Influence of down-regulation of caspase-3 by siRNAs on sodium-butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin.
Sodium butyrate (NaBu) can enhance the expression of foreign protein of recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on foreign protein expression in rCHO cells is compromised by its growth inhibitory and cytotoxic effects. To overcome this cytotoxic effect of NaBu, an expression vector of small interfering RNAs (siRNAs) targeting against caspase-3, a key effector component in apoptosis, was constructed and transfected into rCHO cells producing human thrombopoietin (hTPO). Using this siRNA strategy, rCHO cells (F21 cells) expressing a low level of caspase-3 proenzyme determined by RT-PCR and Western blot analysis were established. Under the condition of 1-5 mM NaBu addition at the exponential growth phase, down-regulation of caspase-3 in F21 cells could not effectively inhibit NaBu-induced apoptotic cell death. This NaBu-induced apoptotic cell death occurred because F21 cells appeared to compensate for the lack of caspase-3 by increasing the active caspase-7 level. These results suggest that the intracellular caspase's interconnectivity should be taken into consideration for the successful inhibition of apoptosis of rCHO cells.
Sung
Yun Hee
YH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Hwang
Su-Jeong
SJ
Lee
Gyun Min
GM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002087:Butyrates / Q000008:administration & dosage*; D016466:CHO Cells; D053148:Caspase 3; D020169:Caspases / Q000235:genetics* / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D015202:Protein Engineering / Q000379:methods; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics
D002087:Butyrates; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D013926:Thrombopoietin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases