Influence of co-down-regulation of caspase-3 and caspase-7 by siRNAs on sodium butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin

Abstract

Previously, the expression of caspase-3 siRNA could not effectively inhibit sodium butyrate (NaBu)-induced apoptotic cell death of recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Caspase-3 siRNA expressing cells appeared to compensate for the lack of caspase-3 by increasing active caspase-7 levels. For the successful inhibition of NaBu-induced apoptosis of rCHO cells, both caspase-3 and caspase-7 were down-regulated using the siRNA expression vector system. Co-down-regulation of caspase-3 and caspase-7 increased cell viability and extended culture longevity in serum-free culture in the presence or absence of 1mM NaBu addition. In the cultures with 1mM NaBu addition, the maximum hTPO concentration in rCHO cells with down-regulation of both caspases was approximately 55% higher than that in rCHO cells without down-regulation of caspases and approximately 16% higher than rCHO cells with down-regulation of only caspase-3. However, in the culture with 3mM NaBu, this strategy could not dramatically enhance the culture longevity and hTPO production, compared to Bcl-2 overexpression. The different result in hTPO production between down-regulation of caspases and Bcl-2 overexpression may be because the down-regulation of caspase-3 and caspase-7, unlike Bcl-2 overexpression, could not maintain mitochondrial membrane potential in the presence of 3mM NaBu. Taken together, co-down-regulation of caspase-3 and caspase-7 is effective in regard to extension of culture longevity and enhancement of hTPO production in a serum-free culture in the presence or absence of 1mM NaBu addition.

In vitro and in vivo RNA interference mediated suppression of Tn‐caspase‐1 for improved recombinant protein production in High FiveTMcell culture with the baculovirus expression vector system

“…Protein quality has also been improved by using RNAi to suppress specific genes, resulting in both increased antibody-dependent cellular cytotoxicity for anticancer therapeutics Kanda et al, 2007;Mori et al, 2004), as well as higher sialic acid content in recombinant glycoproteins (Ngantung et al, 2006). Specific examples of antiapoptosis engineering involving RNAi include enhancing cell viability and protein production in CHO cells through the downregulation of either caspase-3 expression (Lai et al, 2004;Sung et al, 2005) or the expression of both caspase-3 and caspase-7 (Sung et al, 2007) as well as the suppression of the antiapoptotic genes Alg-2 and Requiem (Wong et al, 2006b). The latter study was the second article in a series that used transcriptional profiling to determine potential RNAi targets (Wong et al, 2006a).…”

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

“…When Dual-10 cells were treated with 2.5 mM sodium butyrate, the mean cell number remained similar to the seeding density when compared to Dual-10 cells treated with vehicle, though no significant cell death was seen. The effect of butyrate on cell growth and apoptosis in CHO cells has previously been observed and strategies to improve cell growth are being considered, including media additives such as silk worm hemolymph, down-regulation of BAK, BAX, Caspase 3 and Caspase 7, and overexpression of bcl-2 (Cost et al 2010;Kim et al 2009;Sung et al 2007;Choi et al 2005b;Lee 2002, 2000).…”

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

“…Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in a fresh lysis buffer (1% Nonidet P-40, 0.1% sodium dodecylsulfate [SDS], 0.02% NaN 3 , 50 mM Tris-Cl [pH 8.0], 150 mM NaCl and protease inhibitors [Roche]), as described previously (Hwang and Lee, 2008b;Sung et al, 2007). Approximately, 20 mg of protein extract was loaded after measurement by the Bio-Rad protein assay, as described by the manufacturer's protocol (Bio-Rad, Hercules, CA).…”

“…Until now, apoptosis in rCHO cell culture has been recognized as an important task to be solved, and therefore, there have been numerous studies focusing on the onset of apoptosis and anti-apoptotic gene engineering in rCHO cells (Arden and Betenbaugh, 2004). As a result, it was found that the onset of apoptosis in rCHO cells can be delayed by promoting anti-apoptotic factors as well as by inhibiting the expression of pro-apoptotic factors (Chiang and Sisk, 2005;Figueroa et al, 2007;Kim and Lee, 2000;Lim et al, 2006;Sauerwald et al, 2003;Sung et al, 2007;Tey et al, 2000;Wong et al, 2006;Yun et al, 2007).…”