1. RNA interference (RNAi) is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double-stranded (ds) RNA. Because antisense (AS) mechanisms have similar effects, we compared these two effects in human cancer cell lines, considering a possible application of RNAi for cancer therapy. 2. We tested RNAi effects by transfecting human hepatoma and pancreatic cancer cell lines with AS and sense (S) RNA expression plasmids corresponding to the exogenous luciferase gene or the endogenous c-raf gene in the form of complexes with a cationic lipopolyamine or a tumour-targeting peptide vector we developed. In addition, we compared the effects of small interfering RNA and AS oligoDNA complexed with the peptide vector. 3. From the viewpoint of AS actions, the effect of the AS RNA may be cancelled by the S RNA, although, interestingly, we found that the combination of the AS and S RNA expression plasmids was more effective than the AS RNA expression plasmids alone in reducing target gene expression, whereas the S RNA expression plasmids had no effects. The combination of the luciferase AS and S RNA had no effects on the expression of either the beta-galactosidase gene or the c-raf gene. In the presence of 2-aminopurine (an inhibitor of dsRNA-activated protein kinase), the inhibitory effect of the combination of AS and S RNA on gene expression did not change in the case of the endogenous c-raf gene, but was reduced in the case of the exogenous luciferase gene. The effect of 22 nucleotide RNA duplexes corresponding to the luciferase gene was by one order stronger than that of the phosphorothioate AS DNA. 4. Thus, it is suggested that RNAi may be more potent than AS RNA in reducing target gene expression in human cancer cell lines, regardless of the length of dsRNA. With further studies on the RNAi phenomenon in cancer cells, RNAi could provide a novel approach for cancer gene therapy.
Aims/hypothesis. Considering that increased oxidative stress induced by hyperglycaemia plays a possible role in the pathogenesis of diabetic complications and that mitochondrial DNA (mDNA) is thought to be more vulnerable than nuclear DNA, we investigated what somatic mutations actually occur in the mDNA of diabetic patients. We also studied the relations between those mutations and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) which is known to increase considerably in people with diabetes. Methods. We identified somatic mutations by subcloning and sequencing two segments of mDNA [control region (nt 15996-16401) and the segment encompassing t-RNA Leu(UUR) (nt 3149-3404)] in the peripheral blood cells of six diabetic women and control subjects matched for age and sex. This was done in 20 colonies each. In each case we also assayed urinary 8-OHdG. Results. No difference in the aggregate somatic mutational burden of mDNA was found between patients and control subjects. However, the incidence of somatic transversion mutations in mDNA was significantly higher in diabetic patients than in control subjects (13.93±4.57×10 −5 vs 1.27±1.27×10 −5 mutations per base pair; p=0.031, according to Mann-Whitney U-test). There was no significant difference in transition mutations. A correlation was found between the transversion mutational burden and HbA 1 c values, but not between it and 8-OHdG content in the urine. Conclusions/interpretation. We showed that somatic transversion point mutations of mDNA increase in diabetic patients. Such transversion mutations can become a new biomarker for mDNA damage associated with hyperglycaemia and possibly caused by oxidative stress but not reflected by urinary 8-OHdG. [Diabetologia (2003) 46:1559-1566
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