Ha‐ras mutations in N‐nitrosomorpholine‐induced lesions and inhibition of hepatocarcinogenesis by antisense sequences in rat liver

Abstract

To evaluate the application of Ha-ras mRNA antisense oligonucleotide therapy for liver tumors, we examined the frequency and types of mutation in codon 61 of the Ha-ras oncogene in preneoplastic lesions and hepatocellular carcinomas induced by N-nitrosomorpholine (NNM) in rats. Thirtyseven percent of preneoplastic lesions and 50% of hepatocellular carcinomas contained mutations, mostly CAA-CTA and CAA-AAA transversions. We also investigated the effects on NNM-induced lesions of an antisense 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 twice-weekly i.p. injections of a mutated Ha-ras antisense oligonucleotide with a 5Ј phosphorothioate linkage or a sense oligonucleotide in oligonucleotide-liposome complexes. At week 16, rats that had received the mutated Ha-ras antisense oligonucleotides had significantly fewer and 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 fluorescence in the liver was found to increase with higher doses of mutated, fluorescein-isothiocyanate-labeled antisense or sense oligonucleotides. Moreover, mutated Haras antisense oligonucleotide decreased the expression of mutated Ha-ras mRNA (CAA-CTA). Our findings indicate that mutated Ha-ras antisense oligonucleotide significantly inhibits hepatocarcinogenesis in rats and could be an effective therapy against liver tumors. Int. J. Cancer 72:815-820, 1997. Wiley-Liss, Inc.Point mutations in genes of the ras family, Ha-ras, K-ras and N-ras, have been detected in many human tumors (Bos, 1988;Tomono et al., 1996). In contrast with ras proto-oncogenes, which are expressed by all cells, ras oncogenes possess point mutations in the sequence encoding the active site, the GTP/GDP binding domains, of the ras proteins, most frequently at codons 12, 13 and 61. Even though their frequency in human tumors is estimated to be 20-30% (Bos, 1988), evidence suggests that mutated ras genes are directly involved in cell proliferation and tumorigenicity. In an attempt to improve cancer therapy, mutated ras genes in neoplastic cells have been blocked by antisense techniques. For example, short, modified antisense oligonucleotides directed against Ha-ras point mutations have been reported to induce in vitro selective cleavage of mRNA and to inhibit the proliferation of T24 cells (Saison-Behmoaras et al., 1991). Furthermore, antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles have been found to inhibit specifically cell proliferation and tumorigenicity in mutated Ha-ras-mediated bladder carcinoma in nude mice (Schwab et al., 1994).Antisense oligonucleotides are short sequences that have been used to suppress the expression of oncogenes and growth factors (Calabretta, 1991;Hélène, 1991). In theory, hybridization of the antisense oligonucleotide and the...

ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species

“…Activated ras genes have not been detected in HCC induced in rats by N ‐methyl‐ N ‐nitrosourea, diethylnitrosamine, 2‐acetylaminofluorene, 2‐acetylaminophenanthrene or trans ‐4‐acetylaminostilbene (Sakai and Ogawa, 1990; Bitsch et al ., 1993). Mutations in codon 61 of the Ha‐ ras gene were identified in 3 of 8 Sprague‐Dawley rats N ‐nitrosomorpholine induced‐HCC but 2 types of mutations were detected (2 were CAA→CTA and one CAA→AAA) (Baba et al ., 1997), in contrast with VC‐induced HCC of which 87.5% contained a mutated Ha‐ ras gene with only the codon 61 CAA→CTA mutation.…”

The protective mechanism of Nigella sativa against diethylnitrosamine‐induced hepatocellular carcinoma through its antioxidant effect and EGFR/ERK1/2 signaling

“…Activation of RAF/MAPK/ERK in DENA group may be attributed to a continuous activation of c‐raf, which may be caused by dysregulated overexpression of EGF family ligands or activation of their receptor EGFR . Also, it may be due to the oncogenic mutation of RAS gene that caused by DENA leading to constitutive pathway activation through c‐raf …”

Molecular mechanisms of hepatocellular carcinoma

“…Human ras proteins H‐Ras, N‐Ras, K‐ras4A, and K‐Ras4B are small GTP‐binding proteins that function as molecular switches to influence cell growth, differentiation and apoptosis 45. Single point mutations in codon 13 of H‐ras, codon 12 of N‐ras, and codon 61 of K‐ras were originally observed in HCC caused by various chemicals such as N‐nitrosomorpholine, bleomycin, 1‐nitropyrene, and methyl (acetoxymethyl) nitrosamine 46–52. Ras interacts with a downstream serine/threonine kinase Raf‐1 leading to its activation and downstream signaling, which includes activation of MAPK kinases MEK1 and MEK2, to regulate proliferation and apoptosis 53.…”