Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression.
Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline-regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc.
Welman
Arkadiusz
A
Cancer Research UK, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, United Kingdom. awelman@picr.man.ac.uk
Cawthorne
Christopher
C
Barraclough
Jane
J
Smith
Nigel
N
Griffiths
Gareth J
GJ
Cowen
Rachel L
RL
Williams
Judith C
JC
Stratford
Ian J
IJ
Dive
Caroline
C
D015153:Blotting, Western; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013347:Subcellular Fractions / Q000378:metabolism; D019076:Transgenes
D004318:Doxycycline