Skip to main content
SpringerLink
Log in

Detection of Point Mutations of K-ras Oncogene and p53 Tumor-Suppressor Gene in Sputum Samples

  • Protocol
  • First Online:
Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1105))

Abstract

Mutations in the p53 tumor-suppressor gene and K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage samples of lung cancer patients and also in those of patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these mutations has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells, and, in the case of the p53 gene, inactivating mutations may occur at many codons. This chapter describes methods to identify p53 and K-ras mutations present in low fractions of epithelial cells among the excess of other cell types in sputum samples from lung cancer patients.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300

    Article  PubMed  Google Scholar 

  2. Cancer Facts and Figures 2013 (2013) American Cancer Society

    Google Scholar 

  3. Birrer MJ, Brown PH (1992) Application of molecular genetics to the early diagnosis and screening of lung cancer. Cancer Res 52: 2658s–2664s

    CAS  PubMed  Google Scholar 

  4. Scott A, Salgia R (2008) Biomarkers in lung cancer: from early detection to novel therapeutics and decision-making. Biomark Med 2: 577–586

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Maldonado F, Jett JR (2010) Advances in the diagnosis of lung cancer: contribution of molecular biology to bronchoscopic diagnosis. Curr Opin Pulm Med 16:315–320

    Article  PubMed  Google Scholar 

  6. Vogelstein B, Lane B, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310

    Article  CAS  PubMed  Google Scholar 

  7. Loeb LA, Harris CC (2008) Advances in chemical carcinogenesis: a historical review and prospective. Cancer Res 68:6863–6872

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Suzuki Y, Orita M, Shiraishi M, Hayashi K, Sekiya T (1990) Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 5: 1037–1043

    CAS  PubMed  Google Scholar 

  9. Sugio K, Ishida T, Yokoyama H, Inoue T, Sugimachi K, Sasazuki T (1992) ras gene mutation as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res 52:2903–2906

    CAS  PubMed  Google Scholar 

  10. Rodenhuis S, Slebos RJ (1992) Clinical significance of ras oncogene activation in human lung cancer. Cancer Res 52:2665s–2669s

    CAS  PubMed  Google Scholar 

  11. Husgafvel-Pursianen K, Hackman P, Ridanpaa M, Anttila S, Karjalainen A, Partanen T et al (1993) K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int J Cancer 53:250–256

    Article  Google Scholar 

  12. Keohavong P, DeMichele MA, Melacrinos AC, Landreneau RJ, Weyant RJ, Siegfried JM (1996) Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 2:411–418

    CAS  PubMed  Google Scholar 

  13. Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253:49–53

    Article  CAS  PubMed  Google Scholar 

  14. Greenblatt MS, Bennett WP, Hollstein M, Harris CC (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54: 4855–4878

    CAS  PubMed  Google Scholar 

  15. Yokota J, Wada M, Shimosato Y, Terada M, Sugimura T (1987) Loss of heterozygosity on chromosomes 3, 13, and 17 in small-cell carcinoma and on chromosome 3 in adenocarcinoma of the lung. Proc Natl Acad Sci U S A 84: 9252–9256

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Chiba I, Takahashi T, Nau MM, D’Amico D, Curiel DT, Mitsudomi T et al (1990) Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene 5:1603–1610

    CAS  PubMed  Google Scholar 

  17. Kishimoto Y, Murakami Y, Shiraishi M, Hayashi K, Sekiya T (1992) Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung. Cancer Res 52:4799–4804

    CAS  PubMed  Google Scholar 

  18. Takahashi T, Nau MM, Chiba I, Birrer MJ, Rosenberg RK, Vinocour M et al (1989) p53: a frequent target for genetic abnormalities in lung cancer. Science 246:491–494

    Article  CAS  PubMed  Google Scholar 

  19. Cho Y, Gorina S, Jeffrey PD, Pavletich NP (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265:346–355

    Article  CAS  PubMed  Google Scholar 

  20. Hainaut P, Hollstein M (2000) p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77:81–137

    Article  CAS  PubMed  Google Scholar 

  21. Jassem E, Niklinski J, Rosell R, Niklinska W, Jakobkiewicz J, Monzo M et al (2001) Types and localisation of p53 gene mutations: a report on 332 non-small cell lung cancer patients. Lung Cancer 34:47s–51s

    Article  Google Scholar 

  22. Caron de Fromentel C, Soussi T (1992) TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 4:1–15

    Article  CAS  PubMed  Google Scholar 

  23. de Anta JM, Jassem E, Rosell R, Martinez-Roca M, Jassem J, Martinez-Lopez E et al (1997) TP53 mutational pattern in Spanish and Polish non-small cell lung cancer patients: null mutations are associated with poor prognosis. Oncogene 15:2951–2958

    Article  PubMed  Google Scholar 

  24. Cha RS, Zarbl H, Keohavong P, Thilly WG (1992) Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl 2:14–20

    Article  CAS  PubMed  Google Scholar 

  25. Mitsudomi T, Viallet J, Mulshine JL, Linnoila RI, Minna JD, Gazdar AF (1991) Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene 6:1353–1362

    CAS  PubMed  Google Scholar 

  26. Jiang W, Kahn SM, Guillem JG, Lu SH, Weinstein IB (1989) Rapid detection of ras oncogenes in human tumors: application to colon, esophageal, and gastric cancer. Oncogene 4:923–928

    CAS  PubMed  Google Scholar 

  27. Kumar R, Dunn LL (1989) Designed diagnostic restriction fragment length polymorphism for the detection of point mutations in ras oncogenes. Oncogene Res 1:235–241

    Google Scholar 

  28. Levi S, Urbano-Ispizua A, Gill R, Thomas DM, Gilbertson J, Foster C et al (1991) Multiple K-ras 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. Cancer Res 51: 3497–3502

    CAS  PubMed  Google Scholar 

  29. Kahn SM, Jiang W, Culbertson TA, Weinstein IB, Williams GM, Tomita N et al (1991) Rapid and sensitive non-radioactive detection of mutant K-ras genes via enriched PCR amplification. Oncogene 6:1079–1083

    CAS  PubMed  Google Scholar 

  30. Yakubovskaya MS, Spiegelman V, Luo FC, Malaev S, Salnev A, Zborovskaya I et al (1995) High frequency of K-ras mutations in normal appearing lung tissues and sputum of patients with lung cancer. Int J Cancer 63:810–814

    Article  CAS  PubMed  Google Scholar 

  31. Mills NE, Fishman CL, Scholes J, Anderson SE, Rom WN, Jacobson DR (1995) Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis. J Natl Cancer Inst 87:1056–1060

    Article  CAS  PubMed  Google Scholar 

  32. Mao L, Hruban RH, Boyle JO, Tockman M, Sidransky D (1994) Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 54:1634–1637

    CAS  PubMed  Google Scholar 

  33. Scott FM, Modali R, Lehman TA, Seddon M, Kelly K, Dempsey EC et al (1997) High frequency of K-ras codon 12 mutations in bronchoalveolar lavage fluid of patients at high risk for second primary lung cancer. Clin Cancer Res 3:479–482

    CAS  PubMed  Google Scholar 

  34. Keohavong P, Zhu D, Whiteside TL, Swalsky P, Bakker A, Elder EM et al (1997) Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues. Anal Biochem 247:394–403

    Article  CAS  PubMed  Google Scholar 

  35. Zhang LF, Gao WM, Gealy R, Weissfeld J, Elder E, Whiteside TL et al (2003) Comparison of K-ras gene mutations in tumor and sputum DNA of patients with lung cancer. Biomarkers 8:156–161

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the American Cancer Society (RPG-99-61-01-CNE and RSG-99-161-04-CNE).

Author information

Authors and Affiliations

  1. Department of Environmental Toxicology, The Institute of Environmental and Human Health (TIEHH), Reese Technology Center Building 555, 1207 Gilbert Drive, Lubbock, TX, 79416, USA

    Weimin Gao

  2. Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, 100 Technology Drive, Bridgeside Point, Pittsburgh, PA, 15219, USA

    Phouthone Keohavong

Authors
  1. Weimin Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Phouthone Keohavong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Phouthone Keohavong .

Editor information

Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Humana Press

About this protocol

Cite this protocol

Gao, W., Keohavong, P. (2014). Detection of Point Mutations of K-ras Oncogene and p53 Tumor-Suppressor Gene in Sputum Samples. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-739-6_26

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-738-9

  • Online ISBN: 978-1-62703-739-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation