Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism.
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 ( approximately 19 h compared with approximately 26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO(3) (-)-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.
BK21 HLS, Seoul National University, Seoul, Korea.
Dae-won
DW
Jeong
Il Taeg
IT
Cho
Tae Soo
TS
Kim
Gun Won
GW
Bae
Ik-Hwan
IH
Kim
Ick Young
IY
Kim
D000818:Animals; D017209:Apoptosis; D001639:Bicarbonates / Q000378:metabolism; D016466:CHO Cells; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D005993:Glycerolphosphate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D010084:Oxidation-Reduction; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis
D001639:Bicarbonates; D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005993:Glycerolphosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase; D010959:Tissue Plasminogen Activator