Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures.
In an attempt to investigate the molecular mechanism that leads to apoptotic death in Chinese hamster ovary (CHO) cells in batch and fed-batch cultures, we cloned caspase-2, -8 and -9 from a CHO cDNA library. Recombinant Chinese hamster caspase-2 and -9 expressed in Escherichia coli show highest activities towards commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA, the designated commercial substrates for human caspase-2 and -9, respectively. However, Chinese hamster caspase-8 shows a broad specificity profile and it cleaves the caspase-9 substrate more efficiently than it cleaves the caspase-8 substrate. The commercially available fluoromethyl ketone type of caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk and Z-DEVD-fmk, were shown to completely lack specificity in inhibiting these caspases. The reversible aldehyde form of inhibitors for human caspase-8 and -9, Ac-LEHD-CHO and Ac-IETD-CHO, are equally efficient in inhibiting Chinese hamster caspase-8. Therefore, the wildly used method of utilizing the "caspase-specific" inhibitors to track the role of individual caspases in dying cells can be inaccurate and thus misleading. As an alternative, we stably expressed dominant negative (DN) mutants of Chinese hamster caspase-2, -8 and -9 to specifically inhibit these enzymes in CHO cells. Our results showed that inhibition of either endogenous caspase-8 or caspase-9 enhanced the viability of the CHO cells in both batch and fed-batch suspension cultures, but the inhibition of caspase-2 had minimal effects. These results suggest that caspase-8 and -9 are possibly involved in the apoptotic cell death in batch and fed-batch cultures of CHO cells, whereas caspase-2 is not. These findings can be valuable in the development of strategies for genetically engineering CHO cells to counter apoptotic death in batch and fed-batch cultures.
Yun
Chee Yong
CY
Bioprocessing Technology Institute, Agency for Science, Technology and Research, 20 Biopolis Way, 06-01 Centros, Singapore 138668, Singapore.
Liu
Sen
S
Lim
Sing Fee
SF
Wang
Tianhua
T
Chung
Beatrice Y F
BY
Jiat Teo
Joong
J
Chuan
Kok Hwee
KH
Soon
Allyson S C
AS
Goh
Keng Siong
KS
Song
Zhiwei
Z
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D053181:Caspase 8 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D061945:Caspase Inhibitors; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D053181:Caspase 8; D053453:Caspase 9