Differential Gene Expression of Cytochrome P450 in Immortalised Hepatocyte Cell Lines

RTPCR analysis demonstrated that mRNAs encoding the cytochromes 2B1, 2B2 and 3A1 are present in our immortalised lines although the product amplified appeared larger than that observed with primary hepatocytes. Cytochrome P450 isoenzyme proteins were detected in both freshly isolated and 24h cultured primary hepatocytes with the levels of isoenzyme lower in the latter cells. In addition to giving the expected protein band, multiple bands were identified for the immortalised cells suggesting cross-reactions had occurred. This was unexpected as monoclonal antibodies were used throughout and no cross reaction was observed with other proteins in freshly isolated or primary rat hepatocytes. These proteins were also smaller in the immortalised cells and some degradation of the cytochrome P450 may have occurred. RNA fingerprinting, using RAP-PCR, identified differences in the RNA transcripts from immortalised cell lines compared with those obtained from primary hepatocytes. This suggests the occurrence of differential gene expression in our immortalised hepatocytes and highlights RAP-PCR as a useful screening technique for identifying those immortalised lines which retain the most ‘normal’ phenotype in relation to primary hepatocytes.