Paper
Monday, November 14, 2005
Cigarette Smoke and Gene Expression: Comparative Analysis of Monolayer and Three-Dimensional (3D) Epithelial Cell Cultures
Alison R. Jones, RN, MS1, Homero San Juan, MD, PhD2, Jason W. Beckstead, PhD1, Richard F. Lockey, MD3, and Shyam S. Mohapatra, PhD4. (1) College of Nursing, University of South Florida, Tampa, FL, USA, (2) Universidad del Norte, Barranquilla, Colombia, (3) Joy McCann Culverhouse Airway Disease Center, James A. Haley V.A. Hospital, Tampa, FL, USA, (4) College of Medicine, University of South Florida, Tampa, FL, USA
| Learning Objective #1: State the principles and benefits of microarray analysis as a measure of global gene expression |
| Learning Objective #2: Discuss the impact of cigarette smoke in global gene expression and progression of cigarette-induced pulmonary disease |
Cigarette smoke contains approximately 1,017 oxidants/free radicals and 4,700 chemical compounds and is responsible for approximately one in five deaths in the US. This increases the oxidant burden in smokers and risk of developing pulmonary disease. Most investigations of cigarette exposure utilize monolayer A549 cells, a type II alveolar epithelial cancer cell line which may differ from normal human bronchial epithelial (NHBE) cells. Reports indicate approximately 3,700 genes were altered in NHBE cells exposed to cigarette smoke condensate (CSC). It has been previously shown by our lab that when exposed to the same dosage of CSC, NHBE cells exhibited 20% apoptosis while A549 cells showed only 5% apoptosis. Furthermore, the submerged monolayer of epithelial cells may differ from that of human airway epithelium. The aim of our study was to conduct gene expression analysis to compare the role of CSC in A549 cells and NHBE cells in monolayer and three dimensional (3D) models. In order to develop the 3D model, primary NHBE cells were grown in an air-liquid interface for 21 days. Histopathology analysis indicated a 3D architecture with successful differentiation and development of columnar epithelium similar to normal epithelium in vivo. Gene expression analyses were conducted using Affymetrix oligonucleotide based microarray technology (HG-U133 Plus 2.0 GeneChip). Results revealed expression patterns of approximately 5,000 genes altered after CSC treatment in both NHBE and A549 cell lines. Global biological functions analyses were conducted utilizing Ingenuity Pathways Analysis, a web-based software. Currently, data is being analyzed using cluster analysis techniques. Study of gene expression in these cell lines and the 3D model will provide insight into common in vitro respiratory models and further characterize the impact of cigarette smoke in pulmonary disease progression.