Characterization of the RSL1-dependent conditional expression system in LNCaP prostate cancer cells and development of a single vector format.
BACKGROUND
Conditional expression systems are useful tools for the study of gene function but the use of these systems in prostate cancer cells is limited by the undesired biological effects of the inducing ligands. The RheoSwitch system employs RheoSwitch Ligand 1 (RSL1), a non-steroidal analog of the insect hormone ecdysone, to activate a modified nuclear receptor heterodimer that controls target gene expression via GAL4 response elements. This system has not been tested in prostate cancer cells.


METHODS
We established LNCaP human prostate cancer cell lines that constitutively express RheoSwitch transcription factors to quantify RSL1-dependent expression and assess the effects of RSL1 on cell proliferation and endogenous gene expression. Potential RSL1-responsive genes were identified using Affymetrix microarrays and validated by Northern blot hybridization. A single-vector format was developed to establish cell lines that conditionally produce a recombinant protein.


RESULTS
Stable cell lines displayed tight and potent (over several orders of magnitude) RSL1-dependent regulation of a transiently transfected luciferase reporter gene. RSL1 did not affect basal or androgen-stimulated cell proliferation and exerted minimal effects on the expression of endogenous genes. Cell lines established using the single-vector system also displayed strictly RSL1-dependent production of the recombinant protein encoded by the stably integrated RSL1-responsive expression cassette.


CONCLUSIONS
The RheoSwitch system is well suited for conditional gene expression in prostate cancer cells. The single-vector format should facilitate the production of stable cell lines. This system should be useful for the study of proteins involved in prostate cancer in both cell and animal models of the disease.
Lessard
Julie
J
Molecular Endocrinology and Oncology Research Center, Laval University Medical Research Center (CHUQ), Québec, QC, Canada.
Aicha
Sonia Ben
SB
Fournier
Andréa
A
Calvo
Ezequiel
E
Lavergne
Eliane
E
Pelletier
Mélissa
M
Labrie
Claude
C
D015152:Blotting, Northern; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004440:Ecdysone / Q000031:analogs & derivatives / Q000235:genetics; D020869:Gene Expression Profiling; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008297:Male; D016254:Mutagenesis, Insertional; D009376:Neoplasms, Hormone-Dependent / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D011994:Recombinant Proteins; D004440:Ecdysone