The use of 19F spectroscopy and diffusion-weighted MRI to evaluate differences in gene-dependent enzyme prodrug therapies
The use of 19F spectroscopy and diffusion-weighted MRI to evaluate differences in gene-dependent enzyme prodrug therapies
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DOI：
10.1016/j.ymthe.2004.07.022
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发表时间：
2004-11-01
影响因子：
12.4
通讯作者：
Rehemtulla, A
中科院分区：
文献类型：
--
作者：
Hamstra, DA;Lee, KC;Rehemtulla, A
为了评估基因依赖性酶前药疗法（GDEPT）中基因表达和肿瘤反应的无创性测量，构建了酿酒酵母胞嘧啶脱氨酶（CD）和流感嗜血杆菌尿嘧啶磷酸核糖基转移酶（UPRT）之间的双功能融合基因。 CD 使 5-氟胞嘧啶脱氨（5FQ 为 5-氟尿嘧啶 (5FU)），UPRT 随后将 5FU 转化为氟尿苷单磷酸，并且这两种反应都可以使用 F-19 磁共振波谱 (MRS) 在体外和体内无创监测。瞬时转染后，CD-UPRT 融合蛋白表现出 UPRT 和 CD 酶活性，如 (FMRS)-F-19 所记录。此外，与天然 CD 相比，融合蛋白在 9L 神经胶质瘤细胞中的 CD-UPRT 的稳定表达在体外增加了 5FC 和 5FU 敏感性，体内 CD 和 UPRT 基因功能的非侵入性 F-19 MRS 表明，在携带 CD 表达肿瘤的动物中，5FC 向 5FU 的转化有限，没有可测量的细胞毒性氟化核苷酸的积累。相比之下，表达 CD-UPRT 的肿瘤的 CD 基因活性增加了三倍，并且 F-nuc 的产生显着增加。最后，CD-UPRT 在高级别神经胶质瘤的原位动物模型中产生了更高的功效。更重要的是，通过扩散加权 MRI 测量，细胞水流动性的早期变化可以预测持久的反应和增加的动物存活率。 CD-UPRT GDEPT 与单独的 CD 在生化和临床前模型中进行了比较，并验证 19F MRS 和扩散加权 MRI 作为评估基因功能和治疗效果的工具。
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-fluorcicytosine (5FQ 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 F-19 magnetic resonance spectroscopy (MRS). Following transient transfection the CD-UPRT fusion protein exhibited both UPRT and CD enzymatic activities as documented by (FMRS)-F-19. 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 F-19 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.