A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM.

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and (64)Cu. HT29 (hTERT+) and U2OS (hTERT-) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo.

Tumor-targeted radionuclide imaging and therapy based on human sodium iodide symporter gene driven by a modified telomerase reverse transcriptase promoter.

Human telomerase reverse transcriptase (hTERT) is highly active in most cancer cells and, thus, could be used for tumor targeting. The human sodium iodide symporter (hNIS) gene is being actively researched as a potential radioactive iodine (radioiodine) gene therapy. In this study, we investigated the possibilities of using the hNIS gene driven by the hTERT promoter for molecular imaging and radioiodine gene therapy. Stable cell lines of hTERT-positive cells (Hep3B hepatoma) expressing hNIS, under the control of the 5mmTERT promoter, were generated using a retroviral system. Radioiodine uptake and efflux tests were performed, and a clonogenic assay was used to evaluate the in vitro cytotoxicity of 131I. Finally, scintigraphic, biodistribution, and radioiodine therapy studies were performed in vivo. Radioiodine uptake by 5mmTERT-NIS-transfected Hep3B cells was 22 times higher than by nontransfected Hep3B cells, and 5 times that of 5mmTERT-NIS-transfected U2-OS cells (p < 0.05). Clonogenic assays demonstrated that the survival rate of Hep3B-5mmTERT-NIS cells after 131I incubation was significantly lower than that of Hep3B cells (p < 0.001), and radioiodine accumulations in Hep3B-5mmTERT-NIS tumors were significantly higher than in wild-type tumors. In addition, technetium- 99m scintigraphy clearly visualized Hep3B-5mmTERT-NIS tumors. Moreover, after being treated with 111 MBq of 131I-labeled Hep3B-5mmTERT-NIS, tumor growth was retarded, whereas Hep3B tumor growth progressed. hTERT-positive tumors were successfully targeted by the NIS gene under the control of the 5mmTERT promoter. The described system could be useful for targeted molecular imaging and as a radioiodine gene therapy for cancer.