Tonack S, Patel S, Jalali M, Nedjadi T, Jenkins RE, Goldring C, Neoptolemos J, Costello E. Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression. World J Gastroenterol 2011; 17(15): 1947-1960 [PMID: 21528072 DOI: 10.3748/wjg.v17.i15.1947]
Corresponding Author of This Article
Dr. Eithne Costello, Department of Molecular and Therapeutic Cancer Medicine, 5th Floor UCD Building, Daulby Street, Liverpool, L69 3GA, United Kingdom. ecostell@liv.ac.uk
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World J Gastroenterol. Apr 21, 2011; 17(15): 1947-1960 Published online Apr 21, 2011. doi: 10.3748/wjg.v17.i15.1947
Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression
Sarah Tonack, Sabina Patel, Mehdi Jalali, Taoufik Nedjadi, Rosalind E Jenkins, Christopher Goldring, John Neoptolemos, Eithne Costello
Sarah Tonack, Sabina Patel, Mehdi Jalali, Taoufik Nedjadi, John Neoptolemos, Eithne Costello, Liverpool CR-UK Centre, Department of Molecular and Therapeutic Cancer Medicine, University of Liverpool, Liverpool, L69 3GA, United Kingdom
Rosalind E Jenkins, Christopher Goldring, MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, School of Biomedical Sciences, University of Liverpool, L69 3GA, United Kingdom
John Neoptolemos, Eithne Costello, National Institute for Health Research Pancreatic Biomedical Research Unit, Liverpool, L69 3GA, United Kingdom
Author contributions: Tonack S and Patel S contributed equally to this work; Costello E designed the research; Tonack S, Patel S, Jalali M and Jenkins RE performed the research; Nedjadi T and Goldring C contributed analytical tools; Tonack S and Patel S analyzed the data; Tonack S, Patel S and Costello E wrote the paper; Goldring C and Neoptolemos J revised the manuscript.
Supported by National Institute for Health Research Liverpool Pancreatic Biomedical Research Unit and the Pancreatic Cancer Research Fund (to Nedjadi T)
Correspondence to: Dr. Eithne Costello, Department of Molecular and Therapeutic Cancer Medicine, 5th Floor UCD Building, Daulby Street, Liverpool, L69 3GA, United Kingdom. ecostell@liv.ac.uk
Telephone: +44-151-7064178 Fax:+44-151-7065826
Received: August 25, 2010 Revised: October 19, 2010 Accepted: October 26, 2010 Published online: April 21, 2011
Abstract
AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines.
METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.
RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction.
CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable.
