Smoking-Related Gene Expression in Laser Capture-Microdissected Human Lung

Abstract

PURPOSE: Interindividual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment-specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, GSTP1, and a tumor suppressor gene p16 in laser capture-microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and nonsmokers. EXPERIMENTAL DESIGN: Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. RESULTS: Several correlations of mRNA expression included (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001), (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003), and (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. CONCLUSIONS: The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide interindividual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. (Clin Cancer Res 2009;15(24):7562-70).

Figure 1

LCM from hematoxylin and eosin-stained sections of alveolar (A1-A6) and bronchial epithelial (B1-B6) cells from human lung tissue. Histological sections before capture (A1, A4 and B1, B4); captured cells on transfer film (A2, A5 and B2, B5); and histological sections after capture (A3, A6 and B3, B6).

Figure 2

Box plots for the relative mRNA expression of CYP1B1 and GSTP1 in microdissected alveolar cells (AC) associated with the plasma nicotine and cotinine levels. The Mann-Whitney test was used for testing the differences in median gene expression across the categories of the plasma nicotine and cotinine levels. The vertical axis is log₁₀, such that a range from 0 – 4 relative units represents a 10,000-fold range. Figure 2C, D, and G, H resolve the tobacco exposure further, depicting the significant variance in expression in CYP1B1 and GSTP1, respectively, in finer categories of plasma nicotine and cotinine.

Figure 3

The relative mRNA expression levels of CYP1B1, GSTP1 and p16 in alveolar compartment (AC) and bronchial epithelial compartment (BEC) from paired tissues from 38 subjects. Paired AC and BEC were compared employing the Wilcoxon signed-rank test to determine the differential expression in the paired samples. No overall significant differences were found for the relative mRNA expression of CYP1B1, GSTP1 and p16 between microdissected AC and BEC (P value > 0.05, top panels). However, individual expression patterns (bottom panels) showed ordinal concordance between alveolar and bronchial compartments, for most individuals, particularly for CYP1B1 and GSTP1, with readily apparent exceptions.