Determination of apoptosis in primary rat hepatocytes by real-time quantitative PCR (TaqMan PCR)

Abstract Primary hepatocytes in collagen gel sandwich culture are a well suited model for studying effects like xenobiotic metabolism, hepatotoxicity, apoptosis, and others. Apoptosis is programmed cell death. It plays an essential role in embryonal development as well as in adult tissue for the elimination of undesired cells. The physiologic balance between growth and death can be disturbed by xenobiotics which may induce apoptosis. For the characterization of apoptosis, the expression of two genes, namely bax and p53 was analysed by quantitative real-time PCR. Following incubation of rat primary hepatocytes with camptothecin (CPT), a time- and concentration-dependent increase of mRNA expression was measured for bax at approximately 350% and p53 at approximately 600%. Further, camptothecin and topotecan (TPT) were compared for their effect on bax mRNA expression. Whereas camptothecin induced a continuous increase in bax transcription from 0.1 μ m to 10 μ m , only the highest concentration of topotecan (10 μ m ) led to a comparable increase of bax transcription. Our results demonstrate that apoptosis can be rapidly and simply quantitated in hepatocytes by TaqMan PCR.