title: Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes.
abstract: Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappaB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.
authors.affiliation: Dept. of Pharmacology and Therapeutics, Univ. of Liverpool, Sherrington Bldgs, Ashton St., Liverpool, L69 3GE, Merseyside, UK. c.e.p.goldring@liv.ac.uk
authors.forename: Chris E P
authors.initials: CE
authors.lastname: Goldring
authors.forename: Neil R
authors.initials: NR
authors.lastname: Kitteringham
authors.forename: Rosalind
authors.initials: R
authors.lastname: Jenkins
authors.forename: Cerys A
authors.initials: CA
authors.lastname: Lovatt
authors.forename: Laura E
authors.initials: LE
authors.lastname: Randle
authors.forename: Azman
authors.initials: A
authors.lastname: Abdullah
authors.forename: Andrew
authors.initials: A
authors.lastname: Owen
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Liu
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Butler
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Williams
authors.forename: Peter
authors.initials: P
authors.lastname: Metcalfe
authors.forename: Christian
authors.initials: C
authors.lastname: Berens
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
authors.forename: Brian
authors.initials: B
authors.lastname: Foster
authors.forename: Alec
authors.initials: A
authors.lastname: Simpson
authors.forename: Lesley
authors.initials: L
authors.lastname: McLellan
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics; D004318:Doxycycline; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects* / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics; D022781:Hepatocytes / Q000502:physiology; D006801:Humans; D008099:Liver / Q000166:cytology / Q000502:physiology; D008113:Liver Neoplasms / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D051265:NF-E2-Related Factor 1 / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D052002:NF-kappa B p50 Subunit / Q000235:genetics; D011500:Protein Synthesis Inhibitors; D013752:Tetracycline; D019076:Transgenes / Q000187:drug effects / Q000502:physiology; D015262:Xenobiotics
chemical_list: D000900:Anti-Bacterial Agents; D051265:NF-E2-Related Factor 1; D051267:NF-E2-Related Factor 2; D052002:NF-kappa B p50 Subunit; D011500:Protein Synthesis Inhibitors; D015262:Xenobiotics; D019392:Cytochrome P-450 CYP2E1; D005982:Glutathione Transferase; D013752:Tetracycline; D004318:Doxycycline