Molecular Medicine

, Volume 9, Issue 9–12, pp 200–208 | Cite as

Sampling-Dependent Up-regulation of Gene Expression in Sequential Samples of Human Airway Epithelial Cells

  • Adriana Heguy
  • Ben-Gary Harvey
  • Timothy P O’Connor
  • Neil R Hackett
  • Ronald G Crystal


As part of a study of in vivo gene expression levels in the human airway epithelium in response to chronic cigarette smoking, we have identified a number of genes whose expression levels are altered in a time-dependent fashion resulting from the procedure used to sample epithelial cells. Fiberoptic bronchoscopy and airway epithelium brushing were used to obtain independent samples from a single individual, 1st from the right lung, followed by sampling of the left lung. We observed that a specific subset of early response genes encoding proteins involved in transcription, signal transduction, cell cycle/growth, and apoptosis were significantly up-regulated in the left lung samples (the 2nd region to be sampled) compared with the right lung samples (the 1st region to be sampled). This response was due to the temporal nature of the sampling procedure and not to inherent gene expression differences between airway epithelium of the right and left lungs. When the order of sampling was reversed, with the left airway epithelium sampled 1st, the same subset of genes were up-regulated in the samples obtained from the right airway epithelium. The time-dependent up-regulation of these genes was likely in response to the stress of the procedure and/or the anesthesia used. Sampling-dependent uncertainty of gene expression is likely a general phenomenon relevant to the procedures used for obtaining biological samples, particularly in humans where the sampling procedures are dependent on ensuring comfort and safety.



We thank M Harris for her assistance in the recruitment of volunteers for this study, K Luettich for microarray processing, and N Mohamed for help in preparing this manuscript. These studies were supported, in part, by P01 HL51746 Gene Therapy for Cystic Fibrosis CFF/NIH/NHLBI; Cystic Fibrosis Foundation (Bethesda, MD, USA); Will Rogers Memorial Fund (Los Angeles, CA, USA); and CUMC GCRC: NIH M01RR00047.


  1. 1.
    King HC, Sinha AA. (2001) Gene expression profile analysis by DNA microarrays: promise and pitfalls. JAMA 286:2280–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Schulze A, Downward J. (2001) Navigating gene expression using microarrays—a technology review. Nat. Cell Biol. 3:E190–5.Google Scholar
  3. 3.
    Holloway AJ, van Laar RK, Tothill RW, Bowtell DD. (2002) Options available—from start to finish—for obtaining data from DNA microarrays II. Nat. Genet. 32 Suppl:481–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Hackett NR et al. (2003) Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers. Am. J. Respir. Cell Mol. Biol. 29:331–43.CrossRefPubMedGoogle Scholar
  5. 5.
    Kaplan R, Luettich K, Heguy A, Hackett NR, Harvey BG, Crystal RG. (2003) Monoallelic up-regulation of the imprinted H19 gene in airway epithelium of phenotypically normal cigarette smokers. Cancer Res. 63:1475–82.PubMedGoogle Scholar
  6. 6.
    Malkinson AM, Belinsky SA. (2000) Animal models for studying lung cancer and evaluating novel intervention strategies. In: Lung Cancer: Principles and Practice. Pass HI, Mitchell JB, Johnson DH, Turrisi AT, Minna JD (eds.) Lippincott Williams & Wilkins, Philadelphia, PA, pp. 347–63.Google Scholar
  7. 7.
    Shapiro SD. (2000) Animal models for chronic obstructive pulmonary disease: age of klotho and marlboro mice. Am. J. Respir. Cell Mol. Biol. 22:4–7.CrossRefPubMedGoogle Scholar
  8. 8.
    Shapiro SD. (2000) Animal models for COPD. Chest 117:223S–7S.CrossRefPubMedGoogle Scholar
  9. 9.
    Danel C, Erzurum SC, McElvaney NG, Crystal RG. (1996) Quantitative assessment of the epithelial and inflammatory cell populations in large airways of normals and individuals with cystic fibrosis. Am. J. Respir. Crit. Care Med. 153:362–8.CrossRefPubMedGoogle Scholar
  10. 10.
    Russi TJ, Crystal RG. (1997) Use of bronchoalveolar lavage and airway brushing to investigate the human lung. In: The Lung: Scientific Foundations. Crystal, RG (ed.) Lippencott-Raven, Philadelphia, PA. pp. 371–82.Google Scholar
  11. 11.
    Harvey BG et al. (1999) Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus. J. Clin. Invest. 104:1245–55.CrossRefPubMedGoogle Scholar
  12. 12.
  13. 13.
    Sukhatme VP. (1990) Early transcriptional events in cell growth: the Egr family. J. Am. Soc. Nephrol. 1:859–66.PubMedGoogle Scholar
  14. 14.
    Madden SL, Rauscher FJ. III (1993) Positive and negative regulation of transcription and cell growth mediated by the EGR family of zinc-finger gene products. Ann. N.Y. Acad. Sci. 684:75–84.CrossRefPubMedGoogle Scholar
  15. 15.
    Gashler A, Sukhatme VP. (1995) Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog. Nucleic Acid Res. Mol. Biol. 50:191–224.CrossRefPubMedGoogle Scholar
  16. 16.
    Tamura S, Hanada M, Ohnishi M, Katsura K, Sasaki M, Kobayashi T. (2002) Regulation of stress-activated protein kinase signaling pathways by protein phosphatases. Eur. J. Biochem. 269:1060–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Chavrier P, Zerial M, Lemaire P, Almendral J, Bravo R, Charnay P. (1988) A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells. EMBO J. 7:29–35.CrossRefPubMedGoogle Scholar
  18. 18.
    Keyse SM. (1993) The induction of gene expression in mammalian cells by radiation. Semin. Cancer Biol. 4:119–28.PubMedGoogle Scholar
  19. 19.
    Khachigian LM, Collins T. (1998) Early growth response factor 1: a pleiotropic mediator of inducible gene expression. J. Mol. Med. 76:613–6.CrossRefPubMedGoogle Scholar
  20. 20.
    Nishi H, Nishi KH, Johnson AC. (2002) Early Growth Response-1 gene mediates up-regulation of epidermal growth factor receptor expression during hypoxia. Cancer Res. 62:827–34.PubMedGoogle Scholar
  21. 21.
    Semenza GL. (2000) Oxygen-regulated transcription factors and their role in pulmonary disease. Respir. Res. 1:159–62.CrossRefPubMedGoogle Scholar
  22. 22.
    Landesberg LJ, Ramalingam R, Lee K, Rosengart TK, Crystal RG. (2001) Upregulation of transcription factors in lung in the early phase of postpneumonectomy lung growth. Am. J. Physiol. Lung Cell Mol. Physiol. 281:L1138–49.CrossRefPubMedGoogle Scholar
  23. 23.
    Yan SF et al. (1999) Hypoxia-associated induction of early growth response-1 gene expression. J. Biol. Chem. 274:15030–40.CrossRefPubMedGoogle Scholar
  24. 24.
    Yan SF et al. (2000) Pulmonary expression of early growth response-1: biphasic time course and effect of oxygen concentration. J. Appl. Physiol. 88:2303–9.CrossRefPubMedGoogle Scholar
  25. 25.
    Black AC, Luo J, Chun S, Tabibzadeh S. (1997) Constitutive expression of the HTLV-I pX and env regions in Jurkat T-cells induces differential activation of SRE, CRE and NF kappa B pathways. Virus Genes 15:105–17.CrossRefPubMedGoogle Scholar
  26. 26.
    Akuzawa N, Kurabayashi M, Ohyama Y, Arai M, Nagai R. (2000) Zinc finger transcription factor Egr-1 activates Flt-1 gene expression in THP-1 cells on induction for macrophage differentiation. Arterioscler. Thromb. Vasc. Biol. 20:377–384.CrossRefPubMedGoogle Scholar
  27. 27.
    Krishnaraju K, Hoffman B, Liebermann DA. (2001) Early growth response gene 1 stimulates development of hematopoietic progenitor cells along the macrophage lineage at the expense of the granulocyte and erythroid lineages. Blood 97:1298–305.CrossRefGoogle Scholar
  28. 28.
    Khachigian LM, Anderson KR, Halnon NJ, Gimbrone MA Jr, Resnick N, Collins T. (1997) Egr-1 is activated in endothelial cells exposed to fluid shear stress and interacts with a novel shear-stress-response element in the PDGF A-chain promoter. Arferioscler. Thromb. Vasc. Biol. 17:2280–6.CrossRefGoogle Scholar
  29. 29.
    Shaulian E, Karin M. (2002) AP-1 as a regulator of cell life and death. Naf. Cell Biol. 4:E131–6.Google Scholar
  30. 30.
    Chae HJ et al. (1999) Systemic injection of lidocaine induced expression of c-fos mRNA and protein in adult rat brain. Res. Commun. Mol. Pathol. Pharmacol. 104:31–41.PubMedGoogle Scholar
  31. 31.
    Marota JJ, Crosby G, Uhl GR. (1992) Selective effects of pentobarbital and halothane on c-fos and jun-B gene expression in rat brain. Anesfhesiology 77:365–371.CrossRefGoogle Scholar
  32. 32.
    Hamaya Y, Takeda T, Dohi S, Nakashima S, Nozawa Y. (2000) The effects of pentobarbital, isoflurane, and propofol on immediate-early gene expression in the vital organs of the rat. Anesth. Analg. 90:1177–83.CrossRefPubMedGoogle Scholar
  33. 33.
    Hattori M et al. (1993) Activation of activating protein 1 during hepatic acute phase response. Am. J. Physiol 264:G95–103.PubMedGoogle Scholar
  34. 34.
    Theodosiou A, Ashworth A. (2002) MAP kinase phosphatases. Genome Biol 3:Reviews 1–10.CrossRefGoogle Scholar
  35. 35.
    Keyse SM, Emslie EA. (1992) Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase. Nature 359:644–7.CrossRefPubMedGoogle Scholar
  36. 36.
    Chu Y, Solski PA, Khosravi-Far R, Der CJ, Kelly K. (1996) The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J. Biol. Chem. 271:6497–501.CrossRefPubMedGoogle Scholar
  37. 37.
    Hirsch DD, Stork PJ. (1997) Mitogen-activated protein kinase phosphatases inactivate stress-activated protein kinase pathways in vivo. J. Biol. Chem. 272:4568–75.CrossRefPubMedGoogle Scholar
  38. 38.
    Tanoue T, Moriguchi T, Nishida E. (1999) Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5. J. Biol. Chem. 274:19949–56.CrossRefPubMedGoogle Scholar
  39. 39.
    Kovanen PE et al. (2003) Analysis of gamma c-family cytokine target genes. Identification of dual-specificity phosphatase 5 (DUSP5) as a regulator of mitogen-activated protein kinase activity in interleukin-2 signaling. J. Biol. Chem. 278:5205–13.CrossRefPubMedGoogle Scholar
  40. 40.
    Motosugi H, Quinlan WM, Bree M, Doerschuk CM. (1998) Role of CD11b in focal acid-induced pneumonia and contralateral lung injury in rats. Am. J. Respir. Crit. Care Med. 157:192–8.CrossRefPubMedGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2003

Authors and Affiliations

  • Adriana Heguy
    • 1
  • Ben-Gary Harvey
    • 2
  • Timothy P O’Connor
    • 1
  • Neil R Hackett
    • 3
  • Ronald G Crystal
    • 1
    • 2
    • 3
  1. 1.Department of Genetic MedicineWeill Medical College of Cornell UniversityNew YorkUSA
  2. 2.Division of Pulmonary and Critical Care MedicineWeill Medical College of Cornell UniversityNew YorkUSA
  3. 3.Belfer Gene Therapy Core FacilityWeill Medical College of Cornell UniversityNew YorkUSA

Personalised recommendations