(724a) Redirection of Glycolytic Metabolism By Overexpression of Bcl-2? in CHO Cells

Previous work has shown that overexpressing anti-apoptotic genes in CHO cells leads to lower lactate accumulation and more rapid consumption.  Multiple studies have found that there is a correlation between reduced lactate accumulation and increased antibody titer.  However, little is known as to how apoptotic pathway modulations affect central metabolism.  Accordingly, we conducted a study where Bcl-2Δ was overexpressed by transfecting a commercially available CHO-S line.  Following transfection, we measured Caspase 3/7 expression as a proxy for the overall effectiveness of apoptotic resistance (ApoR) in selected clones.  Two clones were selected, each overexpressing Bcl-2Δ as determined by Western blot.  To better understand how lactate metabolism was perturbed, we conducted metabolic flux analysis (MFA), in an attempt to elucidate the connection between apoptotic and metabolic pathways.  Through MFA, we determined that there was a considerable redirection of glycolytic metabolism at the pyruvate node.  In both ApoR clones, a more substantial portion of pyruvate (nearly 40% in one ApoR clone, whereas only 25% in the control) was directed to the TCA cycle and oxidative metabolism. Consequently, less pyruvate was diverted to lactate.  Interestingly, in the ApoR cell lines, enhanced NADH oxidase enzymatic activity (a 2 to 3-fold increase) was measured, an enzyme known to oxidize NADH in the electron transport chain.  This result, in conjunction with the MFA study, provides a potential explanation for how Bcl-2Δ suppresses lactate production.  We will present the results from this study, as well as the impact of Bcl-2Δ on increasing the integrated viable cell density (IVCD) and improving the overall biomass yield on glucose.