E1A CR2 deletion mutant adenoviral vectors in ovarian cancer: combination with apoptosis inducers and monitoring with [18F] FLT-PET imaging

2992 Introduction. Selectively replicating adenoviral mutants hold great promise as gene therapy vectors. dl922-947 contains a 24 bp deletion in E1A CR2, normally responsible for inactivating pRb. Our data indicate that dl922-947 is capable of replicating in and lysing ovarian cancer cells selectively. However, the mechanism of virus-induced cell death is poorly explained. We have explored the roles of classical and caspase-independent apoptosis in the cytotoxicity of adenoviral E1A CR2 mutants and have attempted to augment anti-tumour effects by co-expression of Smac/DIABLO and by co-treatment with cisplatin. We have also explored the ability of [18F] FLT-PET imaging to monitor the activity of dl922-947 in vivo. Methods. In addition to dl922-947, we have created Ad ΔCR2 and Ad ΔCR2 tSmac. Both contain the same E1A CR2 deletion as dl922-947, whilst Ad ΔCR2 tSmac also contains a Smac expression cassette 3′ to the E1 region. Ovarian carcinoma cells were infected in vitro and monitored for markers of classical and non-classical apoptosis and overall survival. Subcutaneous IGROV1 ovarian carcinoma xenografts were grown in nude female mice and injected with dl922-947 and imaged in a quad HIDAC PET scanner for 60 minutes following injection of 80-100μCi [18F]FLT. Results. E1A CR2 deletion mutant adenoviral vectors induce cleavage of PARP and caspase-3, disappearance of XIAP, cIAP-1, survivin, caspase-9 and caspase-8, loss of mitochondrial membrane potential and the appearance of a sub-G1 population of DNA in ovarian cancer cells, all consistent with classical apoptosis, and enhanced by addition of a Smac gene. The changes were also largely reversible with 50μM zVAD-fmk, a pan-caspase inhibitor. However, annexin V staining did not show increased cell surface phosphatidylserine exposure and acridine orange staining suggested lysosomal leakage, whilst zVAD-fmk treatment only partially reversed virus-induced cytotoxicity. Co-treatment with cisplatin enhanced the cytotoxicity of both Smac-expressing and non-expressing viruses, especially when virus was administered 24 hours after cisplatin. Preliminary [18F] FLT-PET imaging data indicate that a reduction in both fractional FLT retention and SUV60 may be detectable as early as 48 hours after dl922-947 injection, with significant reductions by day 7 compared to control virus-treated animals. These data mirror mean tumour volumes, which were 76% lower on day 7 in dl922-947-treated animals than in control mice. Conclusions. Cell death induced by E1A CR2-deleted adenoviral vectors has features of both classical and non-classical apoptosis, but can be augmented by exposure to apoptosis inducers, such as Smac and cisplatin. [18F] FLT-PET imaging may give an early non-invasive indicator of viral activity in ovarian cancer.