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      Telomere Visualization in Tissue Sections using Pyrrole–Imidazole Polyamide Probes

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          Abstract

          Pyrrole–Imidazole (PI) polyamides bind to specific DNA sequences in the minor groove with high affinity. Specific DNA labeling by PI polyamides does not require DNA denaturation with harsh treatments of heat and formamide and has the advantages of rapid and less disruptive processing. Previously, we developed tandem hairpin PI polyamide probes (TH59 series), which label telomeres in cultured cell lines more efficiently than conventional methods, such as fluorescence in situ hybridization (FISH). Here, we demonstrate that a TH59 derivative, HPTH59-b, along with immunostaining for specifying cell types in the tissues, visualizes telomeres in mouse and human tissue sections. Quantitative measurements of telomere length with single-cell resolution suggested shorter telomeres in the proliferating cell fractions of tumor than in non-tumor tissues. Thus, PI polyamides are a promising alternative for telomere labeling in clinical research, as well as in cell biology.

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          Most cited references33

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines.

            The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential. Telomerase is an enzyme that restores telomeric DNA sequences, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo. Telomerase activity has been detected in 50-100% of tumors of different types, but not in most normal adult somatic tissues. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres). Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.
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              Telomere elongation in immortal human cells without detectable telomerase activity.

              Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                06 July 2016
                2016
                : 6
                : 29261
                Affiliations
                [1 ]Structural Biology Center, National Institute of Genetics, and Department of Genetics, Sokendai (Graduate University for Advanced Studies) , Mishima, Shizuoka 411-8540, Japan
                [2 ]Department of Chemistry, Graduate School of Science, Kyoto University , Sakyo, Kyoto 606-8502, Japan
                [3 ]Department of Pathology, Hospital, National Center for Global Health and Medicine , Shinjuku, Tokyo 162-8655, Japan
                [4 ]Department of Intractable Diseases, Research Institute, National Center for Global Health and Medicine , Shinjuku, Tokyo 162-8655, Japan
                [5 ]Department of Surgery, Hospital, National Center for Global Health and Medicine , Shinjuku, Tokyo 162-8655, Japan
                [6 ]HiPep Laboratories, Nakatsukasa-cho 486-46 , Kamigyo-ku, Kyoto 602-8158, Japan
                [7 ]Division of Brain Function, Department of Integrated Genetics, National Institute of Genetics, and Department of Genetics , Sokendai, Mishima, Shizuoka 411-8540, Japan
                Author notes
                Article
                srep29261
                10.1038/srep29261
                4933941
                27380936
                bc8ca476-e635-489b-819d-033fa6d2f016
                Copyright © 2016, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 12 May 2016
                : 13 June 2016
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