Microarray Analysis of Differentially Expressed Genes in human bronchial cells(BEAS-2B) treated MTX

Methotrexate (MTX) has been widely used for the treatment of a variety of tumors as well as for inflammatory diseases and rheumatoid arthritis (RA). MTX-induced toxicity has been a serious unpredictable side effect of the treatment and an important clinical problem. Possible causes include allergic, cytotoxic or immunologic reactions to this agent. We examined the consequences of the mechanism of MTX-induced pulmonary toxicity gene expression in BEAS-2B cells, huma bronchial cell line, by microarray. The expression of these genes are potential biomarker of methotrexate-induced pulmonary toxicity. Also, We provide a clue about mechanism of pulmonary toxic action by these clinical chemotherapeutic agents. Keywords: 48h treatment, 0.144uM (dose), MTX BEAS-2B cells were seeded and after incubation for 24 h at 37C, the cells were treated with 0.144 μM(IC20) MTX for 48 h. And after total RNA isolation, gene expression analysis was conducted using a 44-k whole human genome microarray. Labeling and hybridization were performed using a FairPlay microarray labeling kit, followed by the coupling of Cy3 (controls) or Cy5 (treated samples) dye. The hybridized slides were scanned using a GenePix 4000B microarray scanner, and the images were analyzed using GenePix 4.1 software to obtain gene expression ratios. The fluorescence intensity of each spot was calculated by local median background subtraction. We then used the robust scatter-plot smoother LOWESS function to perform intensity-dependent normalization of gene expression. Scatter-plot analysis was performed using Microsoft Excel 2000. A significance analysis of microarray (SAM) was performed for genes with significant changes in expression.