Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma

Abstract

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer. Cancer Gene Therapy (2000) 7, 373-383

Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo

“…With the advent of the development of hammerhead ribozymes and its derivatives reported by our institute, 10,11 the Bradeion mRNA containing a characteristic exonexon junction can be specifically targeted and cleaved without damaging a related Bradeion mRNA. Based on the results obtained using ras -inactivating ribozymes by Kijima et al, 7 Yamazaki et al, 8 and Tsuchida et al, 9 we succeeded in constructing and using Bradeion -and -specific ribozymes.…”

“…The doubling time of each cell line was examined and determined as described. 8,9 For flow cytometry analysis, cells were pelleted and resuspended in 1 mL of PBS containing 2 g /mL RNase and 50 mg / mL propidium iodide, and incubated for more than 30 minutes at 378C. The profile of cells in the G0 /G1, S, and G2 /M phases of the cell cycle was analyzed by using EPICS ELITE ESP ( Beckman Instruments, Fullerton, CA ) according to the instructions by manufacturers.…”

“…The parameters used were the doubling time, cell cycle analysis, and RT-PCR of Bradeion genes. 8,9 Inactive ribozyme and maxizyme transfected cells were used as controls. Those ribozymes differed from the active ribozymes by a single G5 -to -A5 mutation in the catalytic core.…”

C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification

“…Because of their simple structures and site-specific cleavage activity, ribozymes have been widely exploited in studies of genes relevant to a broad spectrum of diseases, including cancer development, metastasis, drug resistance and viral infections (reviewed in [1][2][3][4]. Ribozymes can be used as a tool for RNA/gene inactivation in vitro, in cell culture (5)(6)(7)(8) and in animals (9). These studies have significantly improved our knowledge about target-specific optimization, delivery, stability and intracellular localization of ribozymes required for successful clinical application (10)(11)(12).…”

Pancreatic cancer inhibition by specific knockdown of K-ras mutant allele expression

“…10 and 11) to inhibition of RAS expression utilizing anti-sense DNA, ribozymes and more recently RNA inhibition (RNAi) to achieve reduction of RAS protein. [12][13][14][15][16] The approach of inhibiting RAS post-translational processing has the drawback that targeting the prenyltransferases that mediate RAS farnesylation and geranylgeranylation, also inhibits the prenylation of other cellular proteins. On the other hand, this broad spectrum of effect may contribute to anti-tumor activity.…”