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

Abstract

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 {sup 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.more » 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. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.« less

Authors:
 [1];  [2];  [2];  [2];  [1];  [2];  [2];  [2];  [1];  [2];  [1];  [2];  [2];  [1];  [2];  [1];  [2];  [2];  [2]
  1. Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of)
  2. (Korea, Republic of)
Publication Date:
OSTI Identifier:
22606173
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 3; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANIMAL TISSUES; ANTIBODIES; BIOMEDICAL RADIOGRAPHY; COPPER 64; DYES; FLUORESCENCE; IMAGES; IN VITRO; IN VIVO; MEMBRANE PROTEINS; MICE; MICROSCOPY; NEOPLASMS; PEPTIDES; POLYMERASE CHAIN REACTION; POSITRON COMPUTED TOMOGRAPHY; PROBES

Citation Formats

Jung, Kyung oh, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, Tumor Microenvironment Global Core Research Center, Seoul National University, Youn, Hyewon, E-mail: hwyoun@snu.ac.kr, Cancer Research Institute, Seoul National University College of Medicine, Tumor Microenvironment Global Core Research Center, Seoul National University, Cancer Imaging Center, Seoul National University Hospital, Seoul, Kim, Seung Hoo, Cancer Research Institute, Seoul National University College of Medicine, Kim, Young-Hwa, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, Kang, Keon Wook, Cancer Research Institute, Seoul National University College of Medicine, Chung, June-Key, E-mail: jkchung@snu.ac.kr, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, and Tumor Microenvironment Global Core Research Center, Seoul National University. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.068.
Jung, Kyung oh, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, Tumor Microenvironment Global Core Research Center, Seoul National University, Youn, Hyewon, E-mail: hwyoun@snu.ac.kr, Cancer Research Institute, Seoul National University College of Medicine, Tumor Microenvironment Global Core Research Center, Seoul National University, Cancer Imaging Center, Seoul National University Hospital, Seoul, Kim, Seung Hoo, Cancer Research Institute, Seoul National University College of Medicine, Kim, Young-Hwa, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, Kang, Keon Wook, Cancer Research Institute, Seoul National University College of Medicine, Chung, June-Key, E-mail: jkchung@snu.ac.kr, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, & Tumor Microenvironment Global Core Research Center, Seoul National University. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM. United States. doi:10.1016/J.BBRC.2016.06.068.
Jung, Kyung oh, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, Tumor Microenvironment Global Core Research Center, Seoul National University, Youn, Hyewon, E-mail: hwyoun@snu.ac.kr, Cancer Research Institute, Seoul National University College of Medicine, Tumor Microenvironment Global Core Research Center, Seoul National University, Cancer Imaging Center, Seoul National University Hospital, Seoul, Kim, Seung Hoo, Cancer Research Institute, Seoul National University College of Medicine, Kim, Young-Hwa, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, Kang, Keon Wook, Cancer Research Institute, Seoul National University College of Medicine, Chung, June-Key, E-mail: jkchung@snu.ac.kr, Biomedical Sciences, Seoul National University College of Medicine, Cancer Research Institute, Seoul National University College of Medicine, and Tumor Microenvironment Global Core Research Center, Seoul National University. 2016. "A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM". United States. doi:10.1016/J.BBRC.2016.06.068.
@article{osti_22606173,
title = {A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM},
author = {Jung, Kyung oh and Biomedical Sciences, Seoul National University College of Medicine and Cancer Research Institute, Seoul National University College of Medicine and Tumor Microenvironment Global Core Research Center, Seoul National University and Youn, Hyewon, E-mail: hwyoun@snu.ac.kr and Cancer Research Institute, Seoul National University College of Medicine and Tumor Microenvironment Global Core Research Center, Seoul National University and Cancer Imaging Center, Seoul National University Hospital, Seoul and Kim, Seung Hoo and Cancer Research Institute, Seoul National University College of Medicine and Kim, Young-Hwa and Biomedical Sciences, Seoul National University College of Medicine and Cancer Research Institute, Seoul National University College of Medicine and Kang, Keon Wook and Cancer Research Institute, Seoul National University College of Medicine and Chung, June-Key, E-mail: jkchung@snu.ac.kr and Biomedical Sciences, Seoul National University College of Medicine and Cancer Research Institute, Seoul National University College of Medicine and Tumor Microenvironment Global Core Research Center, Seoul National University},
abstractNote = {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 {sup 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. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.},
doi = {10.1016/J.BBRC.2016.06.068},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 477,
place = {United States},
year = 2016,
month = 8
}
  • Human telomerase reverse transcriptase (hTERT) is highly expressed in over 85% of human cancers, which makes it a broadly applicable molecular target for cancer therapy. Several groups have demonstrated that hTERT can efficiently evoke specific cytotoxic T lymphocytes (CTL) responses for malignant tumors. In the present study, we developed a novel virus-like particulate peptide-nucleotide dual vaccine (PNDV) of hTERT, which was composed of a low-affinity epitope variant with encoding full-length gene in the same virus-size particulate. We verified the formation of PNDV by DNA retarding assay, DNase I protection assay and transmission electron microscopy, and confirmed its immunogenicity and transfectionmore » activities in mammalian cells. Furthermore, in vivo immunization of HLA-A2.1 transgenic mice generated efficient IFN-{gamma} secretion and hTERT-specific CTLs which are known to cause selective cell death of telomerase positive gastrointestinal cancer cells. To our knowledge, this represents the first report on collocating a low-affinity epitope variant with a full-length hTERT gene for anti-cancer vaccine design. This novel strategy for vaccine design not only enables enhanced immunity to a universal tumor antigen, but also has the potential to generate CTLs effective in telomerase-positive tumor cells of diverse tissue origins. Therefore, our findings bear significant implications for immunotherapy of human cancers.« less
  • Suppression of apoptosis is thought to contribute to carcinogenesis. Survivin, a member of the inhibitor-of-apoptosis family, blocks apoptotic signaling activated by various cellular stresses. Since elevated expression of survivin observed in human cancers of varied origin was associated with poor patient survival, survivin has attracted growing attention as a potential target for cancer treatment. Immortalization of cells also is required for carcinogenesis; telomere length maintenance by telomerase is required for cancer cells to proliferate indefinitely. Yet how cancer cells activate telomerase remains unclear. We therefore examined possible interrelationships between survivin expression and telomerase activity. Correlation between survivin and human telomerasemore » reverse transcriptase (hTERT) expression was observed in colon cancer tissues, and overexpression of survivin enhanced telomerase activity by up-regulation of hTERT expression in LS180 human colon cancer cells. DNA-binding activities of specificity protein 1 (Sp1) and c-Myc to the hTERT core promoter were increased in survivin gene transfectant cells. Phosphorylation of Sp1 and c-Myc at serine and threonine residues was enhanced by survivin, while total amounts of these proteins were unchanged. Further, 'knockdown' of survivin by a small inhibitory RNA decreased Sp1 and c-Myc phosphorylation. Thus survivin participates not only in inhibition of apoptosis, but also in prolonging cellular lifespan.« less
  • The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breastmore » cancer.« less
  • The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerasemore » reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.« less
  • The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-{gamma} plays a role in cancer development in addition to its role in glucose metabolism. The natural ligand of PPAR-{gamma}, namely, 15-deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} (15d-PGJ{sub 2}), has been shown to possess antineoplastic activity in cancer cells. However, the mechanism underlying its antineoplastic activity remains to be elucidated. Inhibition of the expression of human telomerase reverse transcriptase (hTERT), a major determinant of telomerase activity, reportedly induces rapid apoptosis in cancer cells. In this study, we investigated the effect of 15d-PGJ{sub 2} on hTERT expression. We found that 15d-PGJ{sub 2} induced apoptosis in themore » MIAPaCa-2 pancreatic cancer cells and dose-dependently decreased hTERT mRNA and protein expression. Down-regulation of hTERT expression by hTERT-specific small inhibitory RNA also induced apoptosis. Furthermore, 15d-PGJ{sub 2} attenuated the DNA binding of estrogen receptor (ER). MIAPaCa-2 expressed only ER{beta}, and although its expression did not decrease due to 15d-PGJ{sub 2}, its phosphorylation was suppressed. Additionally, a mitogen-activated protein kinase (MAPK) kinase inhibitor decreased ER{beta} phosphorylation, and 15d-PGJ{sub 2} attenuated MAPK activity. We conclude that hTERT down-regulation by 15d-PGJ{sub 2} plays an important role in the proapoptotic property of the latter. Furthermore, 15d-PGJ{sub 2} inhibits ER{beta}-mediated hTERT gene transcription by suppressing ER{beta} phosphorylation via the inhibition of MAP kinase signaling.« less