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Identification of reliable markers of radiosensitivity and the key molecules that donate susceptibility to anticancer treatments to esophageal cancer cells would be highly desirable. We found that the m RNA expression of insulin-like growth factor 2 m RNA-binding protein 3 ( IGF2BP3) was higher in radioresistant TE-5 and TE-9 cells than in radiosensitive TE-12 clone A1 cells. Conversely, knocking down expression of IGF2BP3 m RNA in TE-5 and TE-9 cells using small interfering RNA significantly enhanced their radiosensitivity. Furthermore, patients with squamous cell esophageal cancers strongly expressing IGF2BP3 tended to respond poorly to chemoradiation. These data suggest that IGF2BP3 may be a key marker of radiosensitivity that diminishes the susceptibility of squamous cell esophageal cancer cells to radiotherapy. IGF2BP3 may, thus, be a useful target for improving radiotherapy for patients with esophageal squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Abstract Background Transforming growth factor (TGF)-β is known to be produced by progressor tumors and to immobilize dendritic cells (DCs) within those tumors. Moreover, although TGF-β1 has been shown to promote tumor progression, there is still no direct, in vivo evidence as to whether TGF-β1 is able to directly induce distant metastasis. Methods To address that issue and investigate the mechanism by which TGF-β1 suppresses DC activity, we subdermally inoculated mouse ears with squamous cell carcinoma cells stably expressing TGF-β1 or empty vector (mock). Results The numbers of DCs within lymph nodes draining the resultant TGF-β1-expressing tumors was significantly lower than within nodes draining tumors not expressing TGF-β1. We then injected fluorescently labeled bone marrow-derived dendritic cells into the tumors, and subsequent analysis confirmed that the tumors were the source of the DCs within the tumor-draining lymph nodes, and that there were significantly fewer immature DCs within the nodes draining TGF-β1-expressing tumors than within nodes draining tumors not expressing TGF-β1. In addition, 14 days after tumor cell inoculation, lymph node metastasis occurred more frequently in mice inoculated with TGF-β1 transfectants than in those inoculated with the mock transfectants. Conclusions These findings provide new evidence that tumor-derived TGF-β1 inhibits migration of DCs from tumors to their draining lymph nodes, and this immunosuppressive effect of TGF-β1 increases the likelihood of metastasis in the affected nodes.

Background: Large non-apoptotic vesicles released from the plasma membrane protrusions are classified as large-EVs (LEVs). However, the triggers of LEV secretion and their functions in tumors remain unknown., Methods: Coculture system of cancer cells, peritoneal mesothelial cells (PMCs), and macrophages (MΦs) was conducted to observe cell-cell contact-mediated LEV secretion. Lineage tracing of PMCs was performed using Wt1 CreERT2 -tdT nu mice to explore the effects of LEVs on PMCs in vivo, and lymphangiogenesis was assessed by qRT-PCR and flow-cytometry., Results: In peritoneal dissemination, cancer cells expressing Ephrin-B (EFNB) secreted LEVs upon the contact with PMCs expressing ephrin type-B (EphB) receptors, which degraded mesothelial barrier by augmenting mesothelial-mesenchymal transition. LEVs were incorporated in subpleural MΦs, and these MΦs transdifferentiated into lymphatic endothelial cells (LEC) and integrated into the lymphatic vessels. LEC differentiation was also induced in PMCs by interacting with LEV-treated MΦs, which promoted lymphangiogenesis. Mechanistically, activation of RhoA-ROCK pathway through EFNB reverse signaling induced LEV secretion. EFNBs on LEVs activated EphB forward signaling in PMC and MΦs, activating Akt, ERK and TGF-β1 pathway, which were indispensable for causing MMT and LEC differentiation. LEVs accelerated peritoneal dissemination and lymphatic invasions by cancer cells. Blocking of EFNBs on LEVs using EphB-Fc-fusion protein attenuated these events., Conclusions: EFNB high cancer cells scattered LEVs when they attached to PMCs, which augmented the local reactions of PMC and MΦ (MMT and lymphangiogenesis) and exaggerated peritoneal dissemination., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The nature of microRNA (miRNA) dysfunction in carcinogenesis remains controversial because of the complex connection between miRNA structural diversity and biological processes. Here, we found that oncofetal IGF2BP3 regulates the selective production of a subset of 3'-isoforms (3'-isomiRs), including miR-21-5p and Let-7 family, which induces significant changes in their cellular seed occupancy and structural components, establishing a cancer-specific gene expression profile. The D-score, reflecting dominant production of a representative miR-21-5p+C (a 3'-isomiR), discriminated between clinical early-stage lung adenocarcinoma (LUAD) cases with low and high recurrence risks, and was associated with molecular features of cell cycle progression, epithelial-mesenchymal transition pressure, and immune evasion. We found that IGF2BP3 controls the production of miR-21-5p+C by directing the nuclear Drosha complex to select the cleavage site. IGF2BP3 was also involved in the production of 3'-isomiRs of miR-425-5p and miR-454-3p. IGF2BP3-regulated these three miRNAs are suggested to be associated with the regulation of p53, TGF-β, and TNF pathways in LUAD. Knockdown of IGF2BP3 also induced a selective upregulation of Let-7 3'-isomiRs, leading to increased cellular Let-7 seed occupancy and broad repression of its target genes encoding cell cycle regulators. The D-score is an index that reflects this cellular situation. Our results suggest that the aberrant regulation of miRNA structural diversity is a critical component for controlling cellular networks, thus supporting the establishment of a malignant gene expression profile in early stage LUAD., Competing Interests: Competing interests statement:The authors declare no competing interest.

Purpose: IDH-wildtype (IDHwt) diffuse gliomas are treated as glioblastoma, however, some of these may show less aggressive clinical courses. The authors investigated the clinical, histopathological, and molecular characteristics of such IDHwt indolent diffuse gliomas (iDGwt), which have not been well documented in the literature., Methods: Adult patients with IDHwt gliomas admitted between 2011 and 2020 were surveyed. In this particular study, the clinical indolence was defined mainly as having a small enhancing lesion and a stable period for more than 1 month before surgery. The current WHO diagnostic criteria were adapted for the diagnoses. Gene mutations and copy number changes in 43 representative glioma-associated genes, MGMT promoter methylation status, and survival data were compared with those of The Cancer Genome Atlas reference cohort., Results: Nine out of 180 surveyed cases (5.0%) fulfilled the present criteria of the iDGwt. Considering the representative regulatory pathways, 8 (88.9%), 4 (44.4%), and 1 (11.1%) case had genetic alterations in the PI3K/MAPK, TP53, and RB pathways, respectively. The frequency of the RB pathway alteration was significantly lower than that in the reference cohort (281 of 362 cases: 77.6%). Two cases (22.2%) showing EGFR amplification met the diagnostic criteria for glioblastoma, and the frequency was significantly lower than that in the reference cohort (412 of 426 cases: 96.7%). The overall survival (median: 37.5 months) in the present series was significantly longer than that in the reference cohort (n = 426, median: 13.9 months)., Conclusions: iDGwt lacked the molecular features of glioblastoma except for the PI3K/MAPK pathway alteration., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The mechanism underlying cancer cell metastasis from the tumor to regional lymph nodes is not yet fully understood. We hypothesized that peritumoral neutrophil accumulation promotes regional lymph node metastasis in thoracic esophageal squamous cell cancer., Methods: Between 2010 and 2019, 126 thoracic esophageal squamous cell cancer patients received curative (R0) esophagectomy without preoperative treatment in our hospital. Using paraffin-embedded resected tumors, we performed immunohistochemical analysis of CD16b-positive neutrophil accumulation in the peritumoral area, which was defined as a 1-mm region centered on the border separating the malignant cell nests from the host tissue. The relationship between the density of peritumoral CD16b staining and pathological lymph node metastasis or 5-year overall survival was evaluated., Results: Although the clinicopathological characteristics of CD16b-high and CD16b-low patients did not differ, greater pathological lymph node metastasis (P < .001) and lymphatic invasion by the tumor (P = .024) and a poorer 5-year survival (P = .010) were seen in CD16b-high patients. Moreover, CD16b-positive neutrophil density was generally higher in the peritumoral area than within the tumor itself. Univariate and multivariate analyses showed that CD16b-positive neutrophil accumulation was an independent factor for lymph node metastasis with an odds ratio >25 (P < .001). On the other hand, blood neutrophil counts did not correlate with lymph node metastasis., Conclusion: Peritumoral accumulation of CD16b-positive neutrophils is an independent factor strongly correlated with lymph node metastasis in thoracic esophageal squamous cell cancer., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Eosinophils are tissue-dwelling immune cells. Accumulating evidence indicates that a type of cell death termed ETosis is an important cell fate involved in the pathophysiology of inflammatory diseases. Although the critical role of eosinophils in eosinophilic granulomatosis with polyangiitis (EGPA; formerly Churg-Strauss syndrome) is well established, the presence of eosinophil ETosis (EETosis) is poorly understood. We undertook this study to better understand the characteristics of EETosis., Methods: In vitro studies using blood-derived eosinophils were conducted to characterize EETosis. The occurrence of EETosis in tissues from patients with EGPA was studied by immunostaining and electron microscopy. Serum concentrations of eosinophil-derived proteins in healthy controls, patients with asthma, and EGPA patients with active disease or with disease in remission (n = 15 per group) were examined., Results: EETosis was reliant on reactive oxygen species and peptidylarginine deiminase type 4-dependent histone citrullination, resulting in the cytolytic release of net-like eosinophil extracellular traps, free galectin-10, and membrane-bound intact granules. The signature of EETosis, including loss of cytoplasmic galectin-10 and deposition of granules, was observed in eosinophils infiltrating various tissues from EGPA patients. Serum eosinophil granule proteins and galectin-10 levels were increased in EGPA and positively correlated with disease activity as assessed by the Birmingham Vasculitis Activity Score (r = 0.8531, P < 0.0001 for galectin-10). When normalized to blood eosinophil counts, this correlation remained for galectin-10 (r = 0.7168, P < 0.0001) but not for granule proteins. Galectin-10 levels in active EGPA positively correlated with serum interleukin-5 levels., Conclusion: Eosinophils infiltrating diseased tissues in EGPA undergo EETosis. Considering the exclusive expression and large pool of cytoplasmic galectin-10 in eosinophils, elevated serum galectin-10 levels in patients with EGPA might reflect the systemic occurrence of cytolytic EETosis., (© 2021, American College of Rheumatology.)

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Pharmacological interventions to enhance fibrinolysis are effective for treating thrombotic disorders. Utilizing the in vitro U937 cell line-based fibrin degradation assay, we had previously found a cyclic pentapeptide malformin A 1 (MA 1 ) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA 1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA 1 -induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA 1 showed that MA 1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA 1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA 1 -induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA 1 -enhanced fibrinolysis in U937 cells. Synthetic active MA 1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA 1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA 1 -induced phosphorylation of RSK1 and ERK1/2, indicating that MA 1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA 1 upregulated the expression of urokinase-type plasminogen activator (uPA) and increased uPA secretion. These inductions were abrogated in RSK1 knockout cells. These results indicate that RSK1 is a key regulator of MA 1 -induced extracellular fibrinolytic activity.

Purpose: To establish whether Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SphK1) contribute to lymph node metastasis in esophageal squamous cell carcinoma., Methods: Immunohistochemical analysis of SphK1 expression was performed using a tissue microarray containing 177 thoracic squamous cell esophageal cancer specimens resected at surgery, to investigate the association between intratumoral SphK1 expression and lymph node metastasis. Serum S1P levels and intratumoral SphK1 mRNA and protein expression were also evaluated in mice with vs. mice without lymph node metastasis in a murine lymph node metastasis model., Results: Among 177 esophageal cancer patients, 127 (72%) were defined as being SphK1-positive. In univariate and multivariate analyses, SphK1 expression status was a significant factor contributing to lymph node metastasis and poorer 5-year overall survival. In the murine lymph node metastasis model, there was no difference in tumor volume or weight between the lymph node metastasis-negative and lymph node metastasis-positive groups. However, levels of SphK1 mRNA and protein and serum S1P levels were all much higher in the metastasis-positive group., Conclusions: S1P/SphK1 may be novel targets for inhibiting lymph node metastasis in esophageal squamous cell carcinoma, and may provide the basis for a therapeutic strategy to suppress lymph node metastasis.

Identification of the key molecules that mediate susceptibility to anticancer treatments would be highly desirable. Based on clinical and cell biological studies, we recently proposed that regenerating gene (REG) Iα may be such a molecule. In the present study, we hypothesized that REG Iα increases radiosensitivity through activation of mitogen-activated protein kinase (MAPK) pathways. To test that idea, we transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and examined its involvement in MAPK signaling and its effect on susceptibility to radiotherapy. We found that REG Iα-expressing cells showed increased expression of c-Jun messenger RNA (mRNA) and phospho-c-Jun protein mediated via the c-Jun N-terminal kinase (JNK) pathway and extracellular signal-regulated kinase (ERK) pathway, as well as increased radiosensitivity. Immunohistochemical analysis confirmed the activation of c-Jun in tumors expressing REG Iα. Collectively, these findings suggest that REG Iα activates c-Jun via the JNK and ERK pathway, thereby enhancing radiosensitivity.

Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 μM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 μM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia., (© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig's epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2'-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21(Cip1) and p27(Kip1) was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.

Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glechoma hederacea L. (Labiatae) has been used in folk medicine to treat various ailments for centuries. We investigated the effects of G. hederacea extract on melanogenesis in B16 melanoma cells. It significantly reduced both the cellular melanin content and tyrosinase activity in a concentration-dependent manner. An MTT assay did not reveal any obvious cytotoxicity. Furthermore, we found that G. hederacea extract decreased tyrosinase and microphthalmia-associated transcription factor protein expression, but did not inhibit tyrosinase-related protein-1 and tyrosinase-related protein-2 expression. RT-PCR analysis indicated that the antimelanogenic effect of G. hederacea extract might be due to inhibition of tyrosinase gene transcription. Moreover, this effect is regulated via suppression of microphthalmia-associated transcription factor protein expression. Our data indicate that G. hederacea extract inhibits melanin synthesis in B16 melanoma cells but is not cytotoxic. Hence it might prove a useful therapeutic agent for treating hyperpigmentation and an effective component of whitening cosmetics.

Zinc is one of the trace elements which induce the proliferation and the differentiation of the osteoblast. In the previous study, we found that zinc ions (Zn(2+) ion)-releasing titanium implants had excellent bone fixation using a rabbit femurs model. In this study, we isolated the Zn(2+) ions (eluted Zn(2+) ion; EZ) released from the implant surface, and evaluated the effect of EZ on the osteogenesis of human bone marrow-derived mesenchymal cells (hBMCs). In the result, it was found that the EZ stimulated cell viability, osteoblast marker gene (type I collagen, osteocalcin (OC), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) expressions and calcium deposition in hBMCs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Both liver epithelial and oval cells are believed to be liver stem cells. We investigated the identification by producing monoclonal antibodies against liver epithelial cells. Monoclonal antibodies against hepatic stem-like cells (HSL cells) have been selected to follow the hepatic stem cells during hepatic regeneration and developmental changes in the liver. Monoclonal antibodies were induced by immunization of BALB/c mice with HSL cells established from the epithelial cells of the adult rat liver. The hybridomas were screened by indirect immunofluorescence staining of HSL cells. We produced a unique monoclonal antibody against HSL cells, MabH, which specifically recognizes liver epithelial cells. MabH did not react with liver parenchymal cells but did react with bile ductule cells under normal conditions in the adult liver. This antibody also reacted with oval cell lines and with the oval cells that appeared during liver regeneration. In addition, fetal liver cells showed immunoreactivity with MabH. Although the level of staining decreased after birth, some cells in the portal area remained highly reactive. These results suggested that liver epithelial cells, oval cells, and fetal liver cells possess a common cell marker of liver stem cells.

We examined age-related changes in the protein expression of carbonic anhydrase III (CAIII) in livers of Long-Evans with a cinnamon-like color (LEC) rats using an agouti color (LEA) rats as controls. The levels of the protein of CAIII in the liver of LEC male rats increased before 20 weeks of age, at the stage of acute hepatitis, and were decreased at 54 weeks of age, while those of CAIII in the liver of LEA male rats were highly expressed at all ages. In the normal LEA rats, CAIII showed sexual dimorphism. The level of CAIII in LEA male rat liver relative to female was four times higher. On the other hand, young LEC rat (at 4-12 weeks) showed a higher protein level of CAIII than LEA rats, and then decreased during development of hepatitis. CAIII mRNA also decreased in the LEC rat liver during hepatocarcinogenesis. The level of CAIII in the tumor region was lower than that in the tumor-free region. Immunohistochemical analysis showed that glutathione S-transferase P (GST-P) was positive and CAIII was negative in the precancerous region. The expression of CAIII was suppressed in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that suppression of CAIII accompanied hepatocarcinogenesis and it is a secondary consequence of the high copper levels in the liver.

Identification of reliable markers of radiosensitivity and the key molecules that enhance the susceptibility of esophageal cancer cells to anticancer treatments would be highly desirable. To identify molecules that confer radiosensitivity to esophageal squamous carcinoma cells, we assessed the radiosensitivities of the TE-5, TE-9 and TE-12 cloneA1 cell lines. TE-12 cloneA1 cells showed significantly greater susceptibility to radiotherapy at 5 and 10Gy than either TE-5 or TE-9 cells. Consistent with that finding, 24h after irradiation (5Gy), TE-12 cloneA1 cells showed higher levels of caspase 3/7 activity than TE-5 or TE-9 cells. When we used DNA microarrays to compare the gene expression profiles of TE-5 and TE-12 cloneA1 cells, we found that the mRNA and protein expression of insulin-like growth factor binding protein 3 (IGFBP3) and Bcl-2-associated athanogene 1 (BAG1) was five or more times higher in TE-12 cloneA1 cells than TE-5 cells. Conversely, knocking down expression of IGFBP3 and BAG1 mRNA in TE-12 cloneA1 cells using small interfering RNA (siRNA) significantly reduced radiosensitivity. These data suggest that IGFBP3 and BAG1 may be key markers of radiosensitivity that enhance the susceptibility of squamous cell esophageal cancer to radiotherapy. IGFBP3 and BAG1 may thus be useful targets for improved and more individualized treatments for patients with esophageal squamous cell carcinoma., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Ialpha and Ibeta and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Ialpha and Ibeta led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-gamma, tumor necrosis factor-alpha, granulocyte-colony stimulating factor or transforming growth factor-beta1. The elevated IL-6 expression seen in REG Ialpha transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.

Contemporary strategies in tooth repair markedly rely on the newest findings on the cellular and biological components of dental development. Among several identified bioactive molecules, neurotrophins were recently proposed to affect tooth germ cell proliferation, differentiation, and cell-extracellular matrix interactions. The present study attempted to explore the effect of nerve growth factor (NGF) on a spontaneously immortalized dental papilla mesenchymal cell line. NGF induced differentiation of odontoblast-lineage cells with subsequent biomineralization in vitro. Here we showed that normalized transcript levels of tissue-specific markers such as DSPP and DMP1 were elevated significantly, indicating cell differentiation and maturation processes. We performed innovative gene expression analysis of TM14, a matricellular protein and novel member of the fibulin family. TM14 expression followed a pattern similar to that of DMP1, which suggests its important role in cell-matrix and intercellular interactions during dentin calcification. Alkaline phosphatase enzyme assay confirmed the extracellular matrix calcifications in NGF-supplemented groups. Thus, NGF was characterized as a potent promoter of mineralization during dentin formation. For the first time, we included TM14 in odontoblast genotype analysis and proved that NGF also promotes in vitro odontoblast differentiation. Collectively, these results highlight the importance of NGF during tooth morphogenesis, as well as urge the elaboration of complex epithelial-mesenchymal tissue cultures, where further elucidation of the signaling factor network could be completed.

Recent progress in cancer biology research has shown that abnormal proliferation in tumor cells can be attributed to aberrations in cell cycle regulation, especially in G₁ phase. During the course of searching for microbial metabolites that affect cell cycle distribution, we have found that simaomicin α, a polycyclic xanthone antibiotic, arrests the cell cycle at G₁ phase. Treatment of T-cell leukemia Jurkat cells with 3 nM simaomicin α induced an increase in the number of cells in G₁ and a decrease in those in G₂–M phase. Cell cycle aberrations induced by simaomicin α were also detected in colon adenocarcinoma HCT15 cells. Simaomicin α had antiproliferative activities in various tumor cell lines with 50% inhibitory concentration values in the range of 0.3–19 nM. Furthermore, simaomicin α induced an increase in cellular caspase-3 activity and DNA fragmentation, indicating that simaomicin α promotes apoptosis. The retinoblastoma protein phosphorylation status of simaomicin α-treated cell lysate was lower than that of control cells, suggesting that the target molecule of simaomicin α is in a pathway upstream of retinoblastoma protein phosphorylation. In the course of evaluating polycyclic xanthone antibiotics structurally related to simaomicin α, we also found that cervinomycin A1 stimulated accumulation of treated cells in G₁ phase. These results indicate that the polycyclic xanthones, including simaomicin α and cervinomycin A1, may be candidate cancer chemotherapeutic agents.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.

Identification of reliable markers of chemo- and radiosensitivity and the key molecules that enhance the susceptibility of squamous esophageal cancer cells to anticancer treatments would be highly desirable. To test whether regenerating gene (REG) I expression enhances chemo- and radiosensitivity in esophageal squamous cell carcinoma cells, we used MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays to compare the chemo- and radiosensitivities of untransfected TE-5 and TE-9 cells with those of cells stably transfected with REG Ialpha and Ibeta. We then used flow cytometry to determine whether REG I expression alters cell cycle progression. No REG I mRNA or protein were detected in untransfected TE-5 and TE-9 cells. Transfection with REG Ialpha and Ibeta led to strong expression of both REG I mRNA and protein in TE-5 and TE-9 cells, which in turn led to significant increases in both chemo- and radiosensitivity. Cell cycle progression was unaffected by REG I expression. REG I thus appears to enhance the chemo- and radiosensitivity of squamous esophageal cancer cells, which suggests that it may be a useful target for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.

Background: A reliable marker of chemoradiosensitivity that would enable appropriate and individualized treatment of thoracic squamous cell esophageal cancer has long been sought. We investigated whether regenerating gene (REG) Ialpha is such a marker., Methods: We assessed expression of REG Ialpha in untreated endoscopic biopsy specimens and examined the correlation between REG Ialpha expression and the clinical responses to definitive chemoradiotherapy and prognosis. We also examined the relationship between REG Ialpha expression in the resected tumor and the prognosis of patients who received esophagectomy for thoracic squamous cell esophageal cancer., Results: Among the 42 patients treated with definitive chemoradiotherapy, 8 of the 23 REG I-positive patients (35%) showed complete responses to chemoradiotherapy, while only one of the 19 REG I-negative patients did so. The survival rate among the REG I-positive patients was significantly better than among the REG I-negative patients. For the 76 patients treated surgically, there was no significant difference in the survival rates among the REG I-positive and REG I-negative patients., Conclusions: REG Ialpha expression in squamous cell esophageal carcinoma may be a reliable marker of chemoradiosensitivity. We anticipate that it will enable us to provide more appropriate and individualized treatment to patients of advanced esophageal squamous cell carcinoma.

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.

To assess the effect of gadolinium (Gd) on the expression of several forms of cytochrome P450 (P450s) and antioxidant enzymes, we treated rats with gadolinium chloride (25 mg as Gd/kg body weight) 4 h after styrene (a multiple P450 inducer) treatment (600 mg/kg). Gd treatment significantly suppressed styrene-inducible cytochrome P4502B1 (CYP2B1), CYP2B2, CYP2E1, and CYP3A2 mRNA expressions to 48.6%, 69.8%, 61.1%, and 38.5%, accompanying with the reduction of proteins expression to 1.42%, 31.2%, 21.1% and 21.1%, respectively, compared with styrene alone treatment. Gd suppressed styrene-inducible CYP1A2 expression, but only at the protein level. On the other hand, styrene treatment caused a decrease in reduced form of glutathione (GSH), as well as increases in lipid peroxide and serum ALT and AST activities, suggesting the occurrence of hepatic damage probably due to styrene-induced oxidative stress in rat liver. Post-treatment of Gd attenuated this styrene-caused hepatic damage. Moreover, mRNA expressions of cellular antioxidant enzymes such as catalase, CuZn-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX) were hardly changed by styrene and/or Gd treatment. In summary, Gd suppressed styrene-inducible expression of not only CYP2B1 but also several forms of P450 at both the mRNA and protein levels, along with attenuation of styrene-caused liver damage. These findings suggested that Gd is a chemo-preventive agent against hepatic damage caused by xenobiotics requiring biotransformation.

Here we report on the spontaneous immortalization upon serial passages of mouse fetal dental papilla cells, which present odontoblast phenotype features. The cells named odontoblast-lineage cell (OLC) produced dentin extracellular matrix proteins, such as DSP and DMP1, and maintained transcripts of various matrix components as osteopontin, BMP-4, procollagen-1, and MEPE. The addition of osteogenic differentiation medium with beta-glycerophosphate and ascorbic acid was effective for inducing calcification and mineralization in vitro in cell cultures for up to 28 days. For the first time, we investigated the expression of Lhx6 and Lhx7 genes during induced biomineralization, since these new members of LIM homeodomain proteins have been recently proposed tracking odontoblastic phenotypes. Our results indicate that beta-glycerophosphate treatment of OLC cultures decreases Lhx6 transcript levels in vitro. Our findings proved odontoblast phenotype-specificity, which demonstrates that this novel odontoblast-lineage cell line is a valuable tool for future experiments in odontology.

Modification of cell surface oligosaccharides by reactive oxygen species (ROS) and the biological effect of such modifications on cell adhesion were investigated. Treatment of HL60, a human promyelocyte leukemia cell line, with ROS, generated by a combination of hypoxanthine and xanthine oxidase (HX/XO), decreased the sialic acid content on the cell surface, as indicated by a flow cytometric analysis involving sialic acid-specific lectins, and a concomitant increase of free sialic acid was observed in the supernatant. A cell adhesion assay showed that the HX/XO treatment of HL60 cells decreases their capability of binding to human umbilical vein endothelial cells (HUVEC), probably because of an impairment of the interaction involving E-selectin, whereas the decrease in the binding was canceled by the addition of superoxide dismutase (SOD) and catalase. In fact, cell surface sialyl lewis x (sLe x), but not lewis x (Le x), was decreased by HX/XO treatment. Thus, it is more likely that the impaired interaction is based on diminished levels of the selectin ligand. Cleavage of sialic acid by ROS was further verified by the degradation of 4MU-Neu5Ac by HX/XO in the presence of hydrogen peroxide and iron ion. These results indicate that glycosidic linkage of sialic acid is a potential target for superoxide and other related ROS. It is well known that ROS cause cellular damages such as lipid peroxidation and protein oxidation, but, as suggested by the findings reported in the literature, ROS may also regulate cell adhesion via the structural alteration of sialylated oligosaccharides on the cell surface.

beta 1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is significantly homologous to both snail beta 1,4GlcNAc transferase and beta1,4Gal transferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT-III, for enzymatic activity. The overexpression of Asp323-substituted, catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of 'dominant negative glycosyltransferase', identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.

We have been successful in generating several lines of transgenic mice and pigs that contain the human beta-d-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galalpha1-3Galbeta1-4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3'-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing beta-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal beta 1-3GalNAc alpha-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO(3)-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1- 4Glc-PA by two-dimensional (1)H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal beta 1-3GlcNAc-R) and type 2 (Gal beta 1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel beta-Gal-3'-sulfotransferase gene family.

The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.

The alpha-Gal epitope (Gal-alpha1-3Gal-beta1-4-GlcNAc-R), which is biosynthesized by the UDP-Gal:alpha1-3-galactosyltransferase (alpha1, 3GT), is highly associated with hyperacute rejection in swine to human xenotransplantation. A variety of strategies have been pursued to reduce or eliminate this epitope from swine tissues. Since swine ES cells are not available at present, the targeted knock out of the alpha1,3GT is restricted. Other strategies, such as enzyme competition of the alpha1,3GT with other glycosyltransferases and/or control of sugar processing by the glycosyltransferases, provide a new insight into the downregulation of the alpha-Gal epitope. This review will focus on this type of strategy, which involves a gene transfection of variety of glycosyltransferases as competitors against alpha1,3GT., (Copyright 1999 Academic Press.)

We previously reported that human lens accumulates gangliosides in association with aging and senile cataract progression. Structural analysis revealed that gangliosides in human cataractous lenses were composed of ganglio-series gangliosides, such as GM3, GM2, GM1 and GD1a, and sialyl-Lewisx-containing neolacto-series gangliosides. Although Lewisx-containing neolacto-series glycolipid was found to accumulate in association with aging and cataract progression, the sialyl-Lewisx gangliosides did not show much accumulation in individual lenses from subjects between 16 and 80 years old. The content of sialyl-Lewisx gangliosides was about two to four times higher than that of Lewisx glycolipids, suggesting the possibility that the increase in Le(x) glycolipid is partly due to the desialylation of sialyl-Le(x) gangliosides. On the other hand, the expression of ganglio-series gangliosides increased in an age-related manner. Thus, age-related changes in lens glycolipids may modify the cell-to-cell interaction induced by cell surface sugar chains, leading to the initiation and progression of cataract.