ベスナリノン ニヨル ヒト ダエキセンガン サイボウ ノ ゾウショク ヨクセイ キコウ ノ カイセキ

唾液腺癌は化学療法、放射線療法に抵抗性で局所再発、遠隔転移の多い予後不良の悪性腫瘍である。ベスナリノン は強心剤として開発されたが、その副作用である無顆粒球症発症の原因究明の過程で多くの悪性腫瘍細胞に対して分 化・アポトーシス誘導活性を有すること明らかにされ、唾液腺癌を含む多くの悪性腫瘍に対して分化誘導療法が試 みられている。本研究ではベスナリノン処理によりG1 arrestが誘導されるヒト唾液腺癌細胞(TYS)を用いてベスナ リノンによる細胞増殖抑制作用の分子機構の解明を試みた。 解析は(1)ベスナリノン処理TYS細胞より調製したmRNAを用いanti-sense oriented expression cDNA library を構築し、そのlibraryをTYS細胞にトランスフェクション後、ベスナリノン耐性株を選択し、挿入されている cDNA断片を回収するanti-sense expression cloning 法と、(2)上記libraryをランダムにシークェンスし分化、増殖 に関与すると考えられる遺伝子のペスナリノンによる発現誘導を検索し、up-regulationがあれば、その分子を中心に そのシグナルを上流、下流へと追っていくcandidate gene searching 法にて行った。 その結果、anti-sense expression cloning 法にてribosomal protein L21 cDNAを得た。ribosomal protein L21 anti-sense cDNAを強制発現させた細胞はベスナリノンに対する感受性が低下していたが、その作用はおそらくある 種のタンパク合成の阻害によるものであり、ribosomal protein L21タンパクの特異的な発現抑制がベスナリノン抵抗 性の原因とは考え難かった。一方、candidate gene searching 法でマウスあるいはラットの細胞においてtransforming growth factor-β1 (TGF-β1)あるいはfollicle-stimulating hormone (FSH)により誘導される遺伝子として報告されて いるTGF-β1-stimulated clone 22 (TSC-22) cDNA断片を得た。TSC-22遺伝子はTYS細胞においてベスナリノン 処理にて明らかな発現誘導が認められたため、ヒトTSC-22cDNAの全長のクローニング、構造解析、機背繍析を試 みた。5'-RACE変法でopen reading frameを含むほほ完全長1.6kbのヒトTSC-22cDNAを得た。全塩基配列を決 定したところ予想されるアミノ酸配列はマウス、ラットに98.6%の相同性が認められた。またロイシンジッパー構 造が認められたが、その近傍にDNA結合領域あるいは核移行シグナルが存在しないことより、bZIP転写因子と結 合しその転写活性を抑制するdominant negative 転写制卸因子である可能性が示唆された。TSC-22遺伝子はTYS 細胞において細胞密度に依存しその発現が増加し、ベスナリノンはその発現を更に増強した。タンパク合成阻害剤シ クロヘキシミドを用いた実験によりベスナリノンによるTSC-22遺伝子の発現増強は間接作用であることが示唆され た。次にTSC-22の上流、下流のシグナルを解析する目的でTGF-β1、p21WAF1の発現を検索した。ベスナリノン はTYS細胞においてTGF-β1をわずかに誘導し、p21WAF1を著明に誘導した。またp21WAF1の誘導は直接作用 であることが明らかとなった。更にTGF-β1はp21WAF1、TSC-22の発現を誘導した。次にTSC-22アンチセン スオリゴヌクレオチドのTYS細胞に対する影響を検索すると、対数増殖期には全く影響を与えないが、細胞密度が 上がり増殖を止めるべき時期になると細胞増殖促進作用が認められた。またTSC-22アンチセンスオリゴヌクレオチ ドはベスナリノンによるTYS細胞の増殖抑制作用を阻害した。 以上の結果より、ベスナリノンはTYS細胞においてTGF-β1あるいは他のタンパク質を介したTSC-22遺伝子の 誘導、更に直接作用あるいはTGF-β1を介したp21WAF1の誘導により細胞周期をG1期に止め、細胞増殖抑制作 用を示すことが示唆された

Carrier cell-mediated cell lysis of squamous cell carcinoma by squamous cell carcinoma antigen 1 promoter-driven oncolytic adenovirus

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. We examined the promoter activity of the 5'-flanking proximal region of the SCCA1 gene. Deletion analysis of SCCA1 promoter identified a 175-bp core promoter region and an enhancer region at -525 to -475 bp upstream of the transcription start site. The transcriptional activity of the SCCA1 promoter was up-regulated in squamous cell carcinoma cells, compared with normal keratinocyte, normal non-keratinocyte and adenocarcinoma cells. Five tandem repeats of enhancer increased SCCA1 promoter activity by 4-fold. Oncolytic adenovirus driven by the SCCA1 promoter with 5 tandem repeats of enhancer specifically killed squamous cell carcinoma cells in vitro and in vivo. A549 carrier cells infected with the oncolytic adenovirus induced complete regression of tumor by overcoming immunogenicity and adenovirus-mGM-CSF augmented the antitumor effect of carrier cells. These findings suggest that SCCA1 promoter is a potential target of gene therapy for squamous cell carcinoma

Circulating fibroblast‐like cells in men with metastatic prostate cancer

BACKGROUND Metastatic prostate cancer is an incurable disease. During the development of this disease, prostate cancer cells enter the bloodstream as single cells or clusters of cells. Prostate fibroblasts, a cancer‐promoting cell type in the prostate cancer microenvironment, could in theory incorporate into these migrating cell clusters or follow cancer cells into the bloodstream through holes in the tumor vasculature. Based on this idea, we hypothesized that fibroblast‐like cells, defined here as cytokeratin 8/18/19 − /DAPI + /CD45 − /vimentin + cells, are present in the blood of men with metastatic prostate cancer. METHODS Veridex's CellSearch® system was used to immunomagnetically capture EpCAM + cells and clusters of cells heterogeneous for EpCAM expression from the blood of men with metastatic prostate cancer, localized cancer, and no known cancer, and immunostain them for the presence of cytokeratins 8/18/19, a nucleus, CD45, and vimentin. Fibroblast‐like cells were then quantified. RESULTS Fibroblast‐like cells were present in 58.3% of men with metastatic prostate cancer but not in any men with localized prostate cancer or no known cancer. The presence of these cells correlated with certain known indicators of poor prognosis: ≥5 circulating tumor cells, defined here as cytokeratin 8/18/19 + /DAPI + /CD45 − cells, per 7.5 ml of blood, and a relatively high serum prostate‐specific antigen level of ≥20 ng/ml. CONCLUSIONS The presence of fibroblast‐like cells in the blood may provide prognostic information as well as information about the biology of metastatic prostate cancer. Prostate 73: 176–181, 2013. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95517/1/22553_ftp.pd

CXCR3+ macrophage in Sjögren's syndrome

Background: Mechanisms underlying immune cells' recruitment and activation into the inflammatory lesions of lip salivary glands (LSGs) from primary Sjögren's syndrome (pSS) patients are incompletely understood. Chemokines play pivotal roles in these processes, so we investigated the clinical significance of chemokine receptor CXCR3 and its ligands in the autoimmune lesions of pSS. Methods: We histologically determined the grade of LSG samples from 22 pSS patients and subjected the samples to immunofluorescence analysis to determine the expressions of CXCR3 and its ligands: CXCL9, CXCL10, and CXCL11. To identify the immune cells expressing CXCR3 in the LSGs, we performed double immunofluorescence analysis using antibodies against CD3 (pan-T cells), CD80 (M1 macrophages), CD163 (M2 macrophage), and CD123 (plasmacytoid dendritic cells: pDCs). The relationship between the grade of lymphocytic infiltration and the number of positively stained cells was analyzed by Spearman's rank correlation test. Results: The expressions of CXCL9 and CXCL10 showed particularly intense staining in the LSG samples' ductal cells. The CXCR3 expression was detected mainly in CD80+ and CD163+ macrophages. The number of CXCR3+CD163+ macrophages inversely correlated with the LSG inflammatory lesions' severity (rs= −0.777, p<0.001). Conclusions: Our results suggest that the enhanced production of CXCL9 and CXCL10 from ductal cells results in the CXCR3+ macrophages' migration. There was an inverse correlation between these two parameters: i.e., the number of CXCR3+CD163+ macrophages decreased as the lymphocytic infiltration grade increased. Although CXCR3 is expressed in all of the innate immune cells, CXCR3+CD163+ M2 macrophages may contribute to the anti-inflammatory functions in pSS lesions

Five functional domains associated with gait performance in Parkinson’s disease and lateral trunk flexion

BackgroundLateral trunk flexion (LTF) is a common symptom of Parkinson’s disease (PD). The sensory re-weighting system and sensory-motor function are poor in patients with PD and LTF, and this may cause gait impairment. However, the specific characteristics of gait impairment in patients with PD and LTF remain unclear. The aim of this study was to compare the characteristics of the gait functional domains between participants with PD with and without LTF.MethodsFifty-eight patients with PD and Hoehn–Yahr grade 2–3 LTF were divided into two groups: the LTF group (n = 22) and the No LTF group (n = 36). The Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS)-Part III score and subjective visual vertical (SVV) angle were measured. The participants walked with a motion sensor on a straight 20 m path at a comfortable speed. Fifteen gait variables (10 gait cycles) were evaluated and categorized into pace, rhythm, asymmetry, variability, and postural control functional domains and were compared between groups.ResultsThe LTF angle, SVV angle; MDS-UPDRS-Part III total, rigidity, and axial scores; and the coefficients of variance for step length, step time, and stance time were significantly higher in the LTF group than in the No LTF group. No other significant differences were observed between the groups.ConclusionParticipants with PD and Hoehn–Yahr severity 2–3 LTF had greater gait variability than those without LTF, but maintained similar pace, rhythm, asymmetry, and postural control domains. Patients with PD and LTF may develop abnormal neural networks causing greater gait variability

Imaging Findings of Localized Lymphoid Hyperplasia of the Pancreas: a Case Report

We report here on a case of localized lymphoid hyperplasia of the pancreas in a 70-year-old man which manifested as double lesions (uncinate process and tail) in the organ. The lesions were incidentally detected as hypoechoic lesions on ultrasonography and they appeared as delayed enhancing lesions on the contrast-enhanced dynamic CT and MRI. Total pancreatectomy was performed, because malignant tumor could not be excluded according to the preoperative imaging studies and the endoscopic ultrasound-guided biopsy failed. Pathology revealed localized lymphoid hyperplasia. The patient had an uneventful postoperative course. He has been alive for 18 months after surgery

Distinct Regulation of CXCL10 Production in Salivary Gland Cells

CXCL10, a CXC chemokine induced by interferon-gamma [IFN-γ], has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions. Although CXCL10 is known to be overexpressed in the salivary glands of individuals with primary Sjögren's syndrome (pSS), it is unclear which cells produce CXCL10 under what types of stimulations. Here we investigated the precise molecular mechanisms by which CXCL10 was produced in human salivary gland ductal (NS-SV-DC) and acinar (NS-SV-AC) cell lines. Our results demonstrated that NS-SV-DC cells produced higher levels of CXCL10 compared to NS-SV-AC cells. In addition, our findings demonstrated that the regulator of the enhancement of CXCL10 was different between NS-SV-DC and NS-SV-AC cells; i.e., interferon-gamma (IFN-γ) had more potential than interferon-alpha (IFN-α), tumor necrosis factor (TNF)-α, and interleukin (IL)1-β in the induction of CXCL10 production in NS-SV-DC cells, whereas TNF-α had potential to induce CXCL10 production in NS-SV-AC cells. A Western blot analysis demonstrated that IFN-γ enhanced the production of CXCL10 via both the JAK/STAT1 pathway and the NF-κB pathway in NS-SV-DC cells, whereas TNF-α enhanced the production of CXCL10 via the NF-κB pathway in NS-SV-AC cells. The results of study suggest that the CXCL10 overexpression in the salivary glands is caused mainly by IFN-γ-stimulated salivary gland ductal cells. The enhanced production of CXCL10 by IFN-γ from ductal cells may result in the inflammation of pSS lesions