Odontology

, Volume 100, Issue 2, pp 149–155

Mutational analysis of HRAS and KRAS genes in oral carcinoma cell lines

  • Sachiko Maemoto
  • Megumi Yumoto
  • Masato Ibata
  • Sho Torizuka
  • Naohumi Ozawa
  • Shunsuke Tatsumi
  • Moeko Hashido
  • Masako Morikawa
  • Genta Maeda
  • Kazushi Imai
Original Article

Abstract

RAS overexpression and its active mutations are involved in malignant tumorigenesis. However, the mutation rates in oral carcinoma cells differ between populations. In the present study, genomic DNA of oral carcinoma cells (HOC313, TSU, HSC2, HSC3, KOSC2, KOSC3, SCCKN, OSC19, Ca9.22, and Ho1u1 cells) or normal gingival fibroblasts (GF12 cells) derived from a Japanese population were amplified by polymerase chain reaction using primer sets, spanning HRAS and KRAS exons. Nucleotide substitutions were analyzed by single strand conformation polymorphism. In contrast to no substitutions in KRAS, nine different substitutions were detected in HRAS. Of the nine, six substitutions were located at intron 1 (HSC2 and HSC3 cells) or intron 2 (HSC3, SCCKN and Ca9.22 cells), and one each of exon 1 (all cells), exon 2 (HOC313, TSU, HSC2 and HSC3 cells) and the 5′ upstream region (all cells). Substitutions at exons 1 and 2 did not affect the amino acid sequence; the exon 1 substitution was positioned at the 5′ untranslated region, which may be a single nucleotide polymorphism (SNP) sequence because all the cells were isolated from a Japanese population, and the mutations at exon 2 was a silent mutation. A substitution at the 5′ upstream region was an SNP. These data demonstrate that SNPs and point mutations observed in HRAS do not change the amino acid sequence, and suggest that the mutations affecting the amino acid sequence may be a rare event in oral carcinomas of the Japanese population.

Keywords

HRAS KRAS Mutation Oral carcinoma SNP 

References

  1. 1.
    Johnson N. Tobacco use and oral cancer: a global perspective. J Dent Educ. 2001;65:328–39.PubMedGoogle Scholar
  2. 2.
    Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. J Dent Res. 2008;87:14–32.PubMedCrossRefGoogle Scholar
  3. 3.
    Vora HH, Shah NG, Patel DD, Trivedi TI, Chikhlikar PR. Prognostic significance of biomarkers in squamous cell carcinoma of the tongue: multivariate analysis. J Surg Oncol. 2003;82:34–50.PubMedCrossRefGoogle Scholar
  4. 4.
    Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:7–13.CrossRefGoogle Scholar
  5. 5.
    Maeda G, Chiba T, Aoba T, Imai K. Epigenetic inactivation of E-cadherin by promoter hypermethylation in oral carcinoma cells. Odontology. 2007;95:24–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Miyazawa J, Mitoro A, Kawashiri S, Chada KK, Imai K. Expression of mesenchyme-specific gene HMGA2 in squamous cell carcinomas of the oral cavity. Cancer Res. 2004;64:2024–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Saranath D, Chang SE, Bhoite LT, Panchai RG, Kerr IB, Mehta AR, Johnson NW, Deo MG. High frequency mutation in codons 12 and 61 of H-ras oncogene in chewing tobacco-related human oral carcinoma in India. Br J Cancer. 1991;63:573–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Hardisson D. Molecular pathogenesis of head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol. 2003;260:502–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Lu SL, Herrington H, Reh D, Weber S, Bornstein S, Wang D, Li AG, Tang CF, Siddiqui Y, Nord J, Andersen P, Corless CL, Wang XJ. Loss of transforming growth factor-β type II receptor promotes metastatic head-and-neck squamous cell carcinoma. Genes Dev. 2006;20:1331–42.PubMedCrossRefGoogle Scholar
  10. 10.
    Kuo MYP, Jeng JH, Chiang CP, Hahn LJ. Mutation of Ki-ras oncogene codon 12 in betel quid chewing-related human oral squamous cell carcinoma in Taiwan. J Oral Pathol Med. 1994;23:70–4.PubMedCrossRefGoogle Scholar
  11. 11.
    Yarbrough WG, Shores C, Witsell DL, Weissler MC, Fidler ME, Gilmer TM. Ras mutations and expression in head and neck squamous cell carcinomas. Laryngoscope. 1994;104:1337–47.PubMedCrossRefGoogle Scholar
  12. 12.
    Huang KH, Huang SF, Chen IH, Liao CT, Wang HM, Hsieh LL. Methylation of RASFF1A, RASFF2A, and HIN-1 is associated with poor outcome after radiotherapy, but not surgery, in oral squamous cell carcinoma. Clin Cancer Res. 2009;15:4174–80.PubMedCrossRefGoogle Scholar
  13. 13.
    Sakata K. Alterations of tumor suppressor genes and the H-ras oncogene in oral squamous cell carcinoma. J Oral Pathol Med. 1996;25:302–7.PubMedCrossRefGoogle Scholar
  14. 14.
    Chang SE, Bhatia P, Johnson NW, Morgan PR, McCormick F, Young B, Hiorns L. Ras mutations in United Kingdom examples of oral malignancies are infrequent. Int J Cancer. 1991;48:409–12.PubMedCrossRefGoogle Scholar
  15. 15.
    Kok SH, Lee JJ, Hsu HC, Chiang CP, Kuo YS, Kuo MYP. Mutations of the adenomatous polyposis coli gene in areca quid and tobacco-associated oral squamous cell carcinomas in Taiwan. J Oral Pathol Med. 2002;31:395–401.PubMedCrossRefGoogle Scholar
  16. 16.
    Quintanilla M, Brown K, Ramsden M, Balmain A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature. 1986;332:78–80.CrossRefGoogle Scholar
  17. 17.
    Das N, Majumder J, DasGupta UB. Ras gene mutations in oral cancer in eastern India. Oral Oncol. 2000;36:76–80.PubMedCrossRefGoogle Scholar
  18. 18.
    Kiaris H, Spandidos DA, Jones AS, Vaughan ED, Field JK. Mutations, expression and genomic instability of the H-ras proto-oncogene in squamous cell carcinomas of the head and neck. Br J Cancer. 1995;72:123–8.PubMedCrossRefGoogle Scholar
  19. 19.
    Azuma M, Furumoto N, Kawamata H, Yoshida H, Yanagawa T, Yura Y, Hayashi Y, Takegawa Y, Sato M. The relation of ras oncogene product p21 expression to clinicopathological status criteria and clinical outcome in squamous cell head and neck cancer. Cancer J. 1987;1:375–80.Google Scholar
  20. 20.
    Tsuji T, Sasaki K, Hiraoka F, Shinozaki F. The immunohistochemical detection of ras p21 and its correlation with differentiation in oral cancers. J Tumour Marker Oncol. 1998;4:415–9.Google Scholar
  21. 21.
    Bernhard EJ, Stanbridge EJ, Gupta S, Gupta AK, Soto D, Bakanauskas VJ, Cerniglia GJ, Muschel RJ, McKenna WG. Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines. Cancer Res. 2000;60:6577–600.Google Scholar
  22. 22.
    Ling CC, Endlich B. Radioresistance induced by oncogenic transformation. Radiat Res. 1989;120:267–79.PubMedCrossRefGoogle Scholar
  23. 23.
    Miller AC, Kario K, Myers CE, Clark EP, Samid D. Increased radioresistance of EJras-transformed human osteosarcoma cells and its modulation by lovastatin, an inhibitor of p21ras isoprenylation. Int J Cancer. 1993;53:302–7.PubMedCrossRefGoogle Scholar
  24. 24.
    Suzuki R, Kohno H, Suzui M, Yoshimi N, Tsuda H, Wakabayashi K, Tanaka T. An animal model for the rapid induction of tongue neoplasms in human c-Ha-ras proto-oncogene transgenic rats by 4-nitroquinoline 1-oxide: its potential use for preclinical chemoprevention studies. Carcinogenesis. 2006;27:619–30.PubMedCrossRefGoogle Scholar
  25. 25.
    Raimondi AR, Molinolo A, Gutkind JS. Rapamycin prevents early onset of tumorigenesis in an oral-specific K-ras and p53 two-hit carcinoma model. Cancer Res. 2009;69:4159–66.PubMedCrossRefGoogle Scholar
  26. 26.
    Sasaki E, Tsuchida N. Most human squamous cell carcinomas in the oral cavity contain mutated p53 tumor-suppressor genes. Oncogene. 1992;7:927–33.Google Scholar
  27. 27.
    Inagaki T, Matsuwari S, Takahashi R, Shimada K, Fujie K, Maeda S. Establishment of human oral-cancer cell lines (KOSC-2 and KOSC-3) carrying p53 and C-myc abnormalities by geneticin treatment. Int J Cancer. 1994;56:301–8.PubMedCrossRefGoogle Scholar
  28. 28.
    Poeta ML, Manola JM, Goldwasser MA, Forastiere A, Benoit N, Califano JA, Ridge JA, Goodwin J, Kenady D, Saunders J, Westra W, Sidransky D, Koch WM. TP53 mutations and survival in squamous-cell carcinoma of the head and neck. N Engl J Med. 2007;357:2552–61.PubMedCrossRefGoogle Scholar
  29. 29.
    Rubin Grandis J, Melhem MF, Gooding WE, Day R, Holst VA, Wagener MM, Drenning SD, Tweardy DJ. Levels of TGF-α and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst. 1998;90:824–32.PubMedCrossRefGoogle Scholar
  30. 30.
    Lo KW, Kwong J, Hui AB, Chan SY, To KF, Chan AS, Chow LS, Teo PM, Johnson PJ, Huang DP. High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma. Cancer Res. 2001;61:3877–81.PubMedGoogle Scholar
  31. 31.
    Weber A, Langhanki L, Sommerer F, Markwarth A, Wittekind C, Tannapfel A. Mutations of the BRAF gene in squamous cell carcinoma of the head and neck. Oncogene. 2003;22:4757–9.PubMedCrossRefGoogle Scholar

Copyright information

© The Society of The Nippon Dental University 2011

Authors and Affiliations

  • Sachiko Maemoto
    • 1
  • Megumi Yumoto
    • 1
  • Masato Ibata
    • 1
  • Sho Torizuka
    • 1
  • Naohumi Ozawa
    • 1
  • Shunsuke Tatsumi
    • 1
  • Moeko Hashido
    • 1
  • Masako Morikawa
    • 1
  • Genta Maeda
    • 1
  • Kazushi Imai
    • 1
  1. 1.Department of BiochemistryThe Nippon Dental University, School of Life Dentistry at TokyoTokyoJapan

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