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Dudi Shneor, Alik Honigman, Jacob Pe'er, Shahar Frenkel; Targeting Uveal Melanoma Response to Hypoxia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4527.
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Tumor hypoxia is considered to be a potential therapeutic problem because it renders solid tumors more resistant to ionizing radiation and chemotherapeutic drugs. We describe a recombinant Murine leukemia virus (MuLV)-based replication-competent retroviruses (RCR) delivery system that infects only growing cells. This results in a persistent viral knockdown of the expression of the regulators of the hypoxia responding genes CREB, HIF1, and HIF2, via shRNA targeting of these genes separately and all together from a single polycistronic RNA.
C918 uveal melanoma cells were stably infected with RCR expressing shRNA targeting CREB, HIF1, and HIF2 separately and all together from a single polycistronic RNA. Knockdown of the target genes was analyzed using qRT-PCR and Western blot. Infected cells co-transfected with either CRE or HRE mediated luciferase (luc) gene expression vector, pCREluc or pHREluc, respectively, served for functional analyses of the transcription factors. Cell viability and caspase 3 activity were determined using the Fluorescent Cell Viability and Caspase-Glo 3/7 Assays (Promega) after 0-72 hours under normoxic and hypoxic (0.5% O2) conditions.
The different RCRs efficiently infect the C918 cells and efficiently knockdown the mRNA and protein levels of CREB, HIF1 and HIF2. Knockdown of these genes by the recombinant RCRs reduced VEGF secretion, reduced tumor cell proliferation, and increased Caspase 3 activity in hypoxia. We found that CREB, more than HIF1 and HIF2, plays a pivotal role in the survival of C918 under hypoxia in vitro while HIF1 affects VEGF in vitro.
MuLV-based RCRs affecting the response of UM to hypoxia, specifically affecting CREB and HIF1, have potential as a novel therapeutic approach for metastatic UM.
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