We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

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Background: Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. Objectives: The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. Methods: The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-ĸB were applied. Results: Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-ĸB. Conclusions: We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions.

Chronic obstructive pulmonary disease (COPD) is a major health problem. The disease is driven by abnormal inflammatory reactions in response to inhaled particles and fumes. Therefore, inflammatory mediators are postulated to be of distinct importance. In the present case-control study, we investigated interleukin (IL)-promoter polymorphisms known to correlate with altered transcription levels of their gene products in patients with COPD. We analyzed tumor necrosis factor-alpha (TNF-alpha)-308, TNF-beta-intron1-252, IL-6-174, IL-10-819, and IL-10-1082 polymorphisms in 469 individuals using restriction fragment length polymorphism-based converted polymerase chain reaction. The study population consisted of 113 patients with COPD based on chronic bronchitis, divided into subgroups by severity (I degrees -III degrees ), 113 matched hospitalized individuals suffering from severe coronary heart disease without pulmonary disease (age-, sex-, and smoking-matched control group), and 243 healthy individuals (population control group). The matched analysis showed no significant differences in genotype distribution of all tested polymorphisms between the matched controls and the COPD patients. However, comparison with the population controls revealed significant differences in IL-10-1082 A/G genotype frequencies (P = 0.0247 for the whole COPD group, P = 0.009 for smokers only), with the genotypes carrying the G allele more common in the COPD cases [odds ratio (OR) = 1.66, 95% confidence interval (CI) 1.01-2.75; P = 0.046]. Interestingly, this shift toward more G alleles was even more pronounced in the matched control group (OR = 2.55, 95% CI 1.47-4.41; P = 0.0007), suggesting both presented groups share corresponding underlying mechanisms. The IL-10-1082_G allele is known to correlate with altered IL-10 levels. Therefore, it might be associated with altered or abnormal inflammatory response, a mechanism that could be postulated to be important in both chronic bronchitis and coronary heart disease.

Apart from cigarette smoking, genetic factors seem to be of importance in the development of lung cancer. The present case-control study investigated frequencies of five inflammatory response gene polymorphisms (TNF-α-308, TNF-β-Intron1-252, IL-6-174, IL-10-819 and IL-10-1082) in patients with lung cancer and controls. The study population consisted of 117 patients with lung cancer (77 patients with NSCLC, including 40 Squamous Cell Carcinoma and 26 Adenocarcinoma, and 40 patients with SCLC), 117 matched controls without pulmonary disease and 243 healthy individuals (population control). Genotype analyses revealed no difference in genotype frequencies using matched-pair analysis. However, in comparison to the population control, the IL-10-1082 genotypes carrying the G allele appeared with higher frequency in the SCLC group (p = 0.006) [SCLC: 84.6%, population controls: 64.6%]. This yields an odds ratio of 3.01 for SCLC (95% CI = [1.21, 7.48]). No associations were seen for all other polymorphisms analysed. The study raises the possibility of a correlation between the IL-10-1082_G allele and the presence of SCLC in a German population. The functional IL-10-1082 polymorphism correlates with altered IL-10 levels and might influence lung cancer susceptibility by altered inflammatory responses in the airways.