Publications by authors named "Masakatsu Takanashi"

38 Publications

Cisplatin induces differentiation in teratomas derived from pluripotent stem cells.

Regen Ther 2021 Dec 8;18:117-126. Epub 2021 Jun 8.

Department of Molecular Pathology, Tokyo Medical University, 6-1-1, Shinjuku, Shinjuku-ku, Tokyo, Japan.

Introduction: Currently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents.

Methods: We first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs . Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks.

Results: Treatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P < 0.05); however, immunohistochemical analyses demonstrated a significantly reduced PCNA labelling index and an increase in an apoptosis marker on immature neural components (P < 0.05) along with emergence of h-Caldesmon mature smooth muscle cells in treated mice. Moreover, newly differentiated components not found in the control group, such as mature adipose tissue, cartilage, and pancreas, as well as striated muscle, salivary glands, gastric mucosa with fundic glands, and hair follicles emerged. The identities of these components were confirmed by immunostaining for specific markers.

Conclusions: Cisplatin has the ability to reduce immature components in ESC/iPSC-derived teratomas, presumably through apoptosis, and also to induce them to differentiate.
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http://dx.doi.org/10.1016/j.reth.2021.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192819PMC
December 2021

WAPL induces cervical intraepithelial neoplasia modulated with estrogen signaling without HPV E6/E7.

Oncogene 2021 May 4;40(21):3695-3706. Epub 2021 May 4.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

Since cervical cancer still afflicts women around the world, it is necessary to understand the underlying mechanism of cervical cancer development. Infection with HPV is essential for the development of cervical intraepithelial neoplasia (CIN). In addition, estrogen receptor signaling is implicated in the development of cervical cancer. Previously, we have isolated human wings apart-like (WAPL), which is expected to cause chromosomal instability in the process of HPV-infected precancerous lesions to cervical cancer. However, the role of WAPL in the development of CIN is still unknown. In this study, in order to elucidate the role of WAPL in the early lesion, we established WAPL overexpressing mice (WAPL Tg mice) and HPV E6/E7 knock-in (KI) mice. WAPL Tg mice developed CIN lesion without HPV E6/E7. Interestingly, in WAPL Tg mice estrogen receptor 1 (ESR1) showed reduction as compared with the wild type, but cell growth factors MYC and Cyclin D1 controlled by ESR1 expressed at high levels. These results suggested that WAPL facilitates sensitivity of ESR1 mediated by some kind of molecule, and as a result, affects the expression of MYC and Cyclin D1 in cervical cancer cells. To detect such molecules, we performed microarray analysis of the uterine cervix in WAPL Tg mice, and focused MACROD1, a co-activator of ESR1. MACROD1 expression was increased in WAPL Tg mice compared with the wild type. In addition, knockdown of WAPL induced the downregulation of MACROD1, MYC, and Cyclin D1 but not ESR1 expression. Furthermore, ESR1 sensitivity assay showed lower activity in WAPL or MACROD1 downregulated cells than control cells. These data suggested that WAPL increases ESR1 sensitivity by activating MACROD1, and induces the expression of MYC and Cyclin D1. Therefore, we concluded that WAPL not only induces chromosomal instability in cervical cancer tumorigenesis, but also plays a key role in activating estrogen receptor signaling in early tumorigenesis.
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http://dx.doi.org/10.1038/s41388-021-01787-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154587PMC
May 2021

Acerola exosome-like nanovesicles to systemically deliver nucleic acid medicine via oral administration.

Mol Ther Methods Clin Dev 2021 Jun 10;21:199-208. Epub 2021 Mar 10.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

Extracellular vesicles derived from mammalian cells could be useful carriers for drug delivery systems (DDSs); however, with regard to clinical application, there are several issues to be overcome. Acerola ( DC.) is a popular health food. In this study, the feasibility of orally administered nucleic acid drug delivery by acerola exosome-like nanoparticles (AELNs) was examined. AELNs were recovered from acerola juice using an affinity column instead of ultracentrifugation. MicroRNA (miRNA) was sufficiently encapsulated in AELNs by 30-min incubation on ice and was protected against RNase, strong acid, and base treatments. The administration of an AELN/miRNA mixture in cells achieved downregulation of the miRNA's target gene, and this mixture showed cytoplasmic localization. AELNs orally delivered small RNA to the digestive system . The target gene-suppressing effect in the small intestine and liver peaked 1 day after administration, indicating potential for use as an oral DDS for nucleic acid in the digestive system.
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http://dx.doi.org/10.1016/j.omtm.2021.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010214PMC
June 2021

Extracellular microvesicles that originated adipose tissue derived mesenchymal stem cells have the potential ability to improve rheumatoid arthritis on mice.

Regen Ther 2020 Dec 9;15:305-311. Epub 2020 Dec 9.

Department of Molecular Pathology, Tokyo Medical University, Japan.

Introduction: Mesenchymal stem cells (MSCs) are promising therapeutic tools in regenerative medicine. In particularly adipose tissue derived MSC (AMSC) has powerful potential for the therapeutics of rheumatoid arthritis (RA) because these cells can control immune balance. RA systemically occurs autoimmune disease. Interestingly, IL-1 receptor antagonist deficient (IL-1ra) mice induce inflammation in joints like RA. In RA therapy, although AMSC improves the inflammation activity, it is little known to play roles of extracellular microvesicles (EV) for improvement of RA. To clarify the MSC-derived EVs are involved amelioration mechanisms for RA by themselves, we examined the functional effects of development for RA by AMSC-EVs.

Methods: We isolated AMSCs derived mice adipose tissue and purified EVs from the culture supernatant of AMSCs. To examine whether EVs can improve RA, we administrated EVs or AMSCs to IL-1ra knockout mice as RA model mice. We analyzed EVs-included factor by western blot methods and RA improvement effect by ELISA.

Results: In this study, we showed that the swellings of joints on mice in wild type AMSC and that in AMSC-EVs decreased than that in IL-1ra mice-AMSC-EVs and in none-treated. We detected IL-1ra expression in AMSC-EVs in wild type mice but not that in IL-1ra mice. Proinflammatory cytokine expression changes in mice showed in AMSCs and AMSC-EVs, but no apparent differences cytokine expressions were detected in IL-1ra mice.

Conclusions: In this study, we concluded that MSCs might improve RA by the transferring of factors such as IL-1ra, which are included their MSC derived- EVs.
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http://dx.doi.org/10.1016/j.reth.2020.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770341PMC
December 2020

Comprehensive miRNA Analysis Using Serum From Patients With Noninfectious Uveitis.

Invest Ophthalmol Vis Sci 2020 09;61(11)

Department of Ophthalmology, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan.

Purpose: MicroRNAs (miRNAs) are noncoding RNAs and have attracted attention as a biomarker in a variety of diseases. However, extensive unbiased miRNAs analysis in patients with uveitis has not been completely explored. In the present study, we comprehensively analyzed the deregulated miRNAs in three major forms of uveitis (Behҫet's disease [BD], sarcoidosis and Vogt-Koyanagi-Harada disease [VKH]) to search for potential biomarkers.

Methods: This study included 10 patients with BD, 17 patients with sarcoidosis, and 13 patients with VKH. Eleven healthy subjects were used as controls. The miRNAs expression levels were studied by microarray using serum samples from patients with uveitis and healthy controls.

Results: A total of 281 upregulated miRNAs and 137 downregulated miRNAs were detected in patients with BD, 35 upregulated miRNAs and 86 downregulated miRNAs in patients with sarcoidosis, and 153 upregulated miRNAs and 35 downregulated miRNAs in patients with VKH. Some deregulated miRNAs were involved in the mitogen-activated protein kinase signaling pathway and inflammatory cytokine pathways. Furthermore, we identified miR-4708-3p, miR-4323, and let-7g-3p as the best predictor miRNAs for BD, sarcoidosis, and VKH, respectively. Panels of miRNAs with diagnostic potential for the three diseases were generated using machine learning.

Conclusions: In this study, comprehensive miRNA analysis identified deregulated miRNAs in three major forms of noninfectious uveitis. This study provides new insights into molecular pathogenetic mechanisms and useful information toward developing novel diagnostic biomarkers and therapeutic targets for BD, sarcoidosis, and VKH.
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http://dx.doi.org/10.1167/iovs.61.11.4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476662PMC
September 2020

Distinctive Tissue and Serum MicroRNA Profile of IgG4-Related Ophthalmic Disease and MALT Lymphoma.

J Clin Med 2020 Aug 5;9(8). Epub 2020 Aug 5.

Department of Ophthalmology, Tokyo Medical University, Tokyo 160-0023, Japan.

The molecular pathogenesis of orbital lymphoproliferative disorders, such as immunoglobulin G4-related ophthalmic disease (IgG4-ROD) and orbital mucosa-associated lymphoid tissue (MALT) lymphoma, remains essentially unknown. Differentiation between the two disorders, which is important since the work-up and treatment can vary greatly, is often challenging due to the lack of specific biomarkers. Although miRNAs play an important role in the regulation of carcinogenesis and inflammation, the relationship between miRNA and orbital lymphoproliferative diseases remains unknown. We performed a comprehensive analysis of 2565 miRNAs from biopsy and serum specimens of 17 cases with IgG4-ROD, where 21 cases with orbital MALT lymphoma were performed. We identified specific miRNA signatures and their miRNA target pathways, as well as the network analysis for IgG4-ROD and orbital MALT lymphoma. Machine-learning analysis identified miR-202-3p and miR-7112-3p as the best discriminators of IgG4-ROD and orbital MALT lymphoma, respectively. Enrichment analyses of biological pathways showed that the longevity-regulating pathway in IgG4-ROD and the mitogen-activated protein kinase (MAPK) signaling pathway in orbital MALT lymphoma was most enriched by target genes of downregulated miRNAs. This is the first evidence of miRNA profiles of biopsy and serum specimens of patients with IgG4-ROD and orbital MALT lymphoma. These data will be useful for developing diagnostic and therapeutic interventions, as well as elucidating the pathogenesis of these disorders.
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http://dx.doi.org/10.3390/jcm9082530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464164PMC
August 2020

High-Throughput MicroRNA Profiling of Vitreoretinal Lymphoma: Vitreous and Serum MicroRNA Profiles Distinct from Uveitis.

J Clin Med 2020 Jun 12;9(6). Epub 2020 Jun 12.

Department of Ophthalmology, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.

Purpose: Vitreoretinal lymphoma (VRL) is a non-Hodgkin lymphoma of the diffuse large B cell type (DLBCL), which is an aggressive cancer causing central nervous system related mortality. The pathogenesis of VRL is largely unknown. The role of microRNAs (miRNAs) has recently acquired remarkable importance in the pathogenesis of many diseases including cancers. Furthermore, miRNAs have shown promise as diagnostic and prognostic markers of cancers. In this study, we aimed to identify differentially expressed miRNAs and pathways in the vitreous and serum of patients with VRL and to investigate the pathogenesis of the disease.

Materials And Methods: Vitreous and serum samples were obtained from 14 patients with VRL and from controls comprising 40 patients with uveitis, 12 with macular hole, 14 with epiretinal membrane, 12 healthy individuals. The expression levels of 2565 miRNAs in serum and vitreous samples were analyzed.

Results: Expression of the miRNAs correlated significantly with the extracellular matrix (ECM) ‒receptor interaction pathway in VRL. Analyses showed that miR-326 was a key driver of B-cell proliferation, and miR-6513-3p could discriminate VRL from uveitis. MiR-1236-3p correlated with vitreous interleukin (IL)-10 concentrations. Machine learning analysis identified miR-361-3p expression as a discriminator between VRL and uveitis.

Conclusions: Our findings demonstrate that aberrant microRNA expression in VRL may affect the expression of genes in a variety of cancer-related pathways. The altered serum miRNAs may discriminate VRL from uveitis, and serum miR-6513-3p has the potential to serve as an auxiliary tool for the diagnosis of VRL.
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http://dx.doi.org/10.3390/jcm9061844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356511PMC
June 2020

miR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species homeostasis and impairment of autophagy.

Lab Invest 2020 06 17;100(6):863-873. Epub 2020 Feb 17.

Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, 160-8402, Japan.

In patients with breast cancer, primary chemotherapy often fails due to survival of chemoresistant breast cancer stem cells (BCSCs) which results in recurrence and metastasis of the tumor. However, the factors determining the chemoresistance of BCSCs have remained to be investigated. Here, we profiled a series of differentially expressed microRNAs (miRNAs) between parental adherent breast cancer cells and BCSC-mimicking mammosphere-derived cancer cells, and identified hsa-miR-27a as a negative regulator for survival and chemoresistance of BCSCs. In the mammosphere, we found that the expression of hsa-miR-27a was downregulated, and ectopic overexpression of hsa-miR-27a reduced both number and size of mammospheres. In addition, overexpression of hsa-miR-27a sensitized breast cancer cells to anticancer drugs by downregulation of genes essential for detoxification of reactive oxygen species (ROS) and impairment of autophagy. Therefore, enhancing the hsa-miR-27a signaling pathway can be a potential therapeutic modality for breast cancer.
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http://dx.doi.org/10.1038/s41374-020-0409-4DOI Listing
June 2020

Fascin as a Useful Marker for Identifying Neural Components in Immature Teratomas of Human Ovary and Those Derived From Murine Embryonic Stem Cells.

Int J Gynecol Pathol 2019 Jul;38(4):377-385

Tokyo Medical University (R.U.) Departments of Molecular Pathology (A.K., M.T., K.F., M.K.) Anatomic Pathology (T.N.), Tokyo Medical University Department of Diagnostic Pathology, NTT Medical Center Tokyo (H.H.), Tokyo, Japan.

Immature teratoma of the human ovary is a rare disease, and its diagnosis and grading are currently based on histologic evaluation of the presence and amount of immature neural components in the tumor. Despite the importance of tumor grading, immature neural components especially without rosette formation are difficult to identify, partly because useful biomarkers for them are not yet available. Toward this goal, we investigated 16 immature teratomas from human ovaries as well as 10 of those derived from murine embryonic stem cells transplanted into immunodeficient mice. Immunohistochemistry was performed for cytokeratin, glial fibrillary acidic protein, S100, and fascin. It was demonstrated that glial fibrillary acidic protein and S100 expression was not observed in the immature neural components of immature teratomas derived from both human ovary and embryonic stem cells, although their expression was detected in mature neural tissues. In contrast, fascin immunopositivity was clearly found in both mature and immature neural components regardless of rosette formation in immature teratomas derived from both human ovary and embryonic stem cells. Assessment of immature neural components by fascin immunostaining yielded the same or slightly increased quantity than quantification based on hematoxylin and eosin staining. These results suggest that fascin immunostaining is useful as a biomarker in correctly diagnosing and grading human immature teratomas. Further, fascin immunostaining may contribute to the development of regenerative medicine through accurate assessment of the maturation status of pluripotent stem cell-derived tumors transplanted into immunodeficient mice.
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http://dx.doi.org/10.1097/PGP.0000000000000528DOI Listing
July 2019

Toll-like receptor 2 activation implicated in oral squamous cell carcinoma development.

Biochem Biophys Res Commun 2018 01 19;495(3):2227-2234. Epub 2017 Dec 19.

Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. Electronic address:

Recent studies have revealed that Toll-like receptors (TLRs) are highly expressed and activated in many types of cancer. Physiologically, TLR2 recognizes bacteria and other microorganisms in the oral cavity; however, the role of TLR2 in oral squamous cell carcinoma (OSCC) is unclear. In this study, we demonstrated that TLR2 is highly expressed in OSCC in comparison with adjacent non-malignant tissue. TLR2 was also expressed in OSCC-derived cell lines, and its expression was activated by ligands derived from bacteria and mycoplasma. Furthermore, to elucidate the mechanism of OSCC progression via TLR2 signal transduction, we focused on microRNAs (miRNAs) that are induced by TLR2 activation. Interestingly, ligand activation of TLR2 induced the expression of miR-146a and we found that downregulation of caspase recruitment domain-containing protein 10 (CARD10) mRNA in OSCC-derived cell lines. Moreover, knockdown of CARD10 induced resistance to cisplatin-induced apoptosis in OSCC cells. These findings suggest that the activation of TLR2 by bacterial components can enhance the progression of OSCC and may be implicated in acquired resistance to cisplatin-induced apoptosis through regulation of the miR-146a pathway.
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http://dx.doi.org/10.1016/j.bbrc.2017.12.098DOI Listing
January 2018

Expression level of microRNA-200c is associated with cell morphology in vitro and histological differentiation through regulation of ZEB1/2 and E-cadherin in gastric carcinoma.

Oncol Rep 2018 Jan 10;39(1):91-100. Epub 2017 Nov 10.

Department of Molecular Pathology, Tokyo Medical University, Tokyo 160-8402, Japan.

Scirrhous type gastric cancer is characterized by diffuse infiltration of poorly differentiated adenocarcinoma cells and poor prognosis. Although association of poorly differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with E-cadherin through regulation of ZEB1/2, has been reported, participation of miR-200c in gastric carcinogenesis is not fully understood. We used 6 cell lines originating from gastric cancers, and investigated levels of miR-200c along with its target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 and E-cadherin protein expression was also assessed via western blotting. Furthermore, we investigated the expression levels of miR‑200c by in situ hybridization, along with the expression of ZEB1 and E-cadherin by immunohistochemistry, in 97 gastric adenocarcinoma tissues. Inverse correlation between miR‑200c and ZEB1 levels were obtained by qRT-PCR in cell lines (P<0.05). Cell lines with low miR-200c and high ZEB1 exhibited low E-cadherin expression in both qRT-PCR and western blotting, and exhibited spindle-shaped morphology, in contrast to round cell morphology in those cell lines with high miR-200c levels. Inverse correlations were also obtained between miR-200c and ZEB1 as well as between ZEB1 and E-cadherin levels in tissue samples (P<0.001). Cancer tissues with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology, in contrast to tubular form in cancers with high miR-200c expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by downregulation of E-cadherin through upregulation of ZEB1, leading to poorly differentiated histology in gastric cancer.
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http://dx.doi.org/10.3892/or.2017.6093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783608PMC
January 2018

Novel form of miR-29b suppresses bleomycin-induced pulmonary fibrosis.

PLoS One 2017 24;12(2):e0171957. Epub 2017 Feb 24.

Department of Molecular Pathology, Tokyo Medical University, Shinjuku, Tokyo, Japan.

MicroRNA 29b (miR-29b) replacement therapy is effective for suppressing fibrosis in a mouse model. However, to develop clinical applications for miRNA mimics, the side effects of nucleic acid drugs have to be addressed. In this study, we focused on miRNA mimics in order to develop therapies for idiopathic pulmonary fibrosis. We developed a single-stranded RNA, termed "miR-29b Psh-match," that has a unique structure to avoid problems associated with the therapeutic uses of miRNAs. A comparison of miR-29b Psh-match and double-stranded one, termed "miR-29b mimic" indicated that the single-stranded form was significantly effective towards fibrosis according to both in vivo and in vitro experiments. This novel form of miR-29b may become the foundation for developing an effective therapeutic drug for pulmonary fibrosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171957PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5325218PMC
September 2017

PRG4 expression in myxoid liposarcoma maintains tumor cell growth through suppression of an antitumor cytokine IL-24.

Biochem Biophys Res Commun 2017 03 10;485(1):209-214. Epub 2017 Feb 10.

Department of Pathology, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama 641-8509, Japan.

PRG4 is one of the downstream molecules of the myxoid liposarcoma (MLS)-specific fusion oncoproteins TLS-CHOP and EWS-CHOP. Exogenous PRG4 expression increases the tumorigenicity of cells injected in nude mice. The molecular functions of PRG4 in tumorigenesis and/or tumor progression of MLS cells, however, still remain unclear. In this report, we demonstrated that siRNA-mediated knockdown of PRG4 suppressed the growth of the MLS-derived cell lines 1955/91 and 2645/94. In addition, PRG4 knockdown promoted adipocytic differentiation in 1955/91 cells. Thus, PRG4 may play essential roles in MLS cell growth and have potential as a therapeutic target. On the other hand, our previous study has revealed that TLS-CHOP suppresses expression of an anti-tumor cytokine IL-24, contributing to tumor cell survival. In this study, we found that double knockdown of PRG4 and IL-24 did not inhibit MLS cell growth, and single knockdown of PRG4 remarkably increased IL-24 expression. These results suggest that the growth inhibitory effect of PRG4 knockdown is caused by induction of IL-24 expression, and PRG4 may contribute to maintain MLS cell growth through repression of IL-24 expression.
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http://dx.doi.org/10.1016/j.bbrc.2017.02.055DOI Listing
March 2017

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.

Exp Anim 2017 May 25;66(2):125-136. Epub 2016 Nov 25.

Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.
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http://dx.doi.org/10.1538/expanim.16-0075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411299PMC
May 2017

Development of Novel Small Hairpin RNAs That do not Require Processing by Dicer or AGO2.

Mol Ther 2016 08 25;24(7):1278-89. Epub 2016 Apr 25.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

The innate cytokine response to nucleic acid is the most challenging problem confronting the practical use of nucleic acid medicine. The degree of stimulation of the innate cytokine response strongly depends on the length of the nucleic acid. In this study, we developed a 30-nucleotide single-strand RNA, termed "guide hairpin RNA (ghRNA, ghR)", that has a physiological function similar to that of miRNA and siRNA. The ghR caused no innate cytokine response either in vitro or in vivo. In addition, its structure does not contain a passenger strand seed sequence, reducing the unwanted gene repression relative to existing short RNA reagents. Systemic and local injection of ghR-form miR-34a (ghR-34a) suppressed tumor growth in a mouse model of RAS-induced lung cancer. Furthermore, Dicer and AGO2 are not required for ghR-34a function. This novel RNA interference (RNAi) technology may provide a novel, safe, and effective nucleic acid drug platform that will increase the clinical usefulness of nucleic acid therapy.
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http://dx.doi.org/10.1038/mt.2016.81DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088761PMC
August 2016

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model.

Mol Ther Nucleic Acids 2015 Oct 20;4:e258. Epub 2015 Oct 20.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.
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http://dx.doi.org/10.1038/mtna.2015.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881762PMC
October 2015

Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells.

Mol Ther 2013 Jan 2;21(1):185-91. Epub 2012 Oct 2.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.
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http://dx.doi.org/10.1038/mt.2012.180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538304PMC
January 2013

TLS-CHOP represses miR-486 expression, inducing upregulation of a metastasis regulator PAI-1 in human myxoid liposarcoma.

Biochem Biophys Res Commun 2012 Oct 18;427(2):355-60. Epub 2012 Sep 18.

Department of Molecular Pathology, Tokyo Medical University, Tokyo 160-8402, Japan.

Myxoid liposarcomas (MLSs) are characterized by t(12;16)(q13;p11) translocation and expression of TLS-CHOP chimeric oncoprotein. However, the molecular functions of TLS-CHOP have not been fully understood. On the other hand, microRNAs (miRNAs) comprise an abundant class of endogenous small non-coding RNAs that negatively regulate the expression of their target genes, and are involved in many biological processes. It is now evident that dysregulation of miRNAs is an important step in the development of many cancers. To our knowledge, however, there have been no reports of the miRNAs involved in MLS tumorigenesis and development. In this study, we have found that miR-486 expression was repressed in TLS-CHOP-expressed NIH3T3 fibroblasts and MLS tissues, and exogenous overexpression of miR-486 repressed growth of MLS cells. Thus, downregulation of miR-486 may be an important process for MLS. In addition, we have identified plasminogen activator inhibitor-1 (PAI-1) as a novel target gene of miR-486. PAI-1 is a unique type of serine protease inhibitor and is known to be one of the key regulators of tumor invasion and metastasis. Furthermore, knockdown of PAI-1 by a specific small interfering RNA (siRNA) inhibited growth of MLS cells, suggesting that increased expression of PAI-1 by miR-486 repression is critical for survival of MLS cells. Collectively, these results suggest a novel essential molecular mechanism that TLS-CHOP activates PAI-1 expression by repression of miR-486 expression in MLS tumorigenesis and development.
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http://dx.doi.org/10.1016/j.bbrc.2012.09.063DOI Listing
October 2012

Isolation of miRNAs that target EGFR mRNA in human lung cancer.

Biochem Biophys Res Commun 2012 Apr 9;420(2):411-6. Epub 2012 Mar 9.

Department of Surgery, Tokyo Medical University, Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.

Lung cancer, predominantly non-small cell lung cancer (NSCLC), remains the leading cause of cancer-related deaths worldwide. Although epidermal growth factor receptor (EGFR) signaling is important and well studied with respect to NSCLC progression, little is known about how miRNAs mediate EGFR signaling to modulate tumorigenesis. To identify miRNAs that target EGFR, we performed a bioinformatics analysis and found that miR-542-5p down-regulates EGFR mRNA and protein expression in human lung cancer cells (H3255, A549, Hcc827). We observed increases in EGFR association with Ago2 in miR-542-5p-transfected cells. Interestingly, we observed an inverse correlation of miR-542-5p expression and EGFR protein levels in human lung cancer tissue samples, suggesting that miR-542-5p directly targets EGFR mRNA. Furthermore, we found that miR-542-5p inhibited the growth of human lung cancer cells. Our findings suggest that miR-542-5p may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel therapeutic target in lung cancer.
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http://dx.doi.org/10.1016/j.bbrc.2012.03.008DOI Listing
April 2012

Identification of a novel role of Septin 10 in paclitaxel-resistance in cancers through a functional genomics screen.

Cancer Sci 2012 Apr 4;103(4):821-7. Epub 2012 Mar 4.

Departments of Molecular Pathology, Tokyo Medical University, Tokyo, USA.

Paclitaxel (also known as taxol) is a member of the taxane class of anticancer agents, which has a well-known mechanism that blocks cell mitosis and kills tumor cells, that is often used in clinics to treat cancer. However, some carcinomas such as breast, ovarian and non-small-cell lung cancers are often resistant to paclitaxel treatment. In this study, we used a lentiviral siRNA library against the entire human genomes to identify genes associated with sensitivity to paclitaxel. We isolated two paclitaxel-resistant clones carrying the siRNA specific to septin 10 (SEPT10) and to budding uninhibited by benzimidazoles 3. The relation of budding uninhibited by benzimidazoles 3 to paclitaxel sensitivity has already been established, but that of SEPT10 remains unknown. Interestingly, overexpression of SEPT10 increased cells' sensitivity to paclitaxel; we also found that SEPT10 is an important regulator for microtubule stability. Furthermore, we found that paclitaxel-resistant tumors had decreased expression of SEPT10. Thus, SEPT10 may be a novel candidate molecule that acts as a good indicator of paclitaxel-resistant carcinomas.
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http://dx.doi.org/10.1111/j.1349-7006.2012.02221.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659246PMC
April 2012

miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer.

Cancer Sci 2011 Dec 27;102(12):2264-71. Epub 2011 Sep 27.

Third Department of Surgery , Tokyo Medical University, Tokyo, Japan.

MicroRNAs (miRNAs) belong to a class of endogenously expressed non-coding small RNAs that function primarily as gene regulators. Growing evidence suggests that miRNAs play a significant role in tumor development, making them potential biomarkers for cancer diagnosis and prognosis. The miR-17-92 cluster has emerged as an important locus, being highly overexpressed in several cancers in association with cancer development and progression. The miR-17-92 miRNA cluster generates a single polycistronic primary transcript that yields six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. In colon cancer development, the pathophysiologic roles of these transcripts and their targets are largely unknown. In the present study, we performed copy number analyses of the six miRNAs transcribed from the miR-17-92 cluster in colon tumor tissues. We determined that miR-92a was transcribed at higher levels than the other five miRNAs in both adenomas and carcinoma. In addition, miR-92a directly targeted the anti-apoptotic molecule BCL-2-interacting mediator of cell death (BIM) in colon cancer tissues. An anti-miR-92a antagomir induced apoptosis of colon cancer-derived cell lines. These data indicate that miR-92a plays a pivotal role in the development of colorectal carcinoma.
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http://dx.doi.org/10.1111/j.1349-7006.2011.02081.xDOI Listing
December 2011

Anthocyanidins-enriched bilberry extracts inhibit 3T3-L1 adipocyte differentiation via the insulin pathway.

Nutr Metab (Lond) 2011 Mar 8;8:14. Epub 2011 Mar 8.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

Background: Obesity and metabolic syndrome are important public concerns, and there is increasing demand for effective therapeutic strategies. Flavonoids are expected to improve the risk factors associated with metabolic syndrome. Anthocyanidins are a kind of flavonoids; well known for their anti-oxidative, anti-inflammatory and anti-tumor properties. However, their effects on adipocytes and molecular systems are not well defined. In this study, we examined the effects of anthocyanidins-enriched bilberry extracts on adipocyte differentiation.

Methods: Utilizing 3T3-L1 cell line, we investigated that bilberry extracts and anthocyanidins induced inhibition of lipid accumulation during adipogenesis. To identify what is the most important bilberry mediated-effect, we analyzed the expressions of key transcriptional factors associated with adipocyte differentiation by Real Time (RT)-PCR. From the results of RT-PCR, we hypothesized that bilberry extracts and anthocyanidins blocks insulin signal, we determined the phosphorylation of tyrosine residues of insulin receptor substrate 1 (IRS1) protein by western blotting analysis. In addition, we compared the whole-genome expression profiles of early stage of adipocyte differentiation under four different growth conditions (DMSO, bilberry, two anthocyanidins) by microarray analyses and Gene Set Enrichment Analysis (GSEA).

Results: Exposure to bilberry extracts and anthocyanidins during adipocyte differentiation inhibited 3T3-L1 differentiation. During this period, bilberry extracts and anthocyanidin significantly decreased a key adipocyte differentiation-associated marker, peroxisome proliferator-activated receptor- γ (Ppar γ ) and sterol regulatory element-binding protein 1c (Srebp1c). Western blotting analysis showed that bilberry extracts and anthocyanidin decreased the phosphorylation of tyrosine residues of IRS1. In addition, microarray experiments and GSEA data revealed significantly altered expression of the known genes of the insulin pathway in cells treated with bilberry extracts or anthocyanidins in the early differentiation stages.

Conclusions: Our data demonstrate that anthocyanidin enriched bilberry extracts strongly inhibit the adipocyte differentiation via the insulin pathway. Furthermore, bilberry extracts might be used as a potential complementary treatment for the obese patients with metabolic syndrome.
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http://dx.doi.org/10.1186/1743-7075-8-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063807PMC
March 2011

Deregulation of miR-92a expression is implicated in hepatocellular carcinoma development.

Pathol Int 2010 May;60(5):351-7

Third Department of Surgery, Tokyo Medical University, Tokyo, Japan.

MicroRNAs (miRNAs) belong to a class of the endogenously expressed non-coding small RNAs which primarily function as gene regulators. Growing evidence suggests that miRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. The miR-17-92 cluster especially is markedly overexpressed in several cancers, and is associated with the cancer development and progression. In this study, we have demonstrated that miR-92a is highly expressed in hepatocellular carcinoma (HCC). In addition, the proliferation of HCC-derived cell lines was enhanced by miR-92a and inhibited by the anti-miR-92a antagomir. On the other hand, we have found that the relative amount of miR-92a in the plasmas from HCC patients is decreased compared with that from the healthy donors. Interestingly, the amount of miR-92a was elevated after surgical treatment. Thus, although the physiological significance of the decrease of miR-92a in plasma is still unknown, deregulation of miR-92 expression in cells and plasma should be implicated in the development of HCC.
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http://dx.doi.org/10.1111/j.1440-1827.2010.02526.xDOI Listing
May 2010

USP15 plays an essential role for caspase-3 activation during Paclitaxel-induced apoptosis.

Biochem Biophys Res Commun 2009 Oct 8;388(2):366-71. Epub 2009 Aug 8.

Department of Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.

Paclitaxel (also known as Taxol) is a well-known anticancer agent that blocks cell mitosis and kills tumor cells, and is often used in clinic to treat cancers. Despite the success of Paclitaxel, the development of drug resistance prevents its clinical applicability. Here, we screened an siRNA library against the entire human genomes using HeLa cells, and have find that lack of USP15 (ubiquitin-specific protease 15) causes Paclitaxel resistance. We also observed the decreased expression of USP15 in Paclitaxel-resistant human ovarian cancer samples. In addition, we have demonstrated that USP15 plays an essential role for stability and activity of caspase-3 during Paclitaxel-induced apoptosis. Thus, USP15 may be a candidate diagnostic marker and therapeutic target for Paclitaxel-resistant cancers.
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http://dx.doi.org/10.1016/j.bbrc.2009.08.015DOI Listing
October 2009

Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients.

PLoS One 2009 14;4(5):e5532. Epub 2009 May 14.

Department of Pathology, Tokyo Medical University, Tokyo, Japan.

Background: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma.

Methodology/principal Findings: We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body.

Conclusions/significance: The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005532PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678255PMC
August 2009

Heterochromatin protein 1gamma epigenetically regulates cell differentiation and exhibits potential as a therapeutic target for various types of cancers.

Am J Pathol 2009 Jan 4;174(1):309-16. Epub 2008 Dec 4.

Department of Pathology, Tokyo Medical University, Tokyo, Japan.

Heterochromatin protein 1 (HP1) is a chromosomal protein that participates in both chromatin packaging and gene silencing. Three HP1 isoforms (alpha, beta, and gamma) occur in mammals, but their functional differences are still incompletely understood. In this study, we found that HP1gamma levels are decreased during adipocyte differentiation, whereas HP1alpha and beta levels are expressed constitutively during adipogenesis in cultured preadipocyte cells. In addition, ectopic overexpression of HP1gamma inhibited adipogenesis. Furthermore, we did not detect any HP1gamma protein in the differentiated cells of various normal human tissues. These results suggest that the loss of HP1gamma is required for cell differentiation to occur. On the other hand, the methylation levels of lysine 20 (K20) on histone H4 showed a significant correlation with HP1gamma expression in both these preadipocyte cells and normal tissue samples. However, all cancer tissues examined were positive for HP1gamma but were often negative for trimethylated histone H4 K20. Thus, a dissociation of the correlation between HP1gamma expression and histone H4 K20 trimethylation may reflect the malfunction of epigenetic control. Finally, suppression of HP1gamma expression restrained cell growth in various cancer-derived cell lines, suggesting that HP1gamma may be an effective target for gene therapy against various human cancers. Taken together, our results demonstrate the novel function of HP1gamma in the epigenetic regulation of both cell differentiation and cancer development.
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http://dx.doi.org/10.2353/ajpath.2009.080148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631343PMC
January 2009

Expression of various types of alternatively spliced WAPL transcripts in human cervical epithelia.

Gene 2008 Oct 10;423(1):57-62. Epub 2008 Jul 10.

Department of Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.

We have previously identified a novel gene WAPL that is inducible by human papillomavirus (HPV) E6 and E7 oncoproteins, and is associated with uterine cervical carcinogenesis. A WAPL splice variant named Friend of EBNA2 (FOE) has also been characterized as a binding partner of the Epstein-Barr virus transformation-related protein EBNA2. On the other hand, recent studies have revealed that WAPL is a cohesin-binding protein and promotes sister-chromatid resolution in mitotic prophase. These data demonstrate that WAPL plays an important role in tumorigenesis and cell cycle progression. In this study, we have isolated a large number of additional alternatively spliced WAPL variants from various cervical epithelia. Each variant consists of a variable 5'-terminal region and the conserved remainder. In addition, we have confirmed the genomic organization of the 5'-region of the WAPL gene, and have investigated the characteristic features of the WAPL variants and their products. Furthermore, we have determined the HPV types of the expressed E6/E7 transcripts in the cervical epithelia with a novel PCR protocol. These results should shed light on a novel aspect of WAPL function.
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http://dx.doi.org/10.1016/j.gene.2008.07.001DOI Listing
October 2008

Dioxin interferes in chromosomal positioning through the aryl hydrocarbon receptor.

Biochem Biophys Res Commun 2008 Sep 18;374(2):361-4. Epub 2008 Jul 18.

Department of Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.

Each chromosome occupies its own-specific space called a 'territory' within the interphase nucleus, and the arrangement of chromosome territories (CTs) is important in epigenetic mechanisms. The molecular mechanism to determine the positioning of CTs, however, remains unknown. On the other hand, dioxin is known to be the typical environmental pollutant that affects a wide variety of biological events in many species. Here, we show that dioxin enlarges the minimum distance between chromosome 12 and chromosome 16 territories in human preadipocyte cells, and the alteration of chromosome positioning is canceled by an aryl hydrocarbon receptor (AhR) antagonist alpha-naphthoflavone. Thus, AhR may be a key molecule to regulate chromosome positioning. Our results suggest a novel effect of dioxin toxicity, and demonstrate a clue to reveal the novel molecular mechanism for the arrangement of CTs.
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http://dx.doi.org/10.1016/j.bbrc.2008.07.044DOI Listing
September 2008

Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas.

Am J Surg Pathol 2006 Mar;30(3):351-6

Department of Pathology, Tokyo Medical University, Tokyo, Japan.

The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.
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http://dx.doi.org/10.1097/01.pas.0000194043.01104.ebDOI Listing
March 2006

Detection of fusion genes in sarcomas using paraffin-embedded tissues.

Neuropathology 2005 Sep;25(3):263-8

Department of Pathology, Tokyo Medical University, Tokyo, Japan.

Many sarcomas are characterized by specific recurrent chromosomal translocations resulting in gene fusions. The genes involved in almost all of these translocations have been cloned, greatly changing sarcoma diagnosis. At the biological level, these chromosomal translocations produce highly specific fusion genes that encode key molecules for tumor development. The clinical correlation between these translocation-derived genetic markers and discrete histopathological entities has been remarkable. Today, detection of fusion genes plays a crucial role in the diagnosis of sarcomas that harbor atypical clinical or pathological presentations. The focus of this brief review is the recent impact that cytogenetic and molecular detection of these translocations has had on sarcoma diagnosis using paraffin-embedded sections.
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http://dx.doi.org/10.1111/j.1440-1789.2005.00642.xDOI Listing
September 2005
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