Reovirus infection of cancer cells is not due to activated Ras pathway

Lee and his co-workers1, 2 claimed that reovirus selectively killed cells with an activated Ras pathway: activated Ras or an activated element of the Ras pathway inhibited double-stranded RNA-activated protein kinase (PKR) activation, thereby allowing viral protein synthesis. Reovirus remains actively pursued as a potential anticancer agent. We have examined the reovirus-induced cytopathic effect (CPE) in eight human tumor cell lines. Untransformed NIH/3T3 and LLC-MK2 cell lines served as controls. NIH/3T3 is a mouse fibroblast cell line;3 the LLC-MK2 cell line was derived from kidneys of adult Rhesus monkeys.4 Reovirus serotype 3 can infect LLC-MK2, many human tumor cell lines and several untransformed murine cell lines such as L929,5 but not NIH/3T3. We sought to correlate CPE, and intracellular viral RNA measured by polyacrylamide gel electrophoresis, with relative Ras protein levels in the individual cell lines, measured by western blotting. In addition, attempts were made to correlate virus-induced translational control pathways with CPE by immunoblotting for total and phosphorylated double-stranded RNA-activated protein kinase and phosphorylated eukaryotic initiation factor-2α (eIF-2α).6 We confirmed that the CPE observed in eight tumor cell lines and LLC-MK2 cells was due to the viral infection. NIH/3T3 cells exhibited no detectable viral RNA, correlating with a lack of infectivity. Ras expression levels showed little change before and after the virus infection. Ras protein levels correlated with the degree of CPE (except in NIH/3T3 cells). The level of Ras expression was the same in DND-1A melanoma, 2780 ovarian carcinoma and NIH/3T3 cell lines. The expression of total double-stranded RNA-activated protein kinase or phosphorylated double-stranded RNA-activated protein kinase correlated with neither CPE nor Ras expression. Elevated levels of phosphorylated eukaryotic initiation factor-2α correlated inversely with CPE only in four cell lines (DND-1A, U87MG glioblastoma, NIH/3T3 and LLC-MK2). In the remaining six cell lines (2780, Hep-2 laryngeal squamous cell carcinoma, MCF-7 breast cancer, JAR choriocarcinoma, U87MG.wtEGFR and U87MG.ΔEGFR), there was no correlation with phospho-eukaryotic initiation factor-2α levels and CPE. We believe that activated Ras or an activated element of Ras pathway is not the determinant of a cell's sensitivity to reovirus infection; rather, the lack of reovirus receptor(s) on the cell surface should be the essential reason for a cell's resistance to reovirus infection just as most other viruses and as van Houdt et al.7 reported recently.