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1.  A pivotal role of Krüppel-like factor 5 in regulation of cancer stem-like cells in hepatocellular carcinoma 
Cancer Biology & Therapy  2015;16(10):1453-1461.
In hepatocellular carcinoma (HCC), there exists a highly tumorigenic subset of cells defined by high expression of CD44 and CD133 that has been reported to contain cancer stem-like cells (CSCs). Krüppel-like factor 5 (KLF5) regulates many factors involved in cell cycle, migration, inflammation, angiogenesis and stemness and has cancer-promoting effects in some cancers. While some reports have indicated that KLF5 may have important roles in regulation of CSCs, what role, if any, KLF5 plays in regulation of CSCs in HCC remains to be elucidated. Flow cytometric analysis of CD44 and CD133 in HCC cell lines revealed subpopulations of CD44High/CD133High and CD44Low/CD133Low cells. We subsequently sorted these subpopulations and identified KLF5 as a gene that is significantly upregulated in CD44High/CD44High cells via RNA sequencing using next generation sequencing technology. Moreover, KLF5 overexpression enriched the CD44High/CD133High subpopulation and, consistent with the up-regulation of CD44High/CD133High cells, KLF5 overexpressing cells were more resistant to anti-cancer drugs and displayed enhanced colony-formation capacity. By contrast, knock-down of KLF5 by siRNA diminished the CD44High/CD133High subpopulation. When KLF5 was acetylated by TGF-β1, the KLF5-mediated CD44High/CD133High subpopulation enrichment was abrogated. Oppositely, ectopic expression of an acetylation-deficient KLF5 mutant further increased CD44High/CD133High subpopulations as compared to cell expressing wild-type KLF5. These findings provide novel mechanistic insight into a pivotal role for KLF5 in the regulation of CSCs in HCC.
PMCID: PMC4846134  PMID: 26176896
acetylation; cancer stem cell; CD44; CD133; hepatocellular carcinoma; KLF5
2.  Fluorescent imaging of superficial head and neck squamous cell carcinoma using a γ-glutamyltranspeptidase-activated targeting agent: a pilot study 
BMC Cancer  2016;16:411.
Detecting superficial head and neck squamous cell carcinoma (HNSCC) by endoscopy is challenging because of limited morphological hallmarks, and iodine cannot be applied to head and neck lesions due to severe mucosal irritation. γ-glutamyltranspeptidase (GGT), a cell surface enzyme, is overexpressed in several cancers, and it has been reported that γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), a fluorescent targeting agent which can be enzymatically activated and becomes fluorescent after cleavage of a GGT-specific sequence, can be activated within a few minutes after application to animal models. We investigated whether early HNSCC can be detected by applying gGlu-HMRG to clinical samples.
gGlu-HMRG was applied to four HNSCC cell lines, and fluorescence was observed by fluorescence microscopy and flow cytometry. Immunohistological examination was performed in three recent cases of endoscopic submucosal dissection (ESD) to investigate GGT expression. Fluorescence imaging with gGlu-HMRG in eight clinical samples resected by ESD or surgery was performed, and fluorescence intensity of tumor and normal mucosa regions of interest (ROI) was prospectively measured.
All four gGlu-HMRG-applied cell lines emitted green fluorescence. Immunohistological examination demonstrated that GGT was highly expressed in HNSCC of the recent three ESD cases but barely in the normal mucosa. Fluorescence imaging showed that iodine-voiding lesions became fluorescent within a few minutes after application of gGlu-HMRG in all eight resected tumors. Tumor ROI fluorescence intensity was significantly higher than in the normal mucosa five minutes after gGlu-HMRG application.
Fluorescence imaging with gGlu-HMRG would be useful for early detection of HNSCC.
PMCID: PMC4936014  PMID: 27387955
Fluorescent imaging; γ-Glutamyltranspeptidase; Head and neck squamous cell carcinoma
3.  EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition 
Cancer Biology & Therapy  2015;16(6):933-940.
There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.
PMCID: PMC4623069  PMID: 25897987
cancer stem cell; esophageal squamous cell carcinoma; epithelial-mesenchymal transition; EGFR inhibitor; organotypic 3D culture; ZEB1
4.  Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression 
Oncogene  2015;34(41):5229-5239.
Epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44Low-CD24High (CD44L) epithelial phenotype to a CD44High-CD24-/Low (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT while excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference (RNAi) and flow cytometric approaches, we find that transforming growth factor (TGF)-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. This data provides novel mechanistic insights into the dynamic expression of SOD2 during EMT. Additionally, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.
PMCID: PMC4530096  PMID: 25659582
SOD2; MnSOD; epithelial-mesenchymal transition; esophageal squamous cell carcinoma; reactive oxygen species; CD44
5.  Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4 
Cell Cycle  2015;13(24):3857-3866.
Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.
PMCID: PMC4614677  PMID: 25558829
Barrett's esophagus; KLF4; metaplasia; Notch signaling; transdifferentiation
6.  Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities 
Oncogene  2014;34(18):2347-2359.
Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference (RNAi) experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence, but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as TGF-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.
PMCID: PMC4268095  PMID: 24931169
Notch; Rb; p16; HPV; E7; senescence; squamous cell carcinoma
7.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
8.  Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture 
Nature protocols  2012;7(2):235-246.
This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.
PMCID: PMC3505594  PMID: 22240585
9.  A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors 
Cancer research  2011;71(21):6836-6847.
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1 enriched EMT competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage independent growth and tumor formation in nude mice. RNA interference (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of squamous esophageal cancers.
PMCID: PMC3206139  PMID: 21890822
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
10.  Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma 
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas. Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice. In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the development of a tumor-promoting inflammatory microenvironment.
PMCID: PMC3410579  PMID: 22860235
Esophageal squamous cell carcinoma; organotypic 3D culture; EGFR; P53; cyclin D1; Wnt; Notch; squamous-cell differentiation; invasion; 4-Nitroquinoline 1-oxide
11.  NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network 
Gastroenterology  2010;139(6):2113-2123.
Background & Aims
The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation and proliferation in the hematopoietic system, the intestine, the pancreas and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology remain unknown.
Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic three-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation (ChIP) assays, quantitative reverse-transcription polymerase chain reactions (RT-PCR), Western blotting, immunofluorescence and immunohistochemistry.
NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia.
Notch signaling is important for esophageal epithelial homeostasis. In particular, the crosstalk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
PMCID: PMC2997138  PMID: 20801121
NOTCH1; NOTCH3; esophageal epithelium; squamous differentiation
12.  Insulin-like growth factor-binding protein-3 promotes transforming growth factor-β1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells 
Carcinogenesis  2010;31(8):1344-1353.
Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2–hTERT–EGFR–p53R175H. In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-β target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-β1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-β1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3I56G/L80G/L81G, a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-β1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3I56G/L80G/L81G also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-β1-mediated EMT and cell invasion.
PMCID: PMC2915630  PMID: 20513670
13.  EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors 
Cancer research  2010;70(10):4174-4184.
Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive as to which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-β. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to enrichment of an EMT-competent cellular subpopulation amongst telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by induction of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT upon TGF-β stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in induction of p15INK4B and p16INK4A, reactivating the EGFR-dependent senescence program. Importantly, TGF-β-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.
PMCID: PMC3007622  PMID: 20424117
EGFR; EMT; senescence; ZEB1; ZEB2
14.  Hypoxia activates the cyclooxygenase-2–prostaglandin E synthase axis 
Carcinogenesis  2009;31(3):427-434.
Hypoxia-inducible factors (HIFs), in particular HIF-1α, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1α target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1α by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1α. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E2 (PGE2) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE2 production in a HIF-1α-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1α and IGFBP3. Activation of the COX-2–PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1β and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1α target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.
PMCID: PMC2832548  PMID: 20042640
15.  Sensitive Assay for Quantification of Hepatitis B Virus Mutants by Use of a Minor Groove Binder Probe and Peptide Nucleic Acids ▿ †  
Journal of Clinical Microbiology  2010;48(12):4487-4494.
Lamivudine is the first nucleoside analogue that was shown to have a potent effect on hepatitis B virus (HBV). However, the emergence of mutants resistant or cross-resistant to nucleoside/nucleotide analogues remains a serious problem. Several assays for the detection and quantification of antiviral-resistant mutants have been reported, but it has been difficult to measure the amounts of mutants accurately, especially when the target strain is a minor component of the mixed population. It has been shown that accurate measurement of a minor strain is difficult as long as a matching reaction with a single probe is included in the assay. We developed a new method for the quantification of lamivudine-resistant strains in a mixed-virus population by real-time PCR using minor groove binder probes and peptide nucleic acids, and we achieved a wide and measurable range, from 3 to 10 log10 copies/ml, and high sensitivity, with a discriminative limit of 0.01% of the predominant strain. The clinical significance of measuring substitutions not only of M204 but also of L180 residues of HBV polymerase was demonstrated by this method. This assay increases the versatility of a sensitive method for the quantification of a single-nucleotide mutation in a heterogeneous population.
PMCID: PMC3008448  PMID: 20926703

Results 1-15 (15)