HA-RAS MUTATIONS IN N-NITROSOMORPHOLINE-INDUCED LESIONS AND INHIBITION OF HEPATOCARCINOGENESIS BY ANTISENSE SEQUENCES IN RAT-LIVER

To evaluate the application of Ha-ras mRNA antisense oligonucleotide t herapy for liver tumors, we examined the frequency and types of mutati on in codon 61 of the Ha-ras oncogene in preneoplastic lesions and hep atocellular carcinomas induced by N-nitrosomorpholine (NNM) in rats. T hirty-seven percent of preneoplastic lesions and 50% of hepatocellular carcinomas contained mutations, mostly CAA-CTA and CAA-AAA transversi ons. We also investigated the effects on NNM-induced lesions of an ant isense oligonucleotide directed against a point mutation (CAA-CTA) in codon 61 of Ha-ras mRNA. In this experiment, Sprague-Dawley rats were given free access to water containing NNM for 8 weeks and received twi ce-weekly i.p. injections of a mutated Ha-ras antisense oligonucleotid e with a 5' phosphorothioate linkage or a sense oligonucleotide in oli gonucleotide-liposome complexes. At week 16, rats that had received th e mutated Ha-ras antisense oligonucleotides had significantly fewer an d smaller preneoplastic lesions positive for glutathione-S-transferase , placental type, and had smaller hepatocellular carcinomas than rats that had received the sense oligonucleotide. Mean cellular fluorescenc e in the river was found to increase with higher doses of mutated, flu orescein-isothiocyanate-labeled antisense or sense oligonucleotides. M oreover, mutated Ha-res antisense oligonucleotide decreased the expres sion of mutated Ha-ras mRNA (CAA-CTA), Our findings indicate that muta ted Ha-ras antisense oligonucleotide significantly inhibits hepatocarc inogenesis in rats and could be an effective therapy against liver tum ors. (C) 1997 Wiley-Liss, Inc.