title: Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro.
abstract: To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli- or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60pM). Intermediate-term stimulation (24h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168h) was higher at low concentrations of crhGM-CSF (15-30pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro.
authors.affiliation: Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 1-754, Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. Electronic address: noiredge2007@yahoo.co.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hashimoto
authors.affiliation: Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 1-754, Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. Electronic address: belltatnk@gmail.com.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 1-754, Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. Electronic address: y-itoh@med.niigata-u.ac.jp.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Itoh
authors.affiliation: Towa Environment Science Co., Ltd. Prophoenix Division, 1-24-22 Nanko-kita, Suminoe, Osaka 559-0034, Japan. Electronic address: yamagata@prophoenix.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Yamagata
authors.affiliation: Department of Medical Informatics, Niigata University Medical and Dental Hospital, 1-754, Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. Electronic address: nktmr@m12.alpha-net.ne.jp.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Kitamura
authors.affiliation: Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 1-754, Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. Electronic address: ryushi@med.niigata-u.ac.jp.
authors.forename: Ryushi
authors.initials: R
authors.lastname: Tazawa
authors.affiliation: Laboratory of Infectious Diseases and Immunology, College of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Electronic address: nakagaki@nvlu.ac.jp.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Nakagaki
authors.affiliation: Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 1-754, Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. Electronic address: radical@med.niigata-u.ac.jp.
authors.forename: Koh
authors.initials: K
authors.lastname: Nakata
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D013058:Mass Spectrometry; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D009439:Neuraminidase
keywords: CHO cells; GM-CSF; Oligosaccharide; Sialic acid; TF-1 cells