Analysis of Loss of Nuclear RNA in Azo Dye-induced Hepatoma by DNA-RNA Competitive Hybridization¹

DNA-RNA hybridization studies, using nuclear RNA's (nRNA's) labeled in vivo and in vitro with high specific radioactivities, were performed to compare the nRNA populations of normal rat liver, livers treated with 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), and 3′-Me-DAB-induced hepatomas. The study with normal liver nRNA labeled by i.p. injection of [³H]orotic acid indicated that the nuclei of a 3′-Me-DAB-induced transplanted hepatoma, AH136B, lacked some RNA species present in normal liver nuclei. No qualitative difference in the RNA populations was seen between normal liver and the livers of rats fed a carcinogenic amount of 3′-Me-DAB, either alone or in combination with 4-nitrostilbene which enhanced the azo dye carcinogenesis. Then, nRNA's of both normal liver and AH136B hepatoma were labeled in vitro by phosphorylation with polynucleotide kinase and adenosine 5′-[γ-³²P]triphosphate. The competitive hybridization with ³²P-labeled normal liver nRNA was completed, and the deletion of RNA in the nuclei of AH136B hepatoma or 3′-Me-DAB-induced primary hepatoma was estimated to be 15% or more in the measure of radioactivity of the hybridized normal liver nRNA. ³²P-labeled AH136B hepatoma nRNA was competed completely by liver nRNA's, suggesting that no unique RNA species were present in the hepatoma nuclei.