Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3.
Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.
Kim
No Soo
NS
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea.
Lee
Gyun Min
GM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015152:Blotting, Northern; D015153:Blotting, Western; D002087:Butyrates / Q000037:antagonists & inhibitors; D016466:CHO Cells / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000235:genetics* / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D016000:Cluster Analysis; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004586:Electrophoresis; D006801:Humans; D058610:Isobutyrates; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D008956:Models, Chemical; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009842:Oligopeptides / Q000494:pharmacology; D016372:RNA, Antisense / Q000032:analysis / Q000378:metabolism*; D012680:Sensitivity and Specificity
D002087:Butyrates; D015853:Cysteine Proteinase Inhibitors; D058610:Isobutyrates; D009842:Oligopeptides; D016372:RNA, Antisense; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C020380:isobutyric acid; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases