A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines.
BACKGROUND
The urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines.


RESULTS
A DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension.


CONCLUSIONS
This doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.
Hasanuzzaman
Mohammad
M
LSU Department of Comparative Biomedical Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. Anusha@lsu.edu
Kutner
Robert
R
Agha-Mohammadi
Siamak
S
Reiser
Jakob
J
Sehgal
Inder
I
D023102:Anoikis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D008164:Luminescent Proteins; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; D014568:Urokinase-Type Plasminogen Activator; D004318:Doxycycline