title: Establishment and characterization of hepatocytes from an Immortomouse/SMP30/GNL knockout mouse hybrid lacking vitamin C to study vitamin C transport.
abstract: Senescence marker protein-30 (SMP30) has been identified as the lactone-hydrolysing enzyme gluconolactonase (GNL), which is involved in vitamin C (L-ascorbic acid, AA) biosynthesis. We previously reported the development of SMP30/GNL knockout (KO) mice unable to synthesize AA in vivo. For more efficient study of the liver's AA uptake and as yet uncharacterized efflux system, we established an immortal hepatocyte line derived from a hybrid of SMP30/GNL KO mice and Immortomice. Immortomice express the thermolabile simian virus 40 (SV40) large T antigen tsA58. These SMP30/GNL KO immortal hepatocytes proliferate at the permissive temperature of 33°C but degrade rapidly at the non-permissive temperature of 39°C. Additionally, they are SMP30-/GNL-deficient, express SV40 large T antigen and proliferate steadily at 33°C. However, the cells' proliferation is arrested at 39°C. A phase contrast micrograph revealed that the cells are binucleated with an enlarged cytoplasm similar to that of primary cultured hepatocytes from wild-type mice. Dose-response and time-dependent study of AA uptake revealed that the cells, although unable to synthesize AA, took up AA from the culture medium. This property of our SMP30/GNL immortal hepatocytes makes them extremely useful for studying AA uptake and efflux systems in the liver.
authors: Cellular Genetics, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
authors: Akiko
authors: A
authors: Amano
authors: Setsuko
authors: S
authors: Handa
authors: Toshiro
authors: T
authors: Aigaki
authors: Kazuhiro
authors: K
authors: Shigemoto
authors: Naoki
authors: N
authors: Maruyama
authors: Akihito
authors: A
authors: Ishigami
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism*; D001692:Biological Transport; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002678:Chimera; D003470:Culture Media / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008858:Microscopy, Phase-Contrast; D061251:Primary Cell Culture; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology; D013696:Temperature; D013997:Time Factors
chemical_list: D000952:Antigens, Polyomavirus Transforming; D002135:Calcium-Binding Proteins; D003470:Culture Media; D047908:Intracellular Signaling Peptides and Proteins; C513324:Rgn protein, mouse; D001205:Ascorbic Acid