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First published online March 22, 2006
doi: 10.1242/10.1242/jcs.02854

Journal of Cell Science 119, 1433-1441 (2006)
Published by The Company of Biologists 2006
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A novel function of OLIG2 to suppress human glial tumor cell growth via p27Kip1 transactivation

Kouichi Tabu1,2,*, Akiko Ohnishi3,*, Yuji Sunden1,4, Tadaki Suzuki1, Masumi Tsuda1, Shinya Tanaka1, Toshiyuki Sakai5, Kazuo Nagashima1 and Hirofumi Sawa2,6,{ddagger}

1 Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
2 21st Century COE Program for Zoonosis Control, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
3 Department of Neurosurgery, National Cancer Center, Tokyo 104-0045, Japan
4 Laboratory of Comparative Pathology, Hokkaido University School of Veterinary Medicine, Sapporo 060-0818, Japan
5 Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
6 Department of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 060-0818, Japan


Figure 1
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  		Fig. 1. Induction of OLIG2 expression in U12-1 cells by removal of doxycycline. Cells were incubated in the absence of doxycycline (Tet-off medium) for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to OLIG2. The same membrane was reprobed with antibodies to actin as an internal control.

 

Figure 2
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  		Fig. 2. Inhibition of U12-1 cell growth by OLIG2. (A) Growth curves of U12-1 cells incubated in Tet-on or Tet-off medium. The cell cycle was synchronized by serum deprivation for 24 hours before transfer of the cells to Tet-on or Tet-off medium for the indicated time. Data represent the number of viable cells and are means ± s.d. of values from four independent experiments. *P<0.05, **P<0.01 (Student's t-test) versus the corresponding value for Tet-on cells. (B) OLIG2-induced inhibition of DNA synthesis in U12-1 cells. Cells were incubated for 72 hours in Tet-on or Tet-off medium and then assayed for BrdU incorporation. BrdU-incorporating cells were detected with antibodies for BrdU (green). The nuclei were counterstained with PI (red). Bars, 100 µm. Cell proliferation was determined by counting BrdU-positive nuclei per total nuclei from three random fields. Data are means ± s.d. of values from three independent experiments. ***P<0.001 (Student's t-test). (C) Inhibition of anchorage-independent growth of U12-1 cells by OLIG2. Cells were grown on soft agar plates containing Tet-on or Tet-off medium for 3 weeks, after which the number of colonies with a diameter of 125-250 µm or >250 µm was counted. Representative micrographs of colonies formed by Tet-on cells are shown in the lower panels. Quantitative data (upper panel) are means ± s.d. of values from three independent experiments. *P<0.05 (Student's t-test).

 

Figure 3
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  		Fig. 3. OLIG2-induced upregulation of p27Kip1 mRNA and protein. U12-1 cells were cultured in the absence of doxycycline (Tet-off medium) for the indicated times to induce OLIG2 expression. The amount of p27Kip1 mRNA was then determined by quantitative RT-PCR analysis (A) and that of p27Kip1 protein was evaluated by immunoblot analysis (B). The abundance of p27Kip1 mRNA was normalized by that of GAPDH mRNA and expressed relative to the normalized value for time zero; data are means ± s.d. of values from three independent experiments. The immunoblot was also reprobed with antibodies to OLIG2 and to actin. *P<0.05, **P<0.01 (Student's t-test).

 

Figure 4
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  		Fig. 4. Knockdown of p27Kip1 expression by siRNA abrogated OLIG2-mediated growth inhibition and DNA synthesis in U12-1 cells. (A) Suppression of p27Kip1 by siRNA in U12-1 Tet-off cells. U12-1 cells seeded on 3.5-cm dishes were serum starved for 18 hours and transfected with either p27Kip1 siRNA or negative control siRNA for 6 hours. Thereafter, 1x105 cells were reseeded in Tet-off medium and were lysed at the indicated days. (B) Growth curves of U12-1 cells in the presence of p27Kip1 siRNA. Cells were cultured in Tet-on or Tet-off medium at the indicated days. Data represent the number of viable cells and are means ± s.d. of values from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test) versus the corresponding value for control siRNA-transfected cells. (C) DNA synthesis of U12-1 cells in the presence of p27Kip1 siRNA. Cells were incubated for 72 hours in Tet-on or Tet-off medium, thereafter assayed for BrdU incorporation. DNA synthesis was examined by immunostaining with anti-BrdU antibody 3 days after transfection of siRNA against p27Kip1 in the presence or absence of OLIG2. BrdU-incorporating cells were detected with antibody for BrdU (green). The nuclei were counterstained with PI (red). Bars, 100 µm. Cell proliferation was determined by counting BrdU-positive nuclei per total nuclei from three random fields. Data are means ± s.d. of values from three independent experiments. **P<0.01, ***P<0.001 (Student's t-test). (D) Effect of cell density on the OLIG2-mediated growth inhibition in U12-1 cells. U12-1 cells were seeded at a density of 700 cells/cm2 in 6-cm dishes (low density) and 50,000 cells/cm2 in 12-well plates (high density), respectively, and cultured for 3 days in either Tet-on or Tet-off medium. At intervals of 24 hours, cells in three dishes were separately harvested and their number and viability were determined. Data are means ± s.d. of values from at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).

 

Figure 5
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  		Fig. 5. Delineation of the OLIG2-responsive region of the human p27Kip1 gene promoter. (A) Schematic representation of luciferase reporter plasmids containing a series of 5' deletion mutants of the human p27Kip1 gene promoter. Nucleotide numbers are relative to the transcription start site. (B) Luciferase activities of U12-1 cells transfected with the indicated reporter plasmids and cultured in Tet-on or Tet-off medium. The experimental reporter luciferase activity was calculated by subtracting the intrinsic activity as measured by samples corresponding to pGVB2, and then normalized by that of Renilla luciferase encoded by the cotransfected pRL-TK. Data are means ± s.d. of values from at least three independent experiments. *P<0.05, **P<0.01 (Student's t-test).

 

Figure 6
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  		Fig. 6. Effects of mutation of Sp1 and CTF sites in the OLIG2-responsive region of the p27Kip1 gene promoter on transcriptional activity. (A) Schematic representation of p27PF-based luciferase reporter plasmids containing point mutations in the Sp1-1, Sp1-2 or CTF sites (underlined). (B) Luciferase activities of U12-1 cells transfected with the indicated reporter plasmids and cultured in Tet-on or Tet-off medium. Firefly luciferase activity was normalized using that of pRL-TK plasmid. Data are means ± s.d. of values from at least three independent experiments. *P<0.05, **P<0.01 (Student's t-test).

 

Figure 7
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  		Fig. 7. EMSA analysis of OLIG2 binding to an oligonucleotide containing the CTF site of the p27Kip1 gene promoter. (A) Immunoblot analysis of nuclear extracts prepared from U12-1 cells cultured for 48 hours in Tet-on or Tet-off medium. The same membrane was probed with antibodies to OLIG2, to p27Kip1, and to lamin A/C (control). (B) EMSA was performed with a 32P-labeled oligonucleotide corresponding to positions -533 to -513 of the human p27Kip1 gene promoter. The corresponding unlabeled oligonucleotide was used as a competitor. The arrowhead indicates a specific DNA-protein complex, and the arrow indicates unbound probe.

 

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© The Company of Biologists Ltd 2006