PCR and Flow Cytometric Analysis of Paclitaxel-inhibited Arylamine N-Acetyltransferase Activity and Gene Expression in Human Osteogenic Sarcoma Cells (U-2 OS)

Abstract

Human epidemiological studies suggest an association between N-acetyltransferase (NAT) activity and the incidence of bladder and colorectal cancers. In this study, paclitaxel was selected to examine the inhibition of arylamine NAT activity, gene expression and 2-aminofluorene-DNA adduct formation in a human osteogenic sarcoma cell line (U-2 OS). The activity of NAT was determined by high performance liquid chromatography (HPLC) assay for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and nonacetylated AF and PABA. Human osteogenic sarcoma cell cytosols and intact cells were used to examine the NAT activity, gene expression and AF-DNA adduct formation. The results demonstrated that NAT activity, percent of NAT in examined cells, gene expression (NAT1 mRNA) and AF-DNA adduct formation in human osteogenic sarcoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The results also demonstrated that paclitaxel decreased the apparent values of Km and Vmax from intact human osteogenic sarcoma cells (U-2 OS). Thus, paclitaxel is an uncompetitive inhibitor of the NAT enzyme.