title: Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy.
abstract: Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcomes with transgene expression and will be useful in vector development. Cytosine deaminase (CD) is a microbial gene undergoing clinical trials in gene-directed enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversion of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-fluorouracil (5-FU). The feasibility of this approach is demonstrated in subcutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic fluxes of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodrug conversion (k(1)(app)), 5-FU efflux from the observable tumor volume (k(2)(app)), and formation of cytotoxic fluorinated nucleotides from 5-FU (k(3)(app)) were 0.49 +/- 0.27 min(-1), 0.766 +/- 0.006 min(-1), and 0.0023 +/- 0.0007 min(-1), respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above the in vitro K(m). These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expression in tumors. This capability provides an approach for measuring gene expression that will be useful in clinical gene therapy trials.
authors.affiliation: Department of Radiology and Biological Chemistry, University of Michigan Medical School, 1150 West Medical Center Drive, MSRBIII Room 9301 Ann Arbor, MI 48109-0648, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Stegman
authors.forename: A
authors.initials: A
authors.lastname: Rehemtulla
authors.forename: B
authors.initials: B
authors.lastname: Beattie
authors.forename: E
authors.initials: E
authors.lastname: Kievit
authors.forename: T S
authors.initials: TS
authors.lastname: Lawrence
authors.forename: R G
authors.initials: RG
authors.lastname: Blasberg
authors.forename: J G
authors.initials: JG
authors.lastname: Tjuvajev
authors.forename: B D
authors.initials: BD
authors.lastname: Ross
mesh_terms: D000818:Animals; D002384:Catalysis; D043525:Cytosine Deaminase; D005437:Flucytosine / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D008279:Magnetic Resonance Imaging; D051379:Mice; D009700:Nucleoside Deaminases / Q000235:genetics*; D019076:Transgenes; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
chemical_list: D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil