Sampling-dependent up-regulation of gene expression in sequential samples of human airway epithelial cells

Abstract

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.

Figure 1

Dendrogram illustrating the relationship of global gene expression in right lung airway epithelium and left lung airway epithelium among different individuals. Normalized expression levels for each of the 4227 expressed genes were analyzed using the GeneSpring program by hierarchical clustering with Spearman correlation. Nonsmokers are indicated as “NSX,” and smokers as “SX,” where “X” is the number assigned to the individual. Right lung samples are labeled “RX” and left lung samples “LX,” where X indicates the sampling order. Note that this assessment did not segregate samples based on location (R versus L), order of sampling (R1 versus R2), or smoking status (NS versus S), indicating that there are no effects of the sampling procedure on global patterns of gene expression. As expected, samples from the right and left lungs of the same individual tended to cluster together.

Figure 2

Statistical significance of genes up-regulated in the left airway epithelium compared with the airway epithelium of 20 healthy non-smokers and smokers in whom the right airway epithelium was sampled 1st. The P values of the 267 genes that were up-regulated >2-fold in the left airway epithelium (sampled 2nd) compared with the right airway epithelium (sampled 1st) were calculated using the two-tailed Student t-test, assuming equal variances. Shown on the ordinate is the P value for each gene and on the abscissa, arbitrary gene number from 1 to 267, derived from the alphabetical order of the GenBank or TIGR identifiers, left to right. Note that 23 genes had a P value < 0.01 (▴), while the remainder had a P value > 0.01 (▵).

Figure 3

Ratio of expression levels of the 2nd sampling site compared with the 1st sampling site for 23 genes identified in Table 2. Each data point represents the average ratio of expression level of the 2nd sample compared with the 1st sample for a given gene, in 2 study groups: the group labeled “Right first” consists of 20 individuals whose right lung was sampled 1st, and the group labeled “Left first” consists of the 4 individuals whose left lung was sampled 1st. Note that in the “Right first” individuals, all 23 genes had a ratio > 2 (because this was a criterion for selecting these genes) and in the “Left first” individuals, 18 of 23 genes had a ratio >2, confirming that the up-regulation is dependent on the timing of sample collection and not location.

Figure 4

Expression of representative genes up-regulated in a statistically significant fashion in the airway epithelium of 2nd brushed area compared with the 1st brushed area in individuals whose right lung was brushed 1st (♦) and those whose left lung was brushed 1st (⋄). A: Transcription factors c-fos, jun B, and 3 early growth response genes (EGR1, EGR2, and EGR3). B: Two signal transduction genes, dual specificity phosphatases 2 and 5 (DUSP2 and DUSP 5); 1 gene involved in apoptosis, immediate early response 3 (IER3); and 2 examples of housekeeping genes, glyceraldehyde phosphate dehydrogenase (GAPDH) and TATA-box binding factor IID (TFIID), that did not show significant changes in expression levels between the 1st and 2nd brush in most individuals. The ordinate represents the ratio of the expression level in the 2nd brushed area compared with the 1st brushed area.