Nobuko Sawa

Ribozyme-mediated inactivation of mutant K-ras oncogene in a colon cancer cell line

Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The β-actin promoter-driven KrasRz sequence (pHβ/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett’s test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher’s exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett’s test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression.

Establishment of an adrenocortical carcinoma xenograft with normotensive hyperaldosteronism in vivo

We established a xenograft line of human adrenocortical carcinoma (ADR‐1), and analyzed the hyper‐aldosteronism induced by the xenograft in vivo. Adrenocortical carcinoma specimens from a 25‐year‐old woman were subcutaneously inoculated into nude mice (BALB/c‐nu/nu) followed by serial passages in vivo. ADR‐1 retained the histopathological features (trabecular and sinusoid nests) seen in the primary carcinoma. The patient showed hyperaldosteronism (serum aldosterone >4000 pg/ml) and hypokalemia (serum K 2.1 mEq/1), but did not show hypertension. The nude rat (F344‐rnu/rnu) bearing ADR‐1 showed hyperaldosteronism (serum aldosterone 3320 ± 1420 pg/ml; control 191 ± 130 pg/ml) and hypokalemia (serum K 3.4 ± 0.4 mEq/1; control 5.2 ± 1.0 mEq/1) in vivo, and hypertension was not obvious. ADR‐1 was shown immunohistochemically to retain production of human‐specific corticosteroid synthetase. The xenograft ADR‐1 will be useful to elucidate the regulatory mechanism of normotensive hyperaldosteronism.