Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system: a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro.
BACKGROUND
We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells.


METHODS
We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro.


RESULTS
The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses.


CONCLUSIONS
This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.
Marignol
Laure
L
Department of Haematology and Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine, St James's Hospital and Trinity College Dublin, Dublin, Ireland. marignol@tcd.ie
Foley
Ruth
R
Southgate
Thomas D
TD
Coffey
Mary
M
Hollywood
Donal
D
Lawler
Mark
M
D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use*; D005437:Flucytosine / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D011836:Radiation Tolerance / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D014162:Transfection
D005437:Flucytosine; D043525:Cytosine Deaminase