Advertisement

The Generation of Stable Oxidative Stress-Resistant Phenotypes in Chinese Hamster Fibroblasts Chronically Exposed to Hydrogen Peroxide or Hyperoxia

  • Douglas R. Spitz
  • Shannon J. Sullivan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

With the recognition that chronic exposure to oxidative stress occurs in many disease states and can be deleterious to the human health, great interest has emerged in understanding the mechanisms by which mammalian cells develop resistance to chronic oxidative stress. In order to study the mechanisms of development of resistance to chronic oxidative stress, a model system where Chinese hamster fibroblasts (HA1) are chronically exposed to progressively increasing concentrations of H2O2 (50–800 μM) or hyperoxia (80–95% O2) has been developed. Following >200 d of exposure to H2O2 (or 18 months of exposure to hyperoxia), the cells developed stable H2O2-resistant (or O2-resistant) phenotypes that are characterized by increases in total glutathione, antioxidant enzyme activity, heme oxygenase activity, stress protein gene expression, DNA repair pathways, and resistance to a wide variety of other toxic stress known to cause oxidant injury. In addition, these oxidant-resistant cells exhibited amplification of the gene for catalase and constitutively elevated AP-1 DNA binding activity. Further, beyond 240 d genomic instability as evidenced by chromosomal rearrangements and alterations in ploidy was stably maintained upon removal from the chronic oxidative stress conditions. These results demonstrate the capability of mammalian cells to develop stable oxidative stress-resistant phenotypes in response to both exogenous (H2O2) as well as endogenous (95% O2) oxidative stress. The understanding of mechanisms of resistance to oxidative stress and its possible relevance in various disease states are discussed.

Key words

Oxidative stress hydrogen peroxide hyperoxia adaptive response oxidant injury cellular resistance mammalian cell phenotype antioxidant enzymes 

Notes

Acknowledgments

DRS is supported by NIH R01-CA100045, DOE DE-FG02-02ER63447, and NIEHS P42 ES013661. SJS is supported by the Department of Pediatrics at the University of Iowa.

References

  1. 1.
    Harman, D. (1957) Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 2, 298–300.Google Scholar
  2. 2.
    Oberley, L.W. and Buettner, G.R. (1979) Role of superoxide dismutase in cancer: a review. Cancer Res. 39, 1141–1149.PubMedGoogle Scholar
  3. 3.
    Ames, B.N. (1983) Dietary carcinogens and anticarcinogens: oxygen radicals and degenerative diseases. Science 221, 1256–1262.CrossRefPubMedGoogle Scholar
  4. 4.
    Cerutti, P.A. (1985) Prooxidant states and tumor promotion. Science 227, 375–381.CrossRefPubMedGoogle Scholar
  5. 5.
    Finkel, T. and Holbrook, N.J. (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247.CrossRefPubMedGoogle Scholar
  6. 6.
    Spitz, D.R., Azzam, E.I., Li, J.J., and Gius, D. (2004) Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology. Cancer Metastasis Rev. 23, 311–322.CrossRefPubMedGoogle Scholar
  7. 7.
    Spitz, D.R., Li, G.C., McCormick, M.L., Sun, Y., and Oberley, L.W. (1988) Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity. Radiat. Res. 114, 114–124.CrossRefPubMedGoogle Scholar
  8. 8.
    Spitz, D.R., Mackey, M.A., Li, G.C., Elwell, J.H., McCormick, M.L.,, and Oberley, L.W. (1989) Relationship between changes in ploidy and stable cellular resistance to hydrogen peroxide. J. Cell Physiol. 139, 592–598.CrossRefPubMedGoogle Scholar
  9. 9.
    Spitz, D.R., Malcolm, R.R., and Roberts, R.J. (1990) Cytotoxicity and metabolism of 4-hydroxynonenal and 2-nonenal in H2O2-resistant cell lines: Do aldehydic by-products of lipid peroxidation contribute to oxidative stress?. Biochem. J. 267, 453–459.PubMedGoogle Scholar
  10. 10.
    Spitz, D.R., Elwell, J.H., Sun, Y., Oberley, L.W., Oberley, T.D., Sullivan, S.J., and Roberts, R.J. (1990) Oxygen toxicity in control and H2O2-resistant Chinese hamster fibroblast cell lines. Arch. Biochem. Biophys. 279, 249–260.CrossRefPubMedGoogle Scholar
  11. 11.
    Spitz, D.R., Adams, D.T., Sherman, C.M., and Roberts, R.J. (1992) Mechanisms of cellular resistance to hydrogen peroxide, hyperoxia and 4-hydroxy-2-nonenal toxicity: The significance of increased catalase activity in H2O2-resistant fibroblasts. Arch. Biochem. Biophys. 292, 221–227.CrossRefPubMedGoogle Scholar
  12. 12.
    Sullivan, S.J., Oberley, T.D., Roberts, R.J., and Spitz, D.R. (1992) A stable O2-resistant cell line: Role of lipid peroxidation by-products in O2-mediated injury. Am. J. Physiol. (Lung Cell Mol. Physiol.) 262, 748–756.Google Scholar
  13. 13.
    Spitz, D.R., Dewey, W.C., and Li, G.C. (1987) Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts. J. Cell. Physiol. 131, 364–373.CrossRefPubMedGoogle Scholar
  14. 14.
    Spitz, D.R. and Li, G.C. (1990) Heat-induced cytotoxicity in H2O2-resistant Chinese hamster fibroblasts. J. Cell. Physiol. 142, 255–260.CrossRefPubMedGoogle Scholar
  15. 15.
    Spitz, D.R., Phillips, J.W., Adams, D.T., Sherman, C.M., Deen, D.F., and Li, G.C. (1993) Cellular resistance to oxidative stress is accompanied by resistance to cisplatin: The significance of increased catalase activity and total glutathione in H2O2-resistant fibroblasts. J. Cell. Physiol. 156, 72–79.CrossRefPubMedGoogle Scholar
  16. 16.
    Walker, M.W., Kinter, M.T., Roberts, R.J., and Spitz, D.R. (1995) Nitric oxide induced cytotoxicity: involvement of cellular resistance to oxidative stress and the role of glutathione in protection. Pediat. Res. 37, 41–49.CrossRefPubMedGoogle Scholar
  17. 17.
    Spitz, D.R., Kinter, M.T., and Roberts, R.J. (1995) The contribution of increased glutathione content to mechanisms of oxidative stress resistance in hydrogen peroxide resistant hamster fibroblasts. J. Cell. Physiol. 165, 600–609.CrossRefPubMedGoogle Scholar
  18. 18.
    Guyton, K.Z., Spitz, D.R., and Holbrook, N.J. (1996) Expression of stress response genes GADD153, c-jun, and heme oxygenase-1 in H2O2- and O2-resistant fibroblasts. Free Radic. Biol. Med. 20, 735–741.CrossRefPubMedGoogle Scholar
  19. 19.
    Dennery, P.A., Wong, H.E., Sridhar, K.J., Rodgers, P., Sim, J.E., and Spitz, D.R. (1996) Differences in basal and hyperoxia associated heme oxygenase expression in oxidant resistant hamster fibroblasts. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 271, 672–679.Google Scholar
  20. 20.
    Dennery, P.A., Sridhar, K.J., Lee, C.S., Wong, H.E., Shokoohi, V., Rodgers, P.A., and Spitz, D.R. (1997) Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts. J. Biol. Chem. 272, 14937–14942.CrossRefPubMedGoogle Scholar
  21. 21.
    Hunt, C.R., Sim, J.E., Featherstone, T., Golden, W., Von Kapp-Herr, C., Hock, R.A., Gomez, R.A., Parsian, A.J., and Spitz, D.R. (1998) Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress. Cancer Res. 58, 3986–3992.PubMedGoogle Scholar
  22. 22.
    Bradbury, C.M., Locke, J.E., Wei, S.J., Rene, L.M., Karimpour, S., Hunt, C., Spitz, D.R., and Gius, D. (2001) Increased activator protein 1 activity as well as resistance to heat-induced radiosensitization, hydrogen peroxide, and cisplatin are inhibited by indomethacin in oxidative stress-resistant cells. Cancer Res. 61, 3486–3492.PubMedGoogle Scholar
  23. 23.
    Suzuki, T., Spitz, D.R., Gandhi, P., Lin, H.Y., and Crawford, D.R. (2002) Mammalian resistance to oxidative stress: a comparative analysis. Gene Expr. 10, 179–191.PubMedGoogle Scholar
  24. 24.
    Keightley, J.A., Shang, L., and Kinter, M. (2004) Proteomic analysis of oxidative stress resistant cells: a specific role for aldose reductase overexpression in cytoprotection. Mol. Cell Proteomics 3, 165–175.Google Scholar
  25. 25.
    Grishko, V.I., Rachek, L.I., Spitz, D.R., Wilson, G.L., and LeDoux, S. (2005) Contribution of mitochondrial DNA repair to cell resistance from oxidative stress. J. Biol. Chem. 280, 8901–8905.CrossRefPubMedGoogle Scholar
  26. 26.
    Bojes, H.K., Suresh, P.K., Mills, E.M., Sim, J.E., Spitz, D.R., Sim, J.E., and Kehrer, J.P. (1998) Bcl-2 and Bcl-xL in peroxide resistant A549 and U87MG cells. Toxicol. Sci. 42, 109–116.PubMedGoogle Scholar
  27. 27.
    Kasugai, I. and Yamada, M. (1992) High production of catalase in hydrogen peroxide-resistant human leukemia HL-60 cell lines. Leuk. Res. 16, 173–179.CrossRefPubMedGoogle Scholar
  28. 28.
    Yamada, M., Hashinaka, K., Inazawa, J., and Abe, T. (1991) Expression of catalase and myeloperoxidase genes in hydrogen peroxide-resistant HL-60 cells. DNA Cell Biol. 10, 735–742.CrossRefPubMedGoogle Scholar
  29. 29.
    Kasugai, I. and Yamada, M. (1989) Adaptation of human leukemia HL-60 cells to hydrogen peroxide as oxidative stress. Leuk. Res. 13, 757–762.CrossRefPubMedGoogle Scholar
  30. 30.
    Lin, F., Jackson, V.E., and Girotti, A.W. (1995) Amplification and hyperexpression of the catalase gene in selenoperoxidase-deficient leukemia cells. Arch. Biochem. Biophys. 317, 7–18.CrossRefPubMedGoogle Scholar
  31. 31.
    Cantoni, O., Sestili, P., Palomba, L., Guidarelli, A., Cattabeni, F., and Murray, D. (1996) Isolation and preliminary characterization of a Chinese hamster ovary cell line with high-degree resistance to hydrogen peroxide. Biochem. Pharmacol. 51, 1021–1029.CrossRefPubMedGoogle Scholar
  32. 32.
    Vallis, K.A. and Wolf, C.R. (1996) Relationship between the adaptive response to oxidants and stable menadione-resistance in Chinese hamster ovary cell lines. Carcinogenesis 17, 649–654.CrossRefPubMedGoogle Scholar
  33. 33.
    Cantoni, O., Guidarelli, A., Sestili, P., Mannello, F., Gazzanelli, G., and Cattabeni, F. (1993) Development and characterization of hydrogen peroxide-resistant Chinese hamster ovary cell variants-I. Relationship between catalase activity and the induction/stability of the oxidant-resistant phenotype. Biochem. Pharmacol. 45, 2251–2257.CrossRefPubMedGoogle Scholar
  34. 34.
    Martins, E.A., Mori, L., Birnboim, H.C., and Meneghini, R. (1992) Menadione-resistant Chinese hamster cell variants are cross resistant to hydrogen peroxide and exhibit stable chromosomal and biochemical alterations. Mol. Cell Biochem. 118, 181–189.CrossRefPubMedGoogle Scholar
  35. 35.
    Park, Y.M., Anderson, R.L., Spitz, D.R., and Hahn, G.M. (1992) Hypoxia and resistance to hydrogen peroxide confer resistance to tumor necrosis factor in murine L929 cells. Radiat. Res. 131, 162–168.CrossRefPubMedGoogle Scholar
  36. 36.
    Sagara, Y., Dargusch, R., Chambers, D., Davis, J., Schubert, D., and Maher, P. (1998) Cellular mechanisms of resistance to chronic oxidative stress. Free Radic. Biol. Med. 24, 1375–1389.CrossRefPubMedGoogle Scholar
  37. 37.
    Goligorsky, M.S., Morgan, M.A., Lyubsky, S., Gross, R.W., Adams, D.T., and Spitz, D.R. (1993) Establishment of a hydrogen peroxide resistant variant of renal tubular epithelial cells: Role of calcium-independent phospholipase A2 in cell damage. Arch. Biochem. Biophys. 301, 119–128.CrossRefPubMedGoogle Scholar
  38. 38.
    Laszlo, A., Davidson, T., Harvey, A., Sim, J.E., Malyapa, R.S., Spitz, D.R., and Roti, J.L. (2006) Alterations in heat-induced radiosensitization accompanied by nuclear structure alterations in Chinese Hamster cells. Int. J. Hyperthermia. 22, 43–60.CrossRefPubMedGoogle Scholar
  39. 39.
    Sullivan, S.J., Roberts, R.J., and Spitz, D.R. (1991) Replacement of media in cell culture alters oxygen toxicity: Possible role of lipid aldehydes and glutathione transferases in O2 toxicity. J. Cell. Physiol. 147, 427–433.CrossRefPubMedGoogle Scholar
  40. 40.
    Spitz, D.R., Sullivan, S.J., Kinter, M.T., Adams, D.T., Sherman, C.M., and Roberts, R.J. (1995) Mechanisms of resistance to oxidative stress in O2-resistant cells. In: The Oxygen Paradox (Davies, K.J.A. and Ursini, F., Eds.), CLEUP University Press, Padova, Italy, pp. 405–412.Google Scholar
  41. 41.
    Joenje, H., Gille, J.J., Oostra, A.B., and Van der Valk, P. (1985) Some characteristics of hyperoxia-adapted HeLa cells. A tissue culture model for cellular oxygen tolerance. Lab. Invest. 52, 420–428.PubMedGoogle Scholar
  42. 42.
    van der Valk, P., Gille, J.J., Oostra, A.B., Roubos, E.W., Sminia, T., and Joenje, H. (1985) Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary. Antioxygenic enzyme levels and ultrastructural morphometry of peroxisomes and mitochondria. Cell Tissue Res. 239, 61–68.CrossRefPubMedGoogle Scholar
  43. 43.
    Campian, J.L., Qian, M., Gao, X., and Eaton, J.W. (2004) Oxygen tolerance and coupling of mitochondrial electron transport. J. Biol. Chem. 279, 46580–46587.CrossRefPubMedGoogle Scholar
  44. 44.
    Li, J., Gao, X., Qian, M., and Eaton, J.W. (2004) Mitochondrial metabolism underlies hyperoxic cell damage. Free Radic. Biol. Med. 36, 1460–1470.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Douglas R. Spitz
    • 1
  • Shannon J. Sullivan
    • 2
  1. 1.Departments of Radiation Oncology, Holden Comprehensive Cancer CenterThe University of IowaIowa CityUSA
  2. 2.Department of PediatricsThe University of IowaIowa CityUSA

Personalised recommendations