Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression.
In an attempt to use the hyperosmotic pressure for improved foreign protein production in recombinant Chinese hamster ovary (rCHO) cells, the response of rCHO cells producing a humanized antibody (SH2-0.32-(Delta)bcl-2 cells) to hyperosmotic pressure was determined in regard to cell growth and death, and antibody production. Further, the feasibility of Bcl-2 overexpression in improving rCHO cell viability under hyperosmotic pressure was also determined by comparing control cells (SH2-0.32-(Delta)bcl-2) with Bcl-2 overexpressing cells (14C6-bcl-2). After 3 days of cultivation in the standard medium (294 mOsm x kg(-1)), the spent medium was exchanged with the fresh media with various osmolalities (294-640 mOsm x kg(-1)). The results obtained show that hyperosmotic pressure inhibited cell growth in a dose-dependent manner, though 14C6-bcl-2 cells were less susceptible to hyperosmotic pressure than SH2-0.32-(Delta)bcl-2 cells. At 522 mOsm x kg(-1), SH2-0.32-(Delta)bcl-2 cells underwent a gradual cell death mainly through apoptosis due to the cytotoxic effect of hyperosmotic pressure. In contrast, Bcl-2 overexpression in 14C6-bcl-2 cells could delay the apoptosis induced by 522 mOsm x kg(-1) by inhibiting caspase-3 activation. Bcl-2 overexpression could also improve the cellular membrane integrity of 14C6-bcl-2 cells. When subjected to hyperosmotic pressure, the specific antibody productivity of SH2-0.32-(Delta)bcl-2 cells and 14C6-bcl-2 cells was increased in a similar extent. As a result, the final antibody concentration achieved in 14C6-bcl-2 cells at 522 mOsm x kg(-1) was 2.5-fold higher than that at 294 mOsm x kg(-1). At 580 mOsm x kg(-1), acute hyperosmotic pressure induced the rapid loss of viability in both SH2-0.32-(Delta)bcl-2 and 14C6-bcl-2 cells through necrosis rather than through apoptosis. Taken together, Bcl-2 overexpression and optimized hyperosmotic pressure could improve the antibody production of rCHO cells.
Kim
No Soo
NS
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, 305-701, Taejon, Republic of Korea.
Lee
Gyun Min
GM
D000818:Animals; D000906:Antibodies / Q000235:genetics; D017209:Apoptosis; D016466:CHO Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D006801:Humans; D009336:Necrosis; D009997:Osmotic Pressure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000906:Antibodies; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases