Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks

<p>Abstract</p> <p>Background</p> <p>An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of <it>CD133 </it>gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express <it>CD133 </it>gene.</p> <p>Results</p> <p>A reporter analysis revealed that P5 promoter, located far upstream in a human <it>CD133 </it>gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of <it>CD133 </it>mRNA expression.</p> <p>Conclusions</p> <p>In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.</p

Efficacy and safety of temporary biliary stent for prevention of post-ERCP cholangitis after endoscopic common bile duct stone removal: a retrospective study

　Although post-endoscopic retrograde cholangiopancreatography (ERCP) cholangitis (PEC) is not as severe as post-ERCP pancreatitis, this complication should not be disregarded. The aim of the present study was to evaluate the efficacy of a temporary biliary stent for prevention of PEC. Between April 2011 and May 2017, 190 patients underwent complete stone removal in a first session of ERCP at our hospital. Using propensity score matching, 72 pairs were enrolled in this study. After common bile duct (CBD) stone removal, the endoscopists decided to insert a temporary biliary stent if necessary. The incident rate of PEC was significantly lower in the stent group than the no-stent group (1% vs. 11%, p = 0.03). The length of hospital stay was also significantly shorter in the stent group than the no-stent group (5 days vs. 7 days, p < 0.01). In the stent group, one case had stent migration into the bile duct and two cases had a mooring stent at the papilla after 1 month. Multivariate analysis identified the pancreatic guide wire technique as a risk factor for PEC. We demonstrated that a temporary biliary stent reduced the incidence of PEC significantly and the outcome of its placement contributed to shortening the hospital stay. Furthermore, the placement of a temporary biliary stent caused fewer adverse effects than expected. Mooring stents were noted in three cases, which were confirmed by plain abdominal X-ray, but the patients had no symptoms. In two cases, the stent remained in the orifice of the papilla, and in one case it migrated into the CBD. All three stents were retrieved by elective endoscopic procedures. In conclusion, a temporary biliary stent can reduce the incidence of PEC and shorten the length of hospital stay without severe adverse outcomes

Two Secreted Proteoglycans, Activators of Urothelial Cell-Cell Adhesion, Negatively Contribute to Bladder Cancer Initiation and Progression.

Osteomodulin (OMD) and proline/arginine-rich end leucine repeat protein (PRELP) are secreted extracellular matrix proteins belonging to the small leucine-rich proteoglycans family. We found that OMD and PRELP were specifically expressed in umbrella cells in bladder epithelia, and their expression levels were dramatically downregulated in all bladder cancers from very early stages and various epithelial cancers. Our in vitro studies including gene expression profiling using bladder cancer cell lines revealed that OMD or PRELP application suppressed the cancer progression by inhibiting TGF-β and EGF pathways, which reversed epithelial-mesenchymal transition (EMT), activated cell-cell adhesion, and inhibited various oncogenic pathways. Furthermore, the overexpression of OMD in bladder cancer cells strongly inhibited the anchorage-independent growth and tumorigenicity in mouse xenograft studies. On the other hand, we found that in the bladder epithelia, the knockout mice of OMD and/or PRELP gene caused partial EMT and a loss of tight junctions of the umbrella cells and resulted in formation of a bladder carcinoma in situ-like structure by spontaneous breakdowns of the umbrella cell layer. Furthermore, the ontological analysis of the expression profiling of an OMD knockout mouse bladder demonstrated very high similarity with those obtained from human bladder cancers. Our data indicate that OMD and PRELP are endogenous inhibitors of cancer initiation and progression by controlling EMT. OMD and/or PRELP may have potential for the treatment of bladder cancer

Combination of shear-wave elastography and liver fibrosis markers predicts severe fibrosis in patients with non-alcoholic steatohepatitis

　非アルコール性脂肪性肝疾患（Non-alcoholic fatty liver disease：NAFLD）の中から予後の悪い線維化が進展した非アルコール性脂肪肝炎（Non-alcoholic steatohepatitis：NASH）を非侵襲的診断法にて拾い上げることが重要である．今回，バイオマーカーやshear wave elastography（以下 SWE）を組み合わせた非侵襲的診断における肝線維化進展症例の診断能の向上について検討を行った．肝生検および SWE を施行し，肝線維化マーカーを測定した NAFLD 患者140名を対象とし，SWE 値と肝線維化マーカーの測定を行い線維化進展例（stage3以上）の診断の拾い上げについて検討した．各種線維化マーカーは stage3-4の線維化進展例で有意に上昇を認め，SWE においてはstage2の段階から上昇し，他の線維化マーカーより早い段階から NASH の線維化の診断ができた．SWE，Ⅳ型コラーゲン7S，WFA+M2BP，P-Ⅲ-P，ヒアルロン酸，FIB4 index における stage3以上の AUC はそれぞれ0.86，0.83，0.79，0.75，0.75，0.77であった．さらに SWE と線維化マーカーを組み合わせたところ，AUC はそれぞれ0.92，0.88，0.86，0.88，0.88で診断能の上昇を認めた．特に SWE とⅣ型コラーゲン7S の診断能が最も優れていた．NASH における SWE は簡便に線維化進展の診断が可能であり，バイオマーカーを組み合わせることで肝線維化診断能が上昇した．以上より線維化の軽度な NASH 症例や非アルコール性脂肪肝（Non-alcoholic fatty liver：NAFL）を識別し，肝生検を減少させる可能性があり，NAFLD の予後の改善に繋がると思われた．　In the recent years, the incidence of nonalcoholic fatty liver disease (NAFLD) is increasing rapidly worldwide. It is important to detect nonalcoholic steatohepatitis (NASH) with a poor prognosis in patients with NAFLD using noninvasive diagnostic methods. Conventional ultrasound (US) is the most common, low-cost technique for NASH diagnosis and improving patient prognosis. We studied the usefulness of US elastography (shear-wave elastography [SWE]) in diagnosing liver fibrosis (LF) with NAFLD and examined the possibility of improving the diagnosis of patients with advanced LF by combining SWE and LF-marker testing. The subjects were 140 patients with NAFLD who underwent liver biopsies, SWE, and LF-marker tests, such as type IV collagen 7S, Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA[+]-M2BP), P-Ⅲ-P, hyaluronic acid, and fibrosis-4 (FIB4) index, at the General Medical Center, Kawasaki Medical School. We evaluated the efficacy of combined SWE and LF-marker tests to diagnose advanced LF (stage ≥3). SWE was performed using 3.75-MHz probes (Canon Aplio 500, JAPAN). There were minimal differences in LF-marker levels for NASH stages 0–2, whereas significantly increased LF-marker levels were observed in patients with advanced LF (stages 3 and 4). SWE showed significantly elevated LF-marker levels at stage 2 compared with stages 0–1, and NASH was detected earlier than other LF markers. The areas under the receiver-operating characteristic curves (AUCs) for SWE, type IV collagen 7S, WFA(+)-M2BP, P-Ⅲ-P, hyaluronic acid, and FIB4 index for stage ≥3 were 0.86, 0.83, 0.79, 0.75, 0.75, and 0.77, respectively. With combined SWE and LF markers, the AUCs increased to 0.92, 0.88, 0.86, 0.88, and 0.88, respectively, showing increased diagnostic ability compared to that of single markers. The diagnostic ability of combined SWE and type IV collagen 7S was superior to that of other combinations. In addition, we detected that most cases were in stage ≥3 on combining SWE and LF markers. SWE for NASH can simply diagnose LF progression; the diagnostic capacity of SWE for LF improves in combination with LF-marker tests. It may be possible to detect the need for liver biopsy and treatment or follow-up, as well as reduce the number of liver biopsies by identifying NAFLD with low LF levels

当院で経験したA 型胃炎の４例

A 型胃炎は稀な疾患で，悪性貧血や胃癌，胃NET の発生母地として知られている．抗胃壁細胞抗体陽性，高ガストリン血症，さらに胃体部を中心とした萎縮性胃炎が診断基準とされている．今回，過去１年に４例のA 型胃炎を診断した．全例で自覚症状は見られなかったが，内視鏡検査での逆萎縮所見からA 型胃炎を疑い，胃生検の病理所見と血液検査で確診した．A 型胃炎が他の自己免疫性疾患に合併することが多いとされているが，本症例にも高齢発症のBasedow 病が１例あり，A 型胃炎は日本でも決してまれな疾患ではないと考えられた．診断には内視鏡所見からA 型胃炎を疑うことが重要で，胃生検や血清ガストリンと抗胃壁細胞抗体の測定を行うことにより確診できる．Type A gastritis is a rare disease and is known as a cause of various conditions including pernicious anaemia, gastric cancer and gastric NETs (Neuroendocrine tumour). The diagnostic criteria of type A gastritis include positive parietal cell antibody, hypergastrinaemia and the presence of atrophic gastritis mainly corpus predominantly atrophic gastritis. We diagnosed four cases of type A gastritis in the past year in our hospital. Although they were all asymptomatic, type A gastritis was suspected by the endoscopic findings (the reverse atrophy) and all confirmed by pathological examination of biopsy specimens and blood test subsequently. It is well known that the patients with autoimmune disease are frequently associated with type A gastritis and there is a case of late onset of Basedow’s disease in our case report. Our study suggests that type A gastritis is not as rare as initially thought in Japan. In order to diagnose type A gastritis, it is important to have a high index of suspicion with endoscopic findings, and to confirm it with gastric biopsy, serum gastrin level and parietal cell antibody

CONTRIBUTION OF CELL SURFACE TO SALT TOLERANCE OF STAPHYLOCOCCUS AUREUS -STUDIES BY FREEZE-ETCHING METHOD-

The role of cell surface in a haloresistant mechanism of S. aureus was analyzed by free-ze-etching method. Our comparative studies were carried out on S. aureus cultivated in 0.5% NaCl containing medium (Normal-Staph) and in 10% NaCl containing medium (10% -Staph), as they were or were treated with L-11 enzyme (cell wall lytic enzyme obtained from Flabobacterium sp.). 1. As shown in photographs of non-treated and enzyme-treated cells, both the cell size and cell wall thickness of 10% -Staph increased to about 1.5 times Normal-Staph. 2. When Normal-Staph was exposed to L-11 enzyme, there occurred a gradual decrease in the OD. In the case of 10% -Staph, the OD showed a down-slope with a shoulder which rose in OD once at about 10 minutes after incubation. 3. The surface pattern of plasma membrane of Normal-Staph appeared to be made up of closely arranged spherical subunits. But in the case of 10% -Staph, the spherical subunits were indistinct and appeared to be sparsely located

A population of BJ fibroblasts escaped from Ras-induced senescence susceptible to transformation

Oncogenic stimuli such as H-Ras induce oncogene-induced senescence (OIS) in fibroblasts to protect against transformation. Here we found that a population of the human diploid fibroblasts can escape from OIS induced by H-RasV12. We designated these OIS-escaped cells as OISEC (OIS-escaped cells). OISEC lost the expression of p16 which plays an important role for cell cycle arrest for induction of senescence, but OISEC preserved the p16 expression machinery and exhibited senescence by the treatment with hydrogen peroxide (H2O2) as stress-induced premature senescence (SIPS). OISEC did not possess anchorage-independent growth potential, and functional disruption of p53 and Rb by SV40 early region encoding large T and small t antigens, induced the aneuploidy phenotype and colony-forming potential of OISEC together with the exhibition of in vivo tumor formation. Finally, we also found that the distinctive feature of OISEC is expression of transcription factors, Oct3/4, SOX2, and Nanog which is closely related to stem-like cell features. This study highlights the presence of a cell population which escaped from OIS, and this OISEC may transform into malignant cancer cells by the additional hits of several genes in vivo