Blockade of the receptor using an anti EGFR antibody to inhibit ligand contain binding or using tyrosine kinase inhibitors to inactivate the signalling capability of the receptor also had no effect on cell survival following infection with reovirus. The activity of the EGFR inhibitors was tested in the context of stimulation by EGF. Both ICR62 and IressaGefitinib effectively inhibited phos phorylation of EGFR, but Tyrphostin AG99 was inactive. HN5 exhibited a previously documented sensitivity to IressaGefitinib. It has been reported that activated Ras signalling blocks the anti viral action of PKR and permits increased reoviral replication. Therefore, we tested the effect of EGFR stimulation and inhibition on reoviral growth. Cells were pre incubated with EGF, ICR62 or media alone and then infected with reovirus.

At various time points after infection the cells and their superna tants were harvested and titred Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries by TCID50 assay. Neither stimulation by EGF nor inhibition by ICR62 affected the growth of reovirus in the 4 cell lines tested. This result was further confirmed using gefitinibiressa. Interestingly, all 4 of the cell lines showed the same level of reoviral replication, despite their differing susceptibility to reovirus induced cell death indi cating that high or low replication rates do not account for the range of reovirus sensitivities observed. Reovirus cytotoxicity does not depend Inhibitors,Modulators,Libraries on PI3 K, MAPK or p38MAPK signalling Having examined the influence of EGFR itself on reo viral oncolysis in SCCHN, we went on to determine whether inhibition of downstream signalling effectors could influence sensitivity to reovirus.

We targeted the three major signalling pathways downstream of Ras MAPK, PI3 K and p38MAPK. To inhibit MEK in the MAPK pathway, the specific tyrosine kinase inhibitors PD184352 and U0126 were used. PD was employed at 2 different Inhibitors,Modulators,Libraries concentrations, 2 uM to tar get MEK12 only and 10 uM for blockade of MEK12 and MEK5. LY294002 and wortmannin were utilised to block PI3 K, and p38MAPK was inhib ited by SB202190. Following incubation with inhibitors, cells were infected with reovirus and cell sur vival was analysed. Inhibitor activity was confirmed by western analysis for all pathways except p38MAPK, where the many isoforms of p38MAPK makes this type of analysis unsuitable. Instead, we confirmed p38MAPK blockade by SB by means of ELISA.

Reoviral Inhibitors,Modulators,Libraries cytotoxicity in SCCHN was not abrogated by blockade selleck chemical in any of the 3 pathways tested, with cell survival being equal to or less than reovirus infection alone. For p38MAPK inhibition by SB, in all cell lines the agent had little single agent cyto toxic activity and did not reduce reovirus induced cell kill. For PI3K inhibition, LY induced significant cell death in 3 of the 4 cell lines but this was not the case with wortmannin. Again, there was no evidence that either LY or wortman nin was capable of abrogating the cytotoxicity of reovirus.