TY - JOUR
T1 - Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism
AU - Jeong, Dae won
AU - Cho, Il Taeg
AU - Kim, Tae Soo
AU - Bae, Gun Won
AU - Kim, Ik Hwan
AU - Kim, Ick Young
N1 - Funding Information:
This work was supported by a grant from the BioGreen 21 Program, from the Rural Development Administration, Republic of Korea.
PY - 2006/3
Y1 - 2006/3
N2 - 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 2∼6 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.
AB - 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 2∼6 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.
KW - Acidosis
KW - Apoptosis
KW - Cellgrowth
KW - Glycolysis
KW - Oxidoresistance
KW - Recombinant protein production
KW - Respiration rate
UR - http://www.scopus.com/inward/record.url?scp=33745891791&partnerID=8YFLogxK
U2 - 10.1007/s11010-005-9004-7
DO - 10.1007/s11010-005-9004-7
M3 - Article
C2 - 16477389
AN - SCOPUS:33745891791
SN - 0300-8177
VL - 284
SP - 1
EP - 8
JO - Molecular and Cellular Biochemistry
JF - Molecular and Cellular Biochemistry
IS - 1-2
ER -