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Tumor Biology

, Volume 32, Issue 1, pp 87–98 | Cite as

Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines

  • Gabor Tarjan
  • G. Kenneth HainesIII
  • Benjamin J. Vesper
  • Jiaping Xue
  • Michael B. Altman
  • Yaroslav R. Yarmolyuk
  • Huma Khurram
  • Kim M. Elseth
  • John C. Roeske
  • Bulent Aydogan
  • James A. Radosevich
Research Article

Abstract

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated (“parent”) cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.

Keywords

Nitric oxide (NO) Glutathione S-transferase (GST) Apurinic/apyrimidinic endonuclease (APE1) Nitric oxide synthase (NOS) Squamous cell carcinoma of the tongue Checkpoint kinases 

Notes

Acknowledgments

The authors wish to thank the UIC Flow Cytometry facility (Drs. Karen Hagen and Jewel Graves) for their assistance with the FACS study. This work was supported by a Veterans Affairs merit review grant (J.A.R.) and a generous gift from The Cherry Family Foundation.

References

  1. 1.
    Campbell WJ, de la Torre J (2008) Head and neck cancer—squamous cell carcinoma. Medscape. http://emedicine.medscape.com/article/1289986-overview. Accessed 26 March 2010
  2. 2.
    De Ridder M, Verellen D, Verovski V, Storme G. Hypoxic tumor cell radiosensitization through nitric oxide. Nitric Oxide. 2008;19:164–9.CrossRefPubMedGoogle Scholar
  3. 3.
    Singh S, Cowen RL, Chinje EC, Stratford IJ. The impact of intracellular generation of nitric oxide on the radiation response of human tumor cells. Radiat Res. 2009;171:572–80.CrossRefPubMedGoogle Scholar
  4. 4.
    Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994;78:915–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Bentz BG, Haines III GK, Lingen MW, Pelzer HJ, Hanson DG, Radosevich JA. Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck. Ann Otol Rhinol Laryngol. 1999;108:781–7.PubMedGoogle Scholar
  6. 6.
    Bentz BG, Haines III GK, Radosevich JA. Increased protein nitrosylation in head and neck squamous cell carcinoma. Head Neck. 2000;22:64–70.CrossRefPubMedGoogle Scholar
  7. 7.
    Bentz BG, Haines III GK, Hanson DG, Radosevich JA. Endothelial constitutive nitric oxide synthase (ecNOS) localization in normal and neoplastic salivary tissue. Head Neck. 1998;20:304–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Chandra R, Haines III GK, Bentz BG, Shah P, Robinson AM, Radosevich JA. Expression of nitric oxide synthase type 3 in reflux-induced esophageal lesions. Otolaryngol Head Neck Surg. 2001;124:442–7.CrossRefPubMedGoogle Scholar
  9. 9.
    Chen YK, Hsue SS, Lin LM. Increased expression of inducible nitric oxide synthase for human buccal squamous-cell carcinomas: immunohistochemical, reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR studies. Head Neck. 2002;24:925–32.CrossRefPubMedGoogle Scholar
  10. 10.
    Chen YK, Hsue SS, Lin LM. Correlation between inducible nitric oxide synthase and p53 expression for DMBA-induced hamster buccal-pouch carcinomas. Oral Dis. 2003;9:227–34.CrossRefPubMedGoogle Scholar
  11. 11.
    Radosevich JA, Elseth KM, Vesper BJ, Tarjan G, Haines III GK. Long-term adaptation of lung tumor cell lines with increasing concentrations of nitric oxide donor. Open Lung Cancer J. 2009;2:35–44.CrossRefGoogle Scholar
  12. 12.
    Vesper BJ, Elseth KM, Tarjan G, Haines III GK, Radosevich JA. Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide. Tumor Biol. 2010;31:267–75.CrossRefGoogle Scholar
  13. 13.
    Yarmolyuk Y, Elseth KM, Vesper BJ, Tarjan G, Haines GK III, Radosevich JA (2010) Part I. A model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell lines. Tumor Biol. doi:  10.1007/s13277-010-0101-1.
  14. 14.
    Miller LP (2007) Quantifying western blots without expensive commercial quantification software. http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html. Accessed 20 May 2010
  15. 15.
    Altman MB, Vesper BJ, Smith BD, Stinauer MA, Pelizzari CA, Aydogan B, et al. Characterization of a novel phantom for three-dimensional in vitro cell experiments. Phys Med Biol. 2009;54:N75–82.CrossRefPubMedGoogle Scholar
  16. 16.
    Natarajan N, Shambaugh III GE, Elseth KM, Haines IGK, Radosevich JA. Adaptation of the diphenylamine (DPA) assay to a 96-well plate tissue culture format and comparison with the MTT assay. Biotechniques. 1994;17:166–71.PubMedGoogle Scholar
  17. 17.
    Fritz G, Kaina B. Phosphorylation of the DNA repair protein ape/ref-1 by CKII affects redox regulation of ap-1. Oncogene. 1999;18:1033–40.CrossRefPubMedGoogle Scholar
  18. 18.
    Yacoub A, Kelley MR, Deutsch WA. The DNA repair activity of human redox/repair protein ape/ref-1 is inactivated by phosphorylation. Cancer Res. 1997;57:5457–9.PubMedGoogle Scholar
  19. 19.
    Bhakat KK, Izumi T, Yang SH, Hazra TK, Mitra S. Role of acetylated human ap-endonuclease (ape1/ref-1) in regulation of the parathyroid hormone gene. EMBO J. 2003;22:6299–309.CrossRefPubMedGoogle Scholar
  20. 20.
    Busso CS, Iwakuma T, Izumi T. Ubiquitination of mammalian ap endonuclease (ape1) regulated by the p53-mdm2 signaling pathway. Oncogene. 2009;28:1616–25.CrossRefPubMedGoogle Scholar
  21. 21.
    Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, et al. Chk1 is an essential kinase that is regulated by Atr and required for the g(2)/m DNA damage checkpoint. Genes Dev. 2000;14:1448–59.CrossRefPubMedGoogle Scholar
  22. 22.
    Walworth NC, Bernards R. Rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science. 1996;271:353–6.CrossRefPubMedGoogle Scholar
  23. 23.
    Wan S, Capasso H, Walworth NC. The topoisomerase I poison camptothecin generates a chk1-dependent DNA damage checkpoint signal in fission yeast. Yeast. 1999;15:821–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Zhou J, Schmid T, Brune B. Hif-1alpha and p53 as targets of no in affecting cell proliferation, death and adaptation. Curr Mol Med. 2004;4:741–51.CrossRefPubMedGoogle Scholar
  25. 25.
    Ahn JY, Schwarz JK, Piwnica-Worms H, Canman CE. Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of chk2 in response to ionizing radiation. Cancer Res. 2000;60:5934–6.PubMedGoogle Scholar
  26. 26.
    Melchionna R, Chen XB, Blasina A, McGowan CH. Threonine 68 is required for radiation-induced phosphorylation and activation of cds1. Nat Cell Biol. 2000;2:762–5.CrossRefPubMedGoogle Scholar
  27. 27.
    Reinhardt HC, Yaffe MB. Kinases that control the cell cycle in response to DNA damage: chk1, chk2, and mk2. Curr Opin Cell Biol. 2009;21:245–55.CrossRefPubMedGoogle Scholar
  28. 28.
    Schwarz JK, Lovly CM, Piwnica-Worms H. Regulation of the chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation. Mol Cancer Res. 2003;1:598–609.PubMedGoogle Scholar
  29. 29.
    Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller DG, Morrow M (eds) Ajcc cancer staging manual, 6th, ed. New York: Springer; 2002Google Scholar
  30. 30.
    Blackett NM, Wooliscroft WE, Fielden EM, Lillicrap SC. Radiation modifying effect of the free radical norpseudopelletierene-n-oxyl on normal bone marrow stem cells in vitro and in vivo. Radiat Res. 1974;58:361–72.CrossRefPubMedGoogle Scholar
  31. 31.
    Cooke BC, Fielden EM, Johnson M. Polyfunctional radiosensitizers. I. Effects of a nitroxyl biradical on the survival of mammalian cells in vitro. Radiat Res. 1976;65:152–62.CrossRefPubMedGoogle Scholar
  32. 32.
    Griffin RJ, Makepeace CM, Hur WJ, Song CW. Radiosensitization of hypoxic tumor cells in vitro by nitric oxide. Int J Radiat Oncol Biol Phys. 1996;36:377–83.PubMedGoogle Scholar
  33. 33.
    Jordan BF, Beghein N, Aubry M, Gregoire V, Gallez B. Potentiation of radiation-induced regrowth delay by isosorbide dinitrate in FSaII murine tumors. Int J Cancer. 2003;103:138–41.CrossRefPubMedGoogle Scholar
  34. 34.
    Millar BC, Jenkins TC, Fielden EM. Polyfunctional radiosensitizers. V. Sensitization of hypoxic Chinese hamster cells, v.79–753b, in vitro by a series of bifunctional nitroxyl compounds. Radiat Res. 1982;90:271–83.CrossRefPubMedGoogle Scholar
  35. 35.
    Brennan PA, Palacios-Callender M, Zaki GA, Spedding AV, Langdon JD. Type II nitric oxide synthase (NOS2) expression correlates with lymph node status in oral squamous cell carcinoma. J Oral Pathol Med. 2001;30:129–34.CrossRefPubMedGoogle Scholar
  36. 36.
    Wang YZ, Cao YQ, Wu JN, Chen M, Cha XY. Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA. World J Gastroenterol. 2005;11:46–50.PubMedGoogle Scholar
  37. 37.
    Loibl S, Buck A, Strank C, von Minckwitz G, Roller M, Sinn HP, et al. The role of early expression of inducible nitric oxide synthase in human breast cancer. Eur J Cancer. 2005;41:265–71.CrossRefPubMedGoogle Scholar
  38. 38.
    Tell G, Quadrifoglio F, Tiribelli C, Kelley MR. The many functions of ape1/ref-1: not only a DNA repair enzyme. Antioxid Redox Signal. 2009;11:601–20.CrossRefPubMedGoogle Scholar
  39. 39.
    Tell G, Damante G, Caldwell D, Kelley MR. The intracellular localization of ape1/ref-1: more than a passive phenomenon? Antioxid Redox Signal. 2005;7:367–84.CrossRefPubMedGoogle Scholar
  40. 40.
    Puglisi F, Aprile G, Minisini AM, Barbone F, Cataldi P, Tell G, et al. Prognostic significance of ape1/ref-1 subcellular localization in non-small cell lung carcinomas. Anticancer Res. 2001;21:4041–9.PubMedGoogle Scholar
  41. 41.
    Yoo DG, Song YJ, Cho EJ, Lee SK, Park JB, Yu JH, et al. Alteration of ape1/ref-1 expression in non-small cell lung cancer: the implications of impaired extracellular superoxide dismutase and catalase antioxidant systems. Lung Cancer. 2008;60:277–84.CrossRefPubMedGoogle Scholar
  42. 42.
    Moore DH, Michael H, Tritt R, Parsons SH, Kelley MR. Alterations in the expression of the DNA repair/redox enzyme ape/ref-1 in epithelial ovarian cancers. Clin Cancer Res. 2000;6:602–9.PubMedGoogle Scholar
  43. 43.
    Qing Y, Wang D, Lei X, Xiang DB, Li MX, Li ZP, et al. The expression of ape1 and its correlation with prognostic significance after 252cf radiotherapy in cervical cancer. Sichuan Da Xue Xue Bao Yi Xue Ban. 2009;40:125–8.PubMedGoogle Scholar
  44. 44.
    Puglisi F, Barbone F, Tell G, Aprile G, Pertoldi B, Raiti C, et al. Prognostic role of ape/ref-1 subcellular expression in stage I–III breast carcinomas. Oncol Rep. 2002;9:11–7.PubMedGoogle Scholar
  45. 45.
    Russo D, Arturi F, Bulotta S, Pellizzari L, Filetti S, Manzini G, et al. Ape1/ref-1 expression and cellular localization in human thyroid carcinoma cell lines. J Endocrinol Invest. 2001;24:RC10–2.PubMedGoogle Scholar
  46. 46.
    Tell G, Pellizzari L, Pucillo C, Puglisi F, Cesselli D, Kelley MR, et al. Tsh controls ref-1 nuclear translocation in thyroid cells. J Mol Endocrinol. 2000;24:383–90.CrossRefPubMedGoogle Scholar
  47. 47.
    Di Maso V, Avellini C, Croce LS, Rosso N, Quadrifoglio F, Cesaratto L, et al. Subcellular localization of ape1/ref-1 in human hepatocellular carcinoma: possible prognostic significance. Mol Med. 2007;13:89–96.PubMedGoogle Scholar
  48. 48.
    Bentz BG, Haines III GK, Radosevich JA. Glutathione s-transferase pi in squamous cell carcinoma of the head and neck. Laryngoscope. 2000;110:1642–7.CrossRefPubMedGoogle Scholar
  49. 49.
    Cullen KJ, Newkirk KA, Schumaker LM, Aldosari N, Rone JD, Haddad BR. Glutathione s-transferase pi amplification is associated with cisplatin resistance in head and neck squamous cell carcinoma cell lines and primary tumors. Cancer Res. 2003;63:8097–102.PubMedGoogle Scholar
  50. 50.
    Shiga H, Heath EI, Rasmussen AA, Trock B, Johnston PG, Forastiere AA, et al. Prognostic value of p53, glutathione s-transferase pi, and thymidylate synthase for neoadjuvant cisplatin-based chemotherapy in head and neck cancer. Clin Cancer Res. 1999;5:4097–104.PubMedGoogle Scholar
  51. 51.
    Dourado DF, Fernandes PA, Ramos MJ. Mammalian cytosolic glutathione transferases. Curr Protein Pept Sci. 2008;9:325–37.CrossRefPubMedGoogle Scholar
  52. 52.
    Mulder TP, Manni JJ, Roelofs HM, Peters WH, Wiersma A. Glutatione s-transferases and glutathione in human head and neck cancer. Carcinogenesis. 1995;16:619–24.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2010

Authors and Affiliations

  • Gabor Tarjan
    • 1
  • G. Kenneth HainesIII
    • 2
  • Benjamin J. Vesper
    • 3
    • 4
  • Jiaping Xue
    • 3
    • 4
  • Michael B. Altman
    • 5
  • Yaroslav R. Yarmolyuk
    • 3
  • Huma Khurram
    • 3
    • 4
  • Kim M. Elseth
    • 3
    • 4
  • John C. Roeske
    • 6
  • Bulent Aydogan
    • 5
    • 7
  • James A. Radosevich
    • 3
    • 4
  1. 1.Department of PathologyJohn H. Stroger, Jr. Hospital of Cook CountyChicagoUSA
  2. 2.Department of PathologyYale University School of MedicineNew HavenUSA
  3. 3.Center for Molecular Biology of Oral Diseases, Department of Oral Medicine and Diagnostic Sciences,College of DentistryUniversity of Illinois at ChicagoChicagoUSA
  4. 4.Jesse Brown VAMCChicagoUSA
  5. 5.Department of Radiation and Cellular OncologyUniversity of ChicagoChicagoUSA
  6. 6.Department of Radiation OncologyLoyola University Medical CenterMaywoodUSA
  7. 7.Department of Radiation OncologyUniversity of Illinois at ChicagoChicagoUSA

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