The role of un-equal division on regulation of neutrophilic differentiation and the participation of G-CSF

• Project/Area Number: 15590091
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: National Institute of Health Sciences
• Principal Investigator: YAMAGUCHI Teruhide National Institute of Health Sciences, 遺伝子細胞医薬部, 部長 (50111117)
• Co-Investigator(Kenkyū-buntansha): UCHIDA Eriko National Institute of Health Sciences, 遺伝子細胞医薬部, 室長 (80176685) ; NAGATA Ryuji National Institute of Health Sciences, 遺伝子細胞医薬部, 主任研究官 (20370942)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,600,000 (Direct Cost: ¥3,600,000); Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000); Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
• Keywords: neutrophilic differentiation / unequal division / aPKC / PKCiota / HL-60 cell / p70S6K / G-CSF / PKCι

Research Abstract

We, previously, reported that the transferrin receptor (Trf-R) positive (Trf-R+) and negative (Trf-R-) cells appeared after treatment with dimethyl sulfoxide (Me2SO) or retinoic acid, and commitment to neutrophilic differentiation and proliferation in Me_2SO-treated HL-60 cells. To clarify the molecular mechanism of commitment to differentiation and proliferation in both-type cells, we compared the protein expression profiles in Trf-R+ and Trf-R- cells using two-dimensional polyacrylamide electrophoresis. While 55kDa and 25kDa spots were more abundant in Trf-R+ cells, the expression of 11kDa spot is apparently increased in Trf-R- cells than that in Trf-R+ cells. The MALDI-MS spectrum analysis and Edman analysis revealed that the 11kDa protein is Calgranulin/S100A8 protein. Knock-down of S100A8 protein using siRNA inhibited the differentiation of HL-60 cells induced by Me_2SO, suggesting that S100A8.
Since atypical PKC(aPKC) has been reported to play an important role on cell polarity, w e focused to clarify the role of aPKC on differentiation and proliferation of neutrophilic maturation. Particularly, we analyzed the participation of aPKC, PKCι/λ in relation to G-CSF-dependent proliferation and S6K1 and PI3K activation. Protein kinase Cι was abundantly expressed in HL-60 cells, but not PKCζ ; the maximum stimulation of PKCι, was observed from 15 min to 30 min after the addition of G-CSF. An immuno-precipitation assay using an anti-PKCι antibody revealed that PKCι formed a complex with S6K1 30 min after the addition of G-CSF. There was a time lag between the activation of PI3K and S6K1 when G-CSF was added to the HL-60 cells. Within 5 to 15 min during this lag time, PKCι was found to translocate from the nucleus to the membrane, and re-translocate to the cytosol, resulting in the association with S6K1. Small interfering RNA for PKCι inhibited G-CSF-induced proliferation and phosphorylation of Thr-389 of S6K1,and promoted differentiation. The dynamic translocation and activation processes of PKCι can be explained as the reason for the time lag in maximum activation between PI3K and S6K1.
These results indicate that while S100A8 plays an important role on differentiation of HL-60 cells., PI3K/PKCι/p70S6K cascade contribute main role on the proliferation of differentiating HL-60 cells.

Research Products

• [Journal Article] HX531, RXR antagonist inhibited the 9-cis retinoic acid-induced binding with cofactor. (2005)
  • Author(s): Kanayasu-Toyoda, T., Fujino, T., Oshizawa, T., Suzuki, T., Nishimaki-Mogami, T., Sato, Y., Sawada, J., Inoue, K., Shudo, K., Yamaguchi, T.
  • Journal Title: J Steroid.Biochem.Molec.Biol. 94 Pages: 305-309
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Approaches to Improving the kinetics of adnovirus-delivered genes and gene products. (2005)
  • Author(s): Xu, Z., Mizuguchi, H., Sakurai, F., Koizumi, N., Hosono, T., Kawabata, K., Watanabe, Y., Yamaguchi, T., Hayakawa, T.
  • Journal Title: Adv Drug Del Rev. 57 Pages: 781-802
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Suppression of proliferation of poliovirus and porcine parvovirus by novel phenoxazine, 2-amino-4, 4a-dihydro-4a-7-dimethyl-3H-phenoxazine and 3-amino-1, 4a- dihydro-4a-8-dimethyl-2H-phenoxazine-2-one. (2005)
  • Author(s): Iwata, A., Sato, K., Yamaguchi, T., Yoshiake, N., Tomoda, A
  • Journal Title: Biol.Pharm.Bull. 28 Pages: 905-907
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Two-dimensional electrophoresisof disease-associated proteins in human cerebrospinal fluid from patients with rheumatoid arthritis (2005)
  • Author(s): Yamamoto, Y., Akita, Y., Tai, S., Fukasaku, S., Yamaguchi, T., Oshizawa, T., Yamaoka, K., Shimamura, M., Hazato, T.
  • Journal Title: J.Elecctrophoresis 49 Pages: 23-27
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] HX531,RXR antagonist inhibited the 9-cis retinoic acid-induced binding with cofactor. (2005)
  • Author(s): Kanayasu-Toyoda, T., Fujino, T., Oshizawa, T., Suzuki, T., Nishimaki-Mogami, T., Sato, Y., Sawada, J., Inoue, K., Shudo, K., Yamaguchi, T.
  • Journal Title: J.Steroid Biochem Mol. 94 Pages: 303-309
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Approaches to Improving the kinetics of adnovirus-delivered genes and gene products. (2005)
  • Author(s): Xu, Z., Mizuguchi, H., Sakurai, F., Koizumi, N., Hosono, T., Kawabata, K, Watanabe, Y., Yamaguchi, T., Hayakawa, T.
  • Journal Title: Adv Drug Deliv Rev 57 Pages: 781-802
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Suppression of proliferation of poliovirus and porcine parvovirus by novel phenoxazine, 2-amino-4,4a-dihydro-4a-7-dimethyl-3H-phenoxazine and 3-amino-1,4a-dihydro-4a-8-dimethyl-2H-phenoxazine-2-one. (2005)
  • Author(s): Iwata, A.Sato, K., Yamaguchi, T., Yoshiake, N., Tomoda, A
  • Journal Title: Biol.Pharm.Bull. 28 Pages: 905-907
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Two-dimensional electrophoresis of disease-associated proteins in human cerebrospinal fluid from patients with rheumatoid arthritis. (2005)
  • Author(s): Yamamoto, Y, Akita, Y, Tai, S., Fukasaku, S., Yamaguchi, T., Oshizawa, T., Yamaoka, K., Shimamura, M, Hazato, T.
  • Journal Title: J.Elecctrophoresis 49 Pages: 23-27
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Adenovirus vector-mediated doxycycline-inducible RNA interference. (2004)
  • Author(s): Hosono T., Mizuguchi H., Katayama K., Xu Z.L., Sakurai F., Ishii-Watabe A., Kawabata K., Yamaguchi T., Nakagawa S., Mayumi T., Hayakawa T.
  • Journal Title: Hum.Gene Ther. 15 Pages: 813-819
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] An improved method for detection of replication competent retrovirus in retrovirus vector products (2004)
  • Author(s): Uchida E, Sato K., Iwata A., Ishii-Watabe A., Mizuguchi H., Hikata M., Murata M., Yamaguchi T., Hayakawa T.
  • Journal Title: Biologicals. 32 Pages: 139-146
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Ribosomal protein S18 identified as a cofilin-binding protein by using phage display library. (2004)
  • Author(s): Kusui K., Sasaki H., Adachi R., Matsui S., Yamamoto K., Yamaguchi T., Kasahara T., Suzuki K.
  • Journal Title: Bio Mol.Cell.Biochem 262 Pages: 187-193
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] An improved method for detection of replication competent retrovirus in retrovirus vector products. (2004)
  • Author(s): Uchida E, Sato K., Iwata A., Ishii-Watabe A., Mizuguchi H., Hikata M., Murata M., Yamaguchi T., Hayakawa T.
  • Journal Title: Biologicals 32 Pages: 139-146
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Ribosomal protein S18 identified as a cofilin-binding protein by using phage display library. (2004)
  • Author(s): Kusui K., Sasaki H., Adachi R., Matsui S., Yamamoto K., Yamaguchi T., Kasahara T., Suzuki K.
  • Journal Title: Mol.Cell.Biochem. 262 Pages: 187-193
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] An improved method for detection of replication competent retrovirus in retrovirus vector products (2004)
  • Author(s): Uchida E., Sato K., Iwata A., Ishii-Watabe A., Mizuguchi H., Hikata M., Murata M., Yamaguchi T., Hayakawa T.
  • Journal Title: Biologicals. 32 Pages: 139-146
• [Journal Article] The role of c-Myc on granulocyte colony-stimulating factor-dependent neutrophilic proliferation and differentiation of HL-60 cells. (2003)
  • Author(s): Kanayasu-Toyoda T, Yamaguchi T, Oshizawa T, Uchida E, Hayakawa T
  • Journal Title: Biochem.Pharmacol. 66 Pages: 133-140
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells. (2003)
  • Author(s): Kanayasu-Toyoda T, Yamaguchi T, Oshizawa T, Hayakawa T
  • Journal Title: J Cell.Physiol. 195 Pages: 119-129
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Possible Involvement of Optimally Phosphorylated L-plastin in Activation of Superoxide Generating NADPH Oxidase. (2003)
  • Author(s): Oshizawa, T., Yamaguchi, T., Suzuki, K., Yamamoto, Y., Hayakawa, T.
  • Journal Title: J.Biochem. 134 Pages: 827-834
  • NAID: 50000393691
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector. (2003)
  • Author(s): Sakurai F., Mizuguchi H., Yamaguchi T., Hayakawa T.
  • Journal Title: Mol.Ther. 8 Pages: 813-821
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Reduction of natural adenovirus tropism to mice liver by fiber-shaft exchange in combination with both CAR- and v integrin-binding ablation. (2003)
  • Author(s): Koizumi, N., Mizuguchi, H., Sakurai, F., Yamaguchi, T., Watanabe, Y., Hayakawa, T.
  • Journal Title: J Virol. 77 Pages: 13062-13072
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Virus Concentration Using Polyethyleneimine- conjugated Magnetic Beads for Improvement of Sensitivity in Nucleic Acid Amplification Tests. (2003)
  • Author(s): Satoh, K., Iwata, A., Murata, M., Hikata, M., Hayakawa, T., Yamaguchi, T.
  • Journal Title: J Virol, Methods 114 Pages: 11-19
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Possible Involvement of Optimally Phosphorylated L-plastin in Activation of Superoxide Generating NADPH Oxidase. (2003)
  • Author(s): Oshizawa, T., Yamaguchi1, T, Suzuki, K., Yamamoto, Y., Hayakawa, T.
  • Journal Title: J.Biochem. 134 Pages: 827-834
  • NAID: 50000393691
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Reduction of natural adenovirus tropism to mice liver by fiber-shaft exchange in combination with both CAR- and v integrin-binding ablation. (2003)
  • Author(s): Koizumi, N., Mizuguchi, H., Sakurai, F, Yamaguchi, T., Watanabe, Y., Hayakawa, T.
  • Journal Title: J Virol. 77 Pages: 13062-13072
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Virus Concentration Using Polyethyleneimine-conjugated Magnetic Beads for Improvement of Sensitivity in Nucleic Acid Amplification Tests. (2003)
  • Author(s): Satoh, K., Iwata, A., Murata, M., Hikata, M., Hayakawa, T., Yamaguchi, T.
  • Journal Title: J Virol, Methods 114 Pages: 11-19
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] HX531, RXR antagonist inhibited the 9-cis retinoic acid-induced binding with cofactor.
  • Author(s): Kanayasu-Toyoda, T., Fujino, T., Oshizawa, T., Suzuki, T., Nishimaki-Mogami T., Sato, Y., Sawada, J., Inoue K., Shudo, K., Yamaguchi, T.
  • Journal Title: J Steroid Biochem.Molec.Biol. (in press)
• [Journal Article] Approaches to Improving the kinetics of adnovirus-delivered genes and gene products.
  • Author(s): Xu, Z., Mizuguchi, H., Sakurai, F., Koizumi, N., Hosono, T., Kawabata, K., Watanabe, Y., Yamaguchi, T., Hayakawa, T.
  • Journal Title: Adv Drug Del Rev. (In press)
• [Journal Article] Suppression of proliferation of poliovirus and porcine, parvovirus by novel phenoxazine, 2-amino-4,4a-dihydro-4a-7-dimethyl-3H-phenoxazine and 3-amino-1,4a-dihydro-4a-8-dimethyl-2H-phenoxazine-2-one.
  • Author(s): Iwata, A., Sato, K., Yamaguchi, T., Yoshiake, N., Tomoda, A
  • Journal Title: Biol.Pharm.Bull. (In press)
• [Publications] Kanayasu-Toyoda, T, Yamaguchi, T, Oshizawa, T, Uchida, E, Hayakawa, T: "The role of c-Myc on granulocyte colony-stimulating factor-dependent neutrophilic proliferation and differentiation of HL-60 cells."Biochem.Pharmacol.. 66. 133-140 (2003)
• [Publications] Kanayasu-Toyoda, T, Yamaguchi, T, Oshizawa, T, Hayakawa, T: "CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells."J.Cell.Physiol.. 195. 119-129 (2003)
• [Publications] Oshizawa, T., Yamaguchi, T., Suzuki, K., Yamamoto, Y., Hayakawa, T: "Possible Involvement of Optimally Phosphorylated L-plastin in Activation of Superoxide Generating NADPH Oxidase."J.Biochem.. 134. 827-834 (2003)