Gene Expression Modulation in A549 Human Lung Cells in Response to Combustion-Generated Nano-Sized Particles

Abstract

High levels of ambient air pollution are associated in humans with aggravation of asthma and of respiratory and cardiopulmonary morbidity; long-term exposures to particulate matter (PM) have been linked to possible increases in lung cancer risk, chronic respiratory disease, and increased death rates. The Biodiagnostics Group of the DLR Institute of Aerospace Medicine develops cellular test systems capable of monitoring the biological consequences of environmental conditions on humans already on cellular and molecular level. Such bioassays rely on the receptor–reporter principle, where cell lines are transfected with plasmids carrying a reporter gene under control of environment-dependent promoters (receptor), which play a key role in regulating gene expressions in response to extracellular signals. We developed the recombinant human lung epithelial cell line A549-NF-κB-EGFP/Neo carrying a genetically encoded fluorescent indicator for monitoring activation of the NF-κB signaling pathway in living cells in response to genotoxic and cytotoxic environmental influences. With this cell line we screened several candidate human radiation-responsive genes (GADD45β, CDKN1A) and NF-κB-dependent genes (IL-6, NFκBIA, and pNF-κB-EGFP) for gene expression changes by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after exposure to combustion generated nano-sized particle samples.