We next attempted to verify the process of GTP reduction that is expected to occur after RBV is incorporated into cells. To this end, we performed a quantitative HPLC analysis using the extract from the OR6 or ORL8 cells treated with 50 µM of
RBV for 8 hours, which is the working time of RBV against HCV RNA replication. Amounts of IMP and GTP were calculated from the peak area obtained by HPLC analysis. Volume of cells was calculated from the mean diameter of cells, and we found 106 cells to be equivalent to 1.1 mm3. We assumed that the extracted nucleotides (nts) were uniformly distributed in the cell aqueous volume. As expected, this website the level of intracellular GTP in ORL8 cells showed a significant (60%) reduction, whereas that in OR6 cells showed only a 27% reduction (Fig. 1A and Supporting Fig. 1A-D). These results support our previous finding that the inhibitory effect of RBV on HCV RNA replication in ORL8 cells
is stronger than that in OR6 cells. In addition, we noticed an unexpected phenomenon: A substantial accumulation of IMP occurred as the Selleck PF-6463922 result of IMPDH inhibition in ORL8 cells, but not in OR6 cells. The IMP level in ORL8 cells became approximately 30 times higher than that in OR6 cells (Fig. 1B and Supporting Fig. 1A-D). However, no additive effect of inosine (up to 100 µM) on HCV RNA replication in ORL8 cells was observed (Supporting Fig. 2). It has been reported that RBV is metabolized in vivo ID-8 through RBV 5′-monophosphate (RMP), a competitive inhibitor of IMPDH, by ADK. Based on our findings, we expected that ADK activity might be able to control the anti-HCV activity of RBV. Indeed, microarray analysis revealed that the actual expression levels of ADK were 764 and 2,840 in OR6c and ORL8c cells, respectively. Quantitative RT-PCR analysis also showed that the mRNA level of ADK in ORL8 cells was 4.5 times higher than that in OR6 cells (Fig. 1C). Furthermore,
we found that the protein level of ADK in ORL8 cells was much higher than that in OR6 cells (Fig. 1D). On the other hand, it is known that ADK has two major isoforms: ADK-long (NM_006721) localized in the nucleus and ADK-short (NM_001123) localized in the cytoplasm. ADK-long differs in the 5′ UTR and initiates translation at an alternative start codon, compared to ADK-short. ADK-long is 17 amino acids longer than ADK-short. We prepared ORL8 cells stably overexpressing ORL8-derived ADK-long or ADK-short using a retroviral gene transfer system and examined its mobility in western blotting analysis. Fortunately, two isoforms were discriminable as 40 (ADK-long) and 38 kDa (ADK-short) (Fig. 1E). Using these isoforms as molecular markers, we performed semiquantitative western blotting analysis by the sample dilution method.