-resistant CHO cells with altered levels of ribonucleotide reductase. Somatic Cell Genet. 1979 Jan;5(1):83–96. doi: 10.1007/BF01538788. [DOI] [PubMed] [Google Scholar]
  16. Littlefield J. W. Hybridization of hamster cells with high and low folate reductase activity. Proc Natl Acad Sci U S A. 1969 Jan;62(1):88–95. doi: 10.1073/pnas.62.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mamont P. S., Duchesne M. C., Grove J., Tardif C. Initial characterization of a HTC cell variant partially resistant to the anti-proliferative effect of ornithine decarboxylase inhibitors. Exp Cell Res. 1978 Sep;115(2):387–393. doi: 10.1016/0014-4827(78)90292-6. [DOI] [PubMed] [Google Scholar]
  18. Owada M., Neufeld E. F. Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? Evidence from I-cell disease. Biochem Biophys Res Commun. 1982 Apr 14;105(3):814–820. doi: 10.1016/0006-291x(82)91042-7. [DOI] [PubMed] [Google Scholar]
  19. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  20. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  21. RADIN N. S. Glycolipide determination. Methods Biochem Anal. 1958;6:163–189. doi: 10.1002/9780470110225.ch7. [DOI] [PubMed] [Google Scholar]
  22. Scher M. G., Jochen A., Waechter C. J. Biosynthesis of glucosylated derivatives of dolichol: possible intermediates in the assembly of white matter glycoproteins. Biochemistry. 1977 Nov 15;16(23):5037–5044. doi: 10.1021/bi00642a015. [DOI] [PubMed] [Google Scholar]
  23. Schimke R. T., Kaufman R. J., Alt F. W., Kellems R. F. Gene amplification and drug resistance in cultured murine cells. Science. 1978 Dec 8;202(4372):1051–1055. doi: 10.1126/science.715457. [DOI] [PubMed] [Google Scholar]
  24. Steiner S., Lester R. L. Studies on the diversity of inositol-containing yeast phospholipids: incorporation of 2-deoxyglucose into lipid. J Bacteriol. 1972 Jan;109(1):81–88. doi: 10.1128/jb.109.1.81-88.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sudo T., Onodera K. Isolation and characterization of tunicamycin resistant mutants from Chinese hamster ovary cells. J Cell Physiol. 1979 Oct;101(1):149–156. doi: 10.1002/jcp.1041010117. [DOI] [PubMed] [Google Scholar]
  26. Suttle D. P., Stark G. R. Coordinate overproduction of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase in hamster cells resistant to pyrazofurin and 6-azauridine. J Biol Chem. 1979 Jun 10;254(11):4602–4607. [PubMed] [Google Scholar]
  27. Takatsuki A., Arima K., Tamura G. Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. J Antibiot (Tokyo) 1971 Apr;24(4):215–223. doi: 10.7164/antibiotics.24.215. [DOI] [PubMed] [Google Scholar]
  28. Tkacz J. S., Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975 Jul 8;65(1):248–257. doi: 10.1016/s0006-291x(75)80086-6. [DOI] [PubMed] [Google Scholar]
  29. Waechter C. J., Harford J. B. Evidence for the enzymatic transfer of N-acetylglucosamine from UDP--N-acetylglucosamine into dolichol derivative and glycoproteins by calf brain membranes. Arch Biochem Biophys. 1977 May;181(1):185–198. doi: 10.1016/0003-9861(77)90497-0. [DOI] [PubMed] [Google Scholar]
  30. Wahl G. M., Padgett R. A., Stark G. R. Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. J Biol Chem. 1979 Sep 10;254(17):8679–8689. [PubMed] [Google Scholar]

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