Study of caspase inhibitors for limiting death in mammalian cell culture

Abstract

Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered.

Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis

“…3) suggesting that C 3 LP activation is necessary for cell death. Indeed, when an irreversible inhibitor of caspase, Z-VAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl (b-methyl ester)-fluoro-methyl ketone) (Kohler et al, 2002;Sauerwald et al, 2003), was applied to cells under shear stress at 8 h and C 3 LP activity measured at 10 h, the activity was reduced to levels similar to that of control cells (Fig. 4).…”

“…Five of these, caspases 3, 6, 7, 8, and 9, are actively involved in the caspase cascade of different forms of apoptosis (Sauerwald et al, 2003). Of these caspases, the presence and involvement of caspase-3-like activity has been previously demonstrated in Xanthomonas campestris (Gautam and Sharma, 2002a).…”

Autophagy and apoptosis in Chinese hamster ovary cell culture

“…Up to now, there have been many approaches to overcome apoptotic cell death during batch cultures by (1) genetically modifying important genes in cell growth, 8,[10][11][12][13][14][15][16][17][18] (2) supplementing essential nutrients, [19][20][21] or (3) adding chemical inhibitors. 22,23 We address each of these approaches below.…”

The effect of Bcl‐2, YAMA, and XIAP over‐expression on apoptosis and adenovirus production in HEK293 cell line

“…Mastrangelo et al (2000) showed that CHO-bcl-xL and BHK-bcl-2 had a lifespan that was double that of the parental cell line postinfection with alphavirus and recombinant CAT showed a twofold increase in production in cell lines over-expressing bcl-2 and bcl-xL, however IL-12 production was greater in CHO-bcl-xL and CHO-bcl-2 whilst reduced in BHK-bcl-xL and BHK-bcl-2. The same group later showed that apoptosis induction by sindbis virus was attenuated in CHO and HEK293 cells using chemical caspase inhibitors and over-expression of XIAP; an anti-apoptosis gene that targets caspases (Sauerwald et al, 2002(Sauerwald et al, , 2003. Vaccina virus induced cell death was limited by over-expression of bcl-2 in immature B-lymphocytes (Baixeras et al, 1998).…”