Development of an SH-SY5Y neuroblastoma model system for studies of ethanol-induced apoptosis
Miller, Kathryn M. (Katie)
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With over 2.5 million related deaths each year, alcohol consumption is a major global health issue. Although ethanol is widely accepted within the scientific community to induce apoptosis and neurodegeneration, the precise mechanisms by which this process occurs are still undefined. In this study we transition our ethanol-induced apoptosis work from the CHO cell line into the more complex and relevant human SH-SY5Y neuroblasoma cell line. We optimized conditions for transfection of GFP:Bcl-2 constructs, co-transfection of Flag-Bcl-2 and Chameleon constructs and established dose responsiveness to ethanol in the SH-SY5Y neuroblastoma line. MTT assay was used to assess cell viability in response to ethanol, as well as ensure no toxicity due to transfection. After optimizing cell culture conditions, we determined the optimal transfection and co-transfection conditions to be 1 μg DNA with 4 μl XG transfection reagent. Transfection efficiency was evaluated under UV light for all constructs except the Flag-Bcl-2, which need to be verified via Western blot. An ethanol dose-response was established at 1000 mM, 1500 mM and 2000 mM concentrations of 100% ethanol for 24 hours. Through this in vitro model system we have developed, we have opened the door to future studies examining the sub-cellular mechanisms involved in ethanol-induced neurodegeneration. Rescue experiments investigating the anti-apoptotic role of the Bcl-2 family could provide more evidence supporting the notion of Bcl-2 as a key mediator for ethanol-induced neurotoxicity. Calcium imaging within the ER and mitochondria, with the Chameleon constructs optimized here, will provide valuable information regarding the sub-cellular localization of calcium in dying cells compared to healthy cells, potentially revealing ethanol-induced apoptosis as a calcium-dependent mechanism.