title: The use of 19F spectroscopy and diffusion-weighted MRI to evaluate differences in gene-dependent enzyme prodrug therapies.
abstract: To evaluate noninvasive measures of gene expression and tumor response in a gene-dependent enzyme prodrug therapy (GDEPT), a bifunctional fusion gene between Saccharomyces cerevisiae cytosine deaminase (CD) and Haemophilus influenzae uracil phosphoribosyltransferase (UPRT) was constructed. CD deaminates 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), and UPRT subsequently converts 5FU to fluorouridine monophosphate, and both of these reactions can be monitored noninvasively in vitro and in vivo using 19F magnetic resonance spectroscopy (MRS). Following transient transfection the CD-UPRT fusion protein exhibited both UPRT and CD enzymatic activities as documented by 19F MRS. In addition, an increase in CD activity and thermal stability was witnessed for the fusion protein compared to native CD. Stable expression of CD-UPRT in 9L glioma cells increased both 5FC and 5FU sensitivity in vitro compared to CD-expressing and wild-type 9L cells. Noninvasive 19F MRS of both CD and UPRT gene function in vivo demonstrated that in animals bearing CD-expressing tumors there was limited conversion of 5FC to 5FU with no measurable accumulation of cytotoxic fluorinated nucleotides (F-nucs). In contrast, CD-UPRT-expressing tumors had increased CD gene activity with a threefold higher intratumoral accumulation of 5FU and significant generation of F-nucs. Finally, CD-UPRT yielded increased efficacy in an orthotopic animal model of high-grade glioma. More importantly, early changes in cellular water mobility, which are felt to reflect cellular death, as measured by diffusion-weighted MRI, were predictive of both durable response and increased animal survival. These results demonstrate the increased efficacy of the CD-UPRT GDEPT compared to CD alone both biochemically and in a preclinical model and validate both 19F MRS and diffusion-weighted MRI as tools to assess gene function and therapeutic efficacy.
authors.affiliation: Department of Radiation Oncology, The University of Michigan Medical Center, Ann Arbor 48109-0582, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Hamstra
authors.forename: Kuei C
authors.initials: KC
authors.lastname: Lee
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Tychewicz
authors.forename: Victor D
authors.initials: VD
authors.lastname: Schepkin
authors.forename: Bradford A
authors.initials: BA
authors.lastname: Moffat
authors.forename: Mark
authors.initials: M
authors.lastname: Chen
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Dornfeld
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Lawrence
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Chenevert
authors.forename: Brian D
authors.initials: BD
authors.lastname: Ross
authors.forename: Juri T
authors.initials: JT
authors.lastname: Gelovani
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000627:therapeutic use; D019677:Artificial Gene Fusion; D001921:Brain / Q000473:pathology; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D038524:Diffusion Magnetic Resonance Imaging; D005437:Flucytosine / Q000032:analysis / Q000378:metabolism* / Q000627:therapeutic use; D005461:Fluorine; D005468:Fluorodeoxyuridylate / Q000032:analysis; D005472:Fluorouracil / Q000032:analysis; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods / Q000592:standards; D009682:Magnetic Resonance Spectroscopy; D009369:Neoplasms / Q000378:metabolism / Q000628:therapy*; D020339:Optic Nerve Glioma / Q000473:pathology; D010430:Pentosyltransferases / Q000235:genetics* / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism* / Q000627:therapeutic use; D051381:Rats; D014161:Transduction, Genetic
chemical_list: D000970:Antineoplastic Agents; D011355:Prodrugs; D005468:Fluorodeoxyuridylate; D005461:Fluorine; D005437:Flucytosine; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D043525:Cytosine Deaminase; D005472:Fluorouracil