Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein.
BACKGROUND
Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function.


METHODS
Expression vectors of CD or CD/UPRT fused to monomeric DsRed (mDsRed) were constructed and rat prostate carcinoma (R3327-AT) cell lines stably expressing either CD/mDsRed or CD/UPRT/mDsRed were generated. The expression of the fusion proteins was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. The function of the fusion protein was confirmed in vitro by assessing 5-FC and 5-FU cytotoxicity. In vivo fluorine-19 magnetic resonance spectroscopy ((19)F MRS) was used to monitor the conversion of 5-FC to 5-FU in mice bearing the R3327-CD/mDsRed and R3327-CD/UPRT/mDsRed tumor xenografts.


RESULTS
Sensitivity to 5-FC and 5-FU was higher in cells stably expressing the CD/UPRT/mDsRed fusion gene than in cells stably expressing CD/mDsRed alone or wild-type cells. Whole tumor (19)F MRS measurements showed rapid conversion of 5-FC to 5-FU within 20 min after 5-FC was administered intravenously in both CD/mDsRed and CD/UPRT/mDsRed tumors with subsequent anabolism to cytotoxic fluoronucleotides (FNucs). CD/UPRT/mDsRed tumor was more efficient in these processes.


CONCLUSIONS
This study demonstrates the utility of these tumor models stably expressing CD or CD/UPRT to non-invasively evaluate the efficacy of the transgene expression/activity by monitoring drug metabolism in vivo using MRS, with potential applications in preclinical and clinical settings.
Xing
Ligang
L
Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York 10065, USA.
Deng
Xuelong
X
Kotedia
Khushali
K
Ackerstaff
Ellen
E
Ponomarev
Vladimir
V
Clifton Ling
C
C
Koutcher
Jason A
JA
Li
Gloria C
GC
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000032:analysis*; D005434:Flow Cytometry; D005462:Fluorine Radioisotopes; D017930:Genes, Reporter / Q000502:physiology; D008164:Luminescent Proteins / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010430:Pentosyltransferases / Q000032:analysis*; D011471:Prostatic Neoplasms; D051381:Rats; D012680:Sensitivity and Specificity; D014162:Transfection; D019076:Transgenes / Q000502:physiology; D000097573:Red Fluorescent Protein
D043525:Cytosine Deaminase; D005462:Fluorine Radioisotopes; D008164:Luminescent Proteins; D010430:Pentosyltransferases; D000097573:Red Fluorescent Protein; C023269:uracil phosphoribosyltransferase