Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice.
BACKGROUND
We aimed to evaluate the efficacy of gene-directed enzyme-prodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'.


METHODS
Stably transformed andogen-independent mouse prostate cancer (PC) cells, RM1-CDUPRT, -GFP or GFP/LacZ cells were used. To assess the local bystander effects of CDUPRT-GDEPT, immunocompetent C57BL/6 mice implanted with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions intraprostatically were treated with 5FC. Pseudo-metastases in the lungs were established by a tail vein injection of untransfected RM1 cells. At necropsy, prostate weight/volume and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry.


RESULTS
CDUPRT expression in RM1-GFP/CDUPRT cells or tumors was confirmed by enzymic conversion of 5FC into 5FU, using HPLC. Treatment of mice bearing intraprostatic RM1-GFP/CDUPRT tumors with 5FC resulted in complete regression of the tumors. A 'local bystander effect' was seen, even though only 20% of the cells expressed CDUPRT. More importantly a significant reduction in pseudo-metastases of RM1 cells in lungs indicated a 'distant bystander effect'. Immunohistochemical evaluation of the treated tumors showed increased necrosis and apoptosis, with decreased tumor vascularity. There was also a significant increase in tumour-infiltration by macrophages, CD4+ T and natural killer cells.


CONCLUSIONS
We conclude that CDUPRT-GDEPT significantly suppressed the aggressive growth of RM1 prostate tumors and lung pseudo-metastases via immune mechanisms involving necrosis and apoptosis.
Khatri
Aparajita
A
Oncology Research Centre, Prince of Wales Hospital Clinical School of Medicine, The University of New South Wales, Randwick, NSW 2031, Australia. a.khatri@unsw.edu.au
Zhang
Bing
B
Doherty
Eboney
E
Chapman
Jane
J
Ow
Kim
K
Pwint
Hnin
H
Martiniello-Wilks
Rosetta
R
Russell
Pamela J
PJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D005437:Flucytosine / Q000378:metabolism / Q000627:therapeutic use; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007763:Lac Operon; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D010430:Pentosyltransferases / Q000235:genetics* / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism / Q000627:therapeutic use; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology / Q000628:therapy*; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
D011355:Prodrugs; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005437:Flucytosine; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D043525:Cytosine Deaminase