HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 290/1/C104    most recent 00133.2005v2 00133.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Goldring, C. E. P. Articles by Park, B. K. Search for Related Content PubMed PubMed Citation Articles by Goldring, C. E. P. Articles by Park, B. K.

METHODS IN CELL PHYSIOLOGY

Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes

¹Department of Pharmacology and Therapeutics, University of Liverpool, United Kingdom; ²Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; and ³Department of Human Anatomy and Cell Biology, University of Liverpool, and ⁴Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, United Kingdom

Submitted 21 March 2005 ; accepted in final form 26 August 2005

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-B]. 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.

liver cells; proteins; CYP2E1; NRF2; GSTP1

This article has been cited by other articles:

				D. W. Nebert Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on "Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes" Am J Physiol Cell Physiol, January 1, 2006; 290(1): C37 - C41. [Full Text] [PDF]