Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large t-antigen gene

Ken Ichi Hosoya, Kazuhiro Tetsuka, Katsuhiko Nagase, Masatoshi Tomi, Shigeki Saeki, Sumio Ohtsuki, Tetsuya Terasaki, Nobuaki Yanai, Masuo Obinata, Akihiko Kikuchi, Teruo Okano, Hitomi Takanaga

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Abstract

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperaturesensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-Mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 ±1.16 mmol/1. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr la and mdr Ib mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 umol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

Original languageEnglish
JournalAAPS Journal
Volume2
Issue number3
Publication statusPublished - 2000
Externally publishedYes

Fingerprint

Simian virus 40
Viral Tumor Antigens
Transgenic Mice
Endothelial Cells
Antigens
Cell Line
Temperature
Brain
P-Glycoprotein
Genes
Cell Culture Techniques
3-O-Methylglucose
Inulin
von Willebrand Factor
Vincristine
Mannitol
Growth
Verapamil
Cyclosporine
Lipoproteins

Keywords

  • Blood-brain barrier
  • GLUT-1
  • Mouse brain capillary endothelial cell line
  • P-glycoprotein
  • Temperature-dependent cell growth

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Medicine(all)

Cite this

Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large t-antigen gene. / Hosoya, Ken Ichi; Tetsuka, Kazuhiro; Nagase, Katsuhiko; Tomi, Masatoshi; Saeki, Shigeki; Ohtsuki, Sumio; Terasaki, Tetsuya; Yanai, Nobuaki; Obinata, Masuo; Kikuchi, Akihiko; Okano, Teruo; Takanaga, Hitomi.

In: AAPS Journal, Vol. 2, No. 3, 2000.

Research output: Contribution to journalArticle

Hosoya, KI, Tetsuka, K, Nagase, K, Tomi, M, Saeki, S, Ohtsuki, S, Terasaki, T, Yanai, N, Obinata, M, Kikuchi, A, Okano, T & Takanaga, H 2000, 'Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large t-antigen gene', AAPS Journal, vol. 2, no. 3.
Hosoya, Ken Ichi ; Tetsuka, Kazuhiro ; Nagase, Katsuhiko ; Tomi, Masatoshi ; Saeki, Shigeki ; Ohtsuki, Sumio ; Terasaki, Tetsuya ; Yanai, Nobuaki ; Obinata, Masuo ; Kikuchi, Akihiko ; Okano, Teruo ; Takanaga, Hitomi. / Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large t-antigen gene. In: AAPS Journal. 2000 ; Vol. 2, No. 3.
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T1 - Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large t-antigen gene

AU - Hosoya, Ken Ichi

AU - Tetsuka, Kazuhiro

AU - Nagase, Katsuhiko

AU - Tomi, Masatoshi

AU - Saeki, Shigeki

AU - Ohtsuki, Sumio

AU - Terasaki, Tetsuya

AU - Yanai, Nobuaki

AU - Obinata, Masuo

AU - Kikuchi, Akihiko

AU - Okano, Teruo

AU - Takanaga, Hitomi

PY - 2000

Y1 - 2000

N2 - Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperaturesensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-Mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 ±1.16 mmol/1. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr la and mdr Ib mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 umol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

AB - Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperaturesensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-Mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 ±1.16 mmol/1. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr la and mdr Ib mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 umol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

KW - Blood-brain barrier

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