CLONING OF THE RAT MDR GENE FAMILY AND REGULATION IN NORMAL AND NEOPLASTIC LIVER

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Multidrug resistance is the phenomenon by which cells become cross- resistant to a range of unrelated compounds in response to exposure to a single agent; this resistance is the result of overexpression of a 170 Kd membrane protein, p-glycoprotein, which is encoded by the mdr gene(s). Previously, we have shown that exposure of rats to xenobiotic agents such as the carcinogens aflatoxin B1, isosafrole and 2-acetylaminofluorene (2-AAF) causes increased expression of mdr mRNA in the liver. To further study the mechanism by which xenobiotics regulate mdr gene expression, we have employed a primary hepatocyte culture system. Exposure of isolated hepatocytes to methycholanthrene (MC), 2-AAF but not 2,3,7,8 - tetrachlorodibenzo-p-dioxin (TCDD) increased the expression of mdr mRNA expression; concomitant increases in cytochrome P4501A gene(s) expression were also observed with these agents. Inhibition of protein synthesis also increased the expression of mdr mRNA in these cells. The enhanced mdr expression caused by these compounds is a result of increased transcription. These data suggest that mdr expression is regulated by a protein that is distinct from the Ah receptor. To further investigate the mechanism of mdr regulation it was necessary to isolate the rat mdr genes. We have identified that the rat mdr gene family is comprised of three members; one of these genes has been isolated and characterized. Sequence analysis of a complete cDNA for a rat mdr cDNA indicated a high degree of identity to the mouse mdrlb gene (mdrl); thus, this rat cDNA was designated the mdrlb gene. Further studies on the 5' promoter and possible 3' mRNA stability regions are being performed. We observed in normal liver a slight but significant zonal difference of distribution of mdr transcripts (zone 1 > zone 3). Significant increase of mdr transcripts during regeneration was observed mainly in zone 1 hepatocytes. Our data suggest that the increased mdr expression might represent a subpopulation of preneoplastic nodules which possibly undergoes malignant transformation more specifically than GST-P positive nodules.