The role of wild-type and mutated N-ras in the malignant transformation of liver cells

Abstract

In order to determine the role of N-ras overexpression and mutation in malignant liver cell transformation, wild-type and mutated N-ras were transfected into the rat liver epithelial cell line OC/CDE 22, and N-ras expression, growth kinetics, growth in soft agar, and tumorigenicity in vivo as well as the involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathway in the expression of the malignant phenotype were analyzed. Although OC/CDE 22 cells transfected with wild-type N-ras showed a high expression of N-ras at the mRNA and protein levels, the cells did not grow in soft agar and were not tumorigenic in vivo. In contrast, OC/CDE 22 cells transfected with mutated N-ras showed anchorage-independent growth and were tumorigenic. When cultured in fetal bovine serum-supplemented medium, OC/CDE 22 cells expressing mutant N-ras showed a higher proliferation rate than nontransfected OC/CDE 22 cells or OC/CDE 22 cells transfected with wild-type N-ras. When held in serum-free medium, untreated OC/CDE 22 cells did not grow at all, while OC/CDE 22 cells transfected with wild-type or mutant N-ras proliferated at a similar rate, which can be explained by the high MAPK activity in these cells. Selective inhibition of the MAPK cascade abolished the growth of OC/CDE 22 cells carrying mutant N-ras in soft agar; furthermore, these cells ceased pile up and formed monolayers on Petri dishes. Thus, activation of the MAPK signaling pathway, though alone not sufficient to malignantly transform liver cells (as shown in liver cells overexpressing wild-type N-ras), is not only essential for growth control but also for the expression of the malignant phenotype (as demonstrated in liver cells transformed by mutated N-ras).

Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse

“…NRAS appears to cooperate with Arf deficiency to promote tumorigenesis in the liver. This is consistent with previous work demonstrating cooperation of the Arf and ras pathways in cellular transformation (34) and the ability of NRAS to transform a nontumorigenic liver epithelial cell line (35). Interestingly, NRAS combined with a heterozygous Arf mutation was sufficient to promote tumorigenesis in this model without observable loss of heterozygosity, although at a much-reduced penetrance.…”

Microarray expression profiling in melanoma reveals a BRAF mutation signature

“…Since all BRAF mutations to date have been reported to occur in exons 11 and 15 (Brose et al, 2002;Davies et al, 2002;Naoki et al, 2002;Yuen et al, 2002), each line was screened for variants in these exons by PCR sequencing. NRAS was also screened for mutations in codons 12, 13 and 61, which have been found previously to activate the potential of NRAS to transform cultured cells (Schleger et al, 2000) and have been found in a variety of human tumors including melanomas (van Elsas et al, 1996). For further information refer to Supporting Text 2.…”

ACB-PCR measurement of K-ras codon 12 mutant fractions in livers of Big Blue(R) rats treated with N-hydroxy-2-acetylaminofluorene

“…The F344 rat rarely develops sporadic liver tumors, so this high MF level raises the question regarding the role of K-ras mutations in the carcinogenic process in rat liver. K-ras mutations may cause a cellular functional change of increased and continuous MAPK pathway activation, which may be a necessary, but not rate-limiting, part of the carcinogenic process (24). Tumor development and carcinogenesis is a complex process in which polyclonal interactions may be a major contributor to overall growth and selection advantage.…”