Exploring Host Factors that Impact Reovirus Replication, Dissemination, and Reovirus-Induced Cell Death in Cancer Versus
Oncolytic viruses, such as reovirus, offer a promising approach to cancer treatment. Concurrently, oncolytic viruses provide a valuable tool for deciphering unique attributes of cancer cells that support superior virus replication, cell death, or virus di
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ntroduction Reovirus is a naturally occurring human virus found to preferentially replicate in cancer cells and possess oncolytic activity both in vitro and in vivo (1–4). While in vivo studies using various murine cancer models and human clinical trials are ongoing to show the efficacy of reovirus as a cancer therapy, we continue to explore the molecular basis of reovirus oncolysis in cell culture systems. Using isogenic cell lines (with and without mutated forms of oncogenes, such as Ras), we found that several steps of reovirus replication
David H. Kirn et al. (eds.), Oncolytic Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 797, DOI 10.1007/978-1-61779-340-0_12, © Springer Science+Business Media, LLC 2012
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M. Shmulevitz and P.W.K. Lee
and spread are enhanced in Ras-transformed cells (5), including (1) reovirus disassembly necessary for entry and onset of infection, (2) production of progeny virions with high infectious capacity, and (3) induction of apoptosis and release of reovirus. Our recent studies show that cell-to-cell spread of reovirus is also greatly enhanced in transformed cells, owing to the impaired ability of transformed cells to express and respond to interferon beta (IFN-E) and establish an antiviral state (6, 7). Understanding the host and viral factors involved in reovirus oncolysis assists in developing combination therapies, allows for best application of reovirus as a therapeutic, and sheds light on other viral- or drug-based cancer therapies. This chapter describes the various methods used to discern the effects of host factors on the efficiency of reovirus infection in cell culture systems. Reovirus replication, apoptosis-mediated release, and cell-to-cell spread can be easily monitored with the methods described in this chapter, including immunohistochemistry (IHC) of reovirus-infected cells (Subheading 3.2), plaque titration of reovirus (Subheading 3.3), and flow cytometric analysis of reovirusinduced cell death (Subheading 3.4). Using these straightforward methods, reovirus replication and spread can be compared between various cell lines and under various tissue culture conditions, thereby providing important information on cellular and viral factors that promote preferential virus replication in transformed or cancer cells. For example, efficiency of reovirus replication and spread can be monitored in cells from various cancer origins, with or without oncogene activation, inhibitors of cell signalling, host gene complementation, siRNA-mediated knock-down, or following infection with various reovirus mutants.
2. Materials 2.1. Preparation of Reovirus-Infected Samples
1. Cell culture medium according to ATCC. 2. Dulbeco’s modified Eagle’s medium (DMEM). 3. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4. 4. U0126 (MEK1/2 inhibitor, Calbiochem): Make stock solution of 10 mg/mL (24.8 mM) by dissolving 1 mg in 100 PL of dimethyl sulfoxide (DMSO). Aliquot and store at −20°C for 1 month. Dilute to 10 PM (final concentration)
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