title: Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants.
abstract: Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines.
authors.affiliation: Department of Chemical Engineering, The Johns Hopkins University, 3400 North Charles St., Baltimore, Maryland 21218, USA.
authors.forename: Tina M
authors.initials: TM
authors.lastname: Sauerwald
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
authors.forename: George A
authors.initials: GA
authors.lastname: Oyler
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics*; D019149:Bioreactors; D016466:CHO Cells; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004791:Enzyme Inhibitors; D017353:Gene Deletion; D006801:Humans; D007668:Kidney / Q000166:cytology; D011506:Proteins / Q000235:genetics / Q000494:pharmacology*; D012845:Sindbis Virus / Q000502:physiology; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011506:Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D020169:Caspases