(Chest. 2004;125:107S-108S.)
© 2004
American College of Chest Physicians
Buccal-Lung Comparison of Quantitative Expression of Carcinogen and Oxidant Metabolism Genes in Human Subjects*
R.J. Jain, PhD;
S. Varma, MS;
G.J. Hurteau, BS and
Simon D. Spivack, MD, MPH, FCCP
* From the Wadsworth Center, New York State Department of Health, State University of New York School of Public Health, and Albany Medical College, Pulmonary and Critical Care Medicine, Albany, NY.
Correspondence to: Simon Spivack, MD, MPH, FCCP, Human Toxicology & Molecular Epidemiology, Wadsworth Center, Biggs E622, New York State Department of Health, SUNY School of Public Health, Empire State Plaza, PO Box 509, Albany, NY 12201-0509; e-mail: spivack{at}wadsworth.org
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Introduction
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The human lung is inaccessible for direct examination. Buccal cells lining the inner cheek of tobacco smokers are carcinogen- and oxidant-exposed, easily accessible, and potentially valuable surrogates for lung tissue. Lung cancer susceptibility phenotyping might include carcinogen-metabolism and oxidant-metabolism gene expression assayed in buccal cells.
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Materials and Methods
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For buccal cell analysis, we adapted known techniques of RNA preservation and extraction. We have employed our novel RNA-specific reverse transcription polymerase chain reaction strategy that is unconfounded by genomic DNA pseudogene sequence encoding target and reference "housekeeper" sequences. All primers were blasted against bacterial sequences to assure human cell specificity of the amplification. Precise real-time quantitation of the expression of these genes employs ratios of target crossover point to reference housekeeper CRO (LightCycler; Roche; Basel, Switzerland). Genes that were analyzed included the aromatic hydrocarbon receptor gene (Ahr), cytochrome P450 genes CYP1A1 and CYP1B1, glutathione-S-transferase genes GSTM1, GSTM3, GSTP1, and GSTT1, nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase (NQO1), copper-zinc superoxide dismutase (CuZn-SOD), catalase (CAT), and glutathione peroxidase (GPx). For any given individual among our initial group of human subjects, we have been able to reliably quantitate gene expression in the buccal cytologic specimens. We have compared buccal gene expression to laser capture microdissected lung gene expression, both by identical techniques of real-time quantitative reverse transcription polymerase chain reaction.
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Results
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We have found close concordance of phase I, phase II, and antioxidant genes in constitutive expression (not expressed, CYP1A1, GSTM1, and GSTM3; all expressed, GSTP1, GSTT1; NQO1, SOD, CAT, and GPx) between the two tissues. There is often buccal-lung discordance for the Ahr gene. However, for all 11 transcripts studied in our first 11 individuals to date, 88 to 96% of the gene-specific buccal-lung expression comparisons have shown qualitatively concordant expression (
2 = 62.4; p < 0.001). The quantitative covariation between the two tissues is under study.
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Conclusion
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We have in preliminary fashion shown that (1) gene expression is quantifiable in cytologically collected buccal cells, and (2) there appears to be good concordance between those carcinogen and oxidant metabolism genes expressed in the buccal mucosa and those expressed in the laser microdissected human lung epithelium. We have enlarged the group undergoing buccal-lung comparison, and have commenced stratification of the expression data for covariation with tobacco exposure, to define each individual’s unique gene expression signature in this cancer-relevant pathway.
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Introduction
Materials and Methods
