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In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr − cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH − anti−LDH − GP3 − CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 − CHO − GP3/anti−LDH − anti−LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time (∼19 h compared with ∼26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO3 −-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of recombinant proteins.
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Jeong, Dw., Cho, I.T., Kim, T.S. et al. Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism. Mol Cell Biochem 284, 1–8 (2006). https://doi.org/10.1007/s11010-005-9004-7
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DOI: https://doi.org/10.1007/s11010-005-9004-7