Padmanabhan Parasuraman, Otero Jesse, Ray Pritha, Paulmurugan Ramasamy, Hoffman Andrew R, Gambhir Sanjiv S, Biswal Sandip, Ulaner Gary A
Department of Radiology and Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California, USA.
J Nucl Med. 2006 Feb;47(2):270-7.
Our goal was to noninvasively measure chemotherapy-induced changes in the expression of critical tumor growth genes. To achieve this goal, we used radionuclide and optical methods to measure changes in human telomerase reverse transcriptase (hTERT) gene expression in tumor cells before and after 5-fluorouracil treatment.
A fusion reporter construct, containing humanized Renilla luciferase (hrl, for bioluminescent imaging), monomeric red fluorescence protein 1 (mrfp1, for fluorescent imaging), and a truncated thymidine kinase (ttk, for imaging of radiolabeled acycloguanosines), was placed under the control of hTERT promoter fragments. These constructs were introduced into tumor cell lines with and without hTERT expression. Transfected cells were treated with 5-fluorouracil, a chemotherapeutic that decreases hTERT gene expression, and treatment-induced changes in hTERT promoter activity were imaged.
When the fusion construct is introduced into cell lines that express hTERT, all 3 reporter systems are highly expressed and hTERT promoter activity can be visualized. Cell lines lacking hTERT transcription show no significant reporter expression. Decreases in hTERT gene expression caused by 5-fluorouracil treatment could be visualized in living 293T cells by both fluorescent microscopy and bioluminescent imaging.
hTERT promoter activity can be monitored by 1 radionuclide and 2 optical reporter systems using a single reporter construct. This in vitro study provides evidence that our multimodality reporter construct can be used to study the expression of a critical tumor growth gene in living subjects.
我们的目标是无创测量化疗引起的关键肿瘤生长基因表达变化。为实现这一目标，我们使用放射性核素和光学方法测量5-氟尿嘧啶治疗前后肿瘤细胞中人端粒酶逆转录酶（hTERT）基因表达的变化。
构建一个融合报告基因载体，包含人源化海肾荧光素酶（hrl，用于生物发光成像）、单体红色荧光蛋白1（mrfp1，用于荧光成像）和截短的胸苷激酶（ttk，用于放射性标记无环鸟苷的成像），将其置于hTERT启动子片段的控制之下。将这些载体导入有或无hTERT表达的肿瘤细胞系。用5-氟尿嘧啶（一种可降低hTERT基因表达的化疗药物）处理转染后的细胞，并对治疗引起的hTERT启动子活性变化进行成像。
当将融合构建体导入表达hTERT的细胞系时，所有3种报告系统均高度表达，hTERT启动子活性可被可视化。缺乏hTERT转录的细胞系未显示出明显的报告基因表达。5-氟尿嘧啶治疗引起的hTERT基因表达降低可通过荧光显微镜和生物发光成像在活的293T细胞中观察到。
使用单个报告基因构建体，可通过1种放射性核素和2种光学报告系统监测hTERT启动子活性。这项体外研究提供了证据，表明我们的多模态报告基因构建体可用于研究活体受试者中关键肿瘤生长基因的表达。
