title: Elucidating the role of requiem in the growth and death of Chinese hamster ovary cells.
abstract: Requiem, a hypothesized transcription factor with apoptosis-related activity, was previously shown to be a potential cell engineering gene target for improving recombinant protein production. Requiem suppression has resulted in improved viable cell density and extended culture viability, leading to an overall improvement in recombinant protein productivity. However, not much is known about the function of requiem. We found that requiem is highly conserved at both nucleotide and amino acid levels in Chinese hamster ovary (CHO) cells when compared to human and mouse sequences, suggesting that requiem's functional role is evolutionary well conserved. Upon inducing requiem over-expression, proliferation rates of CHO cells were significantly decreased with doubling times increased by 26%. Interestingly, the over-expression of requiem did not decrease cell viability and could not induce apoptosis. However, requiem sensitized the cells to increased caspase-9 activities under staurosporine-induced apoptosis, suggesting that it has a role to play in mitochondria-mediated apoptosis under staurosporine treatment. The nuclear localization of REQUIEM in CHO cells and its conserved plant homeodomain (PHD) zinc fingers seem to further support the hypothesis that requiem encodes for a potential transcription factor. Upon requiem over-expression, we found that the differentially expressed genes involved in transcriptional regulation and cell proliferation and growth were associated both upstream and downstream of p53.
authors.affiliation: Department of Chemical Engineering and Chemical Technology, Imperial College London, UK. lim_yiping@bti.a-star.edu.sg
authors.forename: Yiping
authors.initials: Y
authors.lastname: Lim
authors.forename: Valerie X F
authors.initials: VX
authors.lastname: Seah
authors.forename: Athanasios
authors.initials: A
authors.lastname: Mantalaris
authors.forename: Miranda G S
authors.initials: MG
authors.lastname: Yap
authors.forename: Danny C F
authors.initials: DC
authors.lastname: Wong
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000166:cytology* / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D053263:Gene Regulatory Networks; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D021381:Protein Transport / Q000187:drug effects; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D020169:Caspases; D019311:Staurosporine; D004318:Doxycycline