Publications by authors named "June Ho Shin"

29 Publications

  • Page 1 of 1

Mutations Predict Lung Cancer Radiation Resistance That Can Be Targeted by Glutaminase Inhibition.

Cancer Discov 2020 Dec 18;10(12):1826-1841. Epub 2020 Oct 18.

Department of Radiation Oncology, Stanford University, Stanford, California.

Tumor genotyping is not routinely performed in localized non-small cell lung cancer (NSCLC) due to lack of associations of mutations with outcome. Here, we analyze 232 consecutive patients with localized NSCLC and demonstrate that and mutations are predictive of high rates of local recurrence (LR) after radiotherapy but not surgery. Half of LRs occurred in tumors with mutations, indicating that they are major molecular drivers of clinical radioresistance. Next, we functionally evaluate mutations in our radiotherapy cohort and demonstrate that only pathogenic mutations are associated with radioresistance. Furthermore, expression of NFE2L2 target genes does not predict LR, underscoring the utility of tumor genotyping. Finally, we show that glutaminase inhibition preferentially radiosensitizes -mutant cells via depletion of glutathione and increased radiation-induced DNA damage. Our findings suggest that genotyping for mutations could facilitate treatment personalization and provide a potential strategy for overcoming radioresistance conferred by these mutations. SIGNIFICANCE: This study shows that mutations in and predict for LR after radiotherapy but not surgery in patients with NSCLC. Approximately half of all LRs are associated with these mutations and glutaminase inhibition may allow personalized radiosensitization of -mutant tumors..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710558PMC
December 2020

Ebp1 p48 promotes oncogenic activities in human colon cancer cells through regulation of TIF-90-mediated ribosomal RNA synthesis.

J Cell Physiol 2019 08 22;234(10):17612-17621. Epub 2019 Feb 22.

Department of Medical Biotechnology, Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Vietnam.

The ErbB3-binding protein 1 (Ebp1) has been reported as either an oncogenic regulator or a tumor suppressor in a variety of cancers. Here, we show that Ebp1 p48, a predominant expression isoform, is highly expressed in the majority of human colon tumor cells compared with normal adjacent tissues and its expression is required for the oncogenic activities of these cells. Depletion of Ebp1 expression in primary colon cancer cells inhibits cell proliferation, colony forming, and invasion in vitro as well as tumor formation in vivo and enhances cell sensitivity to irradiation. We further demonstrated that Ebp1 interacts with TIF-90, a splice variant of transcription initiation factor IA (TIF-IA) of the RNA polymerase I complex, allowing for regulation of ribosomal RNA (rRNA) synthesis and oncogenesis in human colon cancer cells. Moreover, Ebp1 expression is essential for Akt protected TIF-90 stability by preventing TIF-90's ubiquitination by Mdm2 and hence, its proteasomal degradation. The results of the present study support a mechanism of underlying oncogenic activities by means of Ebp1 through regulation of TIF-90-mediated rRNA synthesis and suggest the potential therapeutic treatment of colon cancer by targeting Ebp1 and its signaling.
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http://dx.doi.org/10.1002/jcp.28385DOI Listing
August 2019

Assessing the Impact of Targeting CEACAM1 in Head and Neck Squamous Cell Carcinoma.

Otolaryngol Head Neck Surg 2018 07 13;159(1):76-84. Epub 2018 Feb 13.

1 Department of Otolaryngology-Head and Neck Surgery, Stanford Cancer Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

Objective In conjunction with advances made in cytotoxic chemotherapy, radiation, and surgery, immunotherapy has emerged as a fourth modality of treatment for head and neck squamous cell carcinoma (HNSCC). Understanding the mechanisms by which HNSCC evades immune-mediated control will aid in the development of new therapies to augment an antitumor immune response. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell surface receptor that is expressed on malignant cells and lymphocytes such as natural killer (NK) cells. We sought to determine whether tumor-derived CEACAM1 inhibits NK cell cytotoxicity and whether blockade of CEACAM1 restores antitumor immunity. Study Design In vitro HNSCC cell line study. Setting Research laboratory. Subject and Methods We utilized a real-time cell analyzer to assess NK cell cytotoxicity against an oral squamous cell carcinoma cell line after modulating CEACAM1 expression by cytokines and shRNA knockdown of CEACAM1 expression. Results NK cells and HNSCC cells both demonstrated cytokine-inducible expression of CEACAM1. Coincubation of NK cells and HNSCC cells resulted in the upregulation of CEACAM1 on the tumor cells. When compared with CEACAM1 cells, CEACAM1 tumor cells exhibited increased cell growth and increased size and number of organoids in 3-dimensional culture. Notably, CEACAM1 HNSCC cells were more resistant to NK cell-mediated killing, but the inhibited expression of CEACAM1 by an shRNA construct restored NK cell cytotoxicity. Conclusion Together, these data indicate that CEACAM1 acts as an inducible checkpoint molecule, and they support the idea that targeting CEACAM1 could serve as a novel immunotherapy approach in HNSCC.
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http://dx.doi.org/10.1177/0194599818756627DOI Listing
July 2018

The aryl hydrocarbon receptor modulates the function of human CD56 NK cells.

Eur J Immunol 2018 05 12;48(5):771-776. Epub 2018 Feb 12.

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.

Human natural killer (NK) cells are divided into two subsets: CD56 and CD56 NK cells, which differ in maturation, function and distribution. Mechanisms regulating NK cell functions are not completely understood. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, that binds to a variety of endogenous and exogenous molecules, and that has recently been shown to modulate the function and differentiation of immune cells. Here, we studied the expression of AhR and its involvement in the regulation of NK cell functions. We found that AhR mRNA is highly expressed in peripheral CD56 NK cells and that AhR mRNA expression gradually decreases as NK cells display a more mature phenotype. CD56 NK cells were highly sensitive to AhR ligands. Specifically, AhR ligands modulated their activation and their expression of NK cell receptors, as well as cytokine secretion which is the major function of these cells. As CD56 NK cells are highly enriched in tissues and in tumors, our observations point to a possible effect of local AhR ligands in the regulation of the function of CD56 tissue-resident or intratumoral NK cells.
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http://dx.doi.org/10.1002/eji.201747289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658132PMC
May 2018

NSD1 inactivation defines an immune cold, DNA hypomethylated subtype in squamous cell carcinoma.

Sci Rep 2017 12 6;7(1):17064. Epub 2017 Dec 6.

Department of Medicine, Stanford Center for Biomedical Informatics Research, Stanford University, Stanford, USA.

Chromatin modifying enzymes are frequently mutated in cancer, resulting in widespread epigenetic deregulation. Recent reports indicate that inactivating mutations in the histone methyltransferase NSD1 define an intrinsic subtype of head and neck squamous cell carcinoma (HNSC) that features pronounced DNA hypomethylation. Here, we describe a similar hypomethylated subtype of lung squamous cell carcinoma (LUSC) that is enriched for both inactivating mutations and deletions in NSD1. The 'NSD1 subtypes' of HNSC and LUSC are highly correlated at the DNA methylation and gene expression levels, featuring ectopic expression of developmental transcription factors and genes that are also hypomethylated in Sotos syndrome, a congenital disorder caused by germline NSD1 mutations. Further, the NSD1 subtype of HNSC displays an 'immune cold' phenotype characterized by low infiltration of tumor-associated leukocytes, particularly macrophages and CD8 T cells, as well as low expression of genes encoding the immunotherapy target PD-1 immune checkpoint receptor and its ligands. Using an in vivo model, we demonstrate that NSD1 inactivation results in reduced T cell infiltration into the tumor microenvironment, implicating NSD1 as a tumor cell-intrinsic driver of an immune cold phenotype. NSD1 inactivation therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy.
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http://dx.doi.org/10.1038/s41598-017-17298-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719078PMC
December 2017

CD271 Confers an Invasive and Metastatic Phenotype of Head and Neck Squamous Cell Carcinoma through the Upregulation of Slug.

Clin Cancer Res 2018 02 5;24(3):674-683. Epub 2017 Dec 5.

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford Cancer Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California.

Head and neck squamous cell carcinoma (HNSCC) is comprised of heterogeneous populations of cells, and CD271 (NGFR; p75NTR) has been associated with a tumor-initiating cell subpopulation. This study assessed the role of CD271 in modulating metastatic behavior in HNSCC. CD271 was overexpressed in murine and human oral squamous cell carcinoma cells to assess the impact of CD271 activation on the invasive and metastatic phenotype of these cells, using and orthotopic modeling. Treatment with human nerve growth factor (NGF) to activate CD271, as well as shRNA knockdown of the CD271-upregulated expression, was used to assess the mechanism of the CD271-induced invasive phenotype. Relevance of CD271 expression in human HNSCC was evaluated in patient-derived xenografts (PDX) and primary human oral cancers, annotated with clinical behavior characteristics and survival data. Forced expression of CD271 resulted in a more invasive and metastatic phenotype. Slug, an epithelial-to-mesenchymal transition (EMT)-related transcription factor, encoded by , was highly expressed in MOC2-CD271 and HSC3-CD271, compared with respective parental cells. CD271 activation by NGF conferred enhanced invasiveness in CD271-overexpressing cells, which was abrogated by knockdown. In PDXs and primary human HNSCC, CD271 expression correlated with higher expression, greater nodal metastasis, and shorter disease-free survival. Activation of CD271 results in upregulation of /Slug, which, in turn, results in a more invasive phenotype and an enhanced capacity for metastasis to regional lymph nodes. These findings point to CD271 as a promising, therapeutic target for oral cancer metastasis. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713647PMC
February 2018

Synthesis of a Phlorin from a Meso-Fused Anthriporphyrin by a Diels-Alder Strategy.

Angew Chem Int Ed Engl 2017 12 27;56(51):16247-16251. Epub 2017 Nov 27.

Department of Chemistry, Inha University, Incheon, 22212, Republic of Korea.

An anthracene-containing meso-fused carbaporphyrin, which has extended π-conjugation pathways as compared to the corresponding naphthalene-containing carbaporphyrin, has been synthesized. The weak global aromaticity of the anthriporphyrin also allowed its use as the diene for a Diels-Alder reaction with dimethyl acetylenedicarboxylate (DMAD). The resulting phlorin contains an interesting bicyclic structure. Moreover, to the best of our knowledge, this phlorin is the first Diels-Alder adduct of a diene forming part of the global π-conjugation pathway of an aromatic porphyrinoid.
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http://dx.doi.org/10.1002/anie.201709026DOI Listing
December 2017

Fluorescent and cooperative ion pair receptor based on tolan for Na (or Li) and HSO: logic AND gate.

Chem Commun (Camb) 2017 Oct 4;53(83):11414-11417. Epub 2017 Oct 4.

Department of Chemistry, Inha University, Incheon, 22212, Republic of Korea.

A tolan derivative was synthesized as a fluorescent and cooperative ion pair receptor. As both Na and HSO ions were complexed to the receptor, only substantial fluorescence was quenched. Thus, it also acts as a logic AND gate.
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http://dx.doi.org/10.1039/c7cc06907cDOI Listing
October 2017

Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor.

Oncotarget 2017 Aug 10;8(32):52345-52356. Epub 2017 Apr 10.

Stanford Cancer Institute, School of Medicine, Stanford University, Stanford, CA, 94305, USA.

Chemoresistant cancer cells express high levels of aldehyde dehydrogenases (ALDHs), particularly in head and neck squamous cell carcinoma (HNSCC). The ALDH family of enzymes detoxify both exogenous and endogenous aldehydes. Since many chemotherapeutic agents, such as cisplatin, result in the generation of cytotoxic aldehydes and oxidative stress, we hypothesized that cells expressing high levels of ALDH may be more chemoresistant due to their increased detoxifying capacity and that inhibitors of ALDHs may sensitize them to these drugs. Here, we show that overall ALDH activity is increased with cisplatin treatment of HNSCC and that ALDH3A1 protein expression is particularly enriched in cells treated with cisplatin. Activation of ALDH3A1 by a small molecule activator (Alda-89) increased survival of HNSCC cells treated with cisplatin. Conversely, treatment with a novel small molecule ALDH inhibitor (Aldi-6) resulted in a marked decrease in cell viability, and the combination of Aldi-6 and cisplatin resulted in a more pronounced reduction of cell viability and a greater reduction in tumor burden than what was observed with cisplatin alone. These data indicate that ALDH3A1 contributes to cisplatin resistance in HNSCC and that the targeting of ALDH, specifically, ALDH3A1, appears to be a promising strategy in this disease.
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http://dx.doi.org/10.18632/oncotarget.17017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581033PMC
August 2017

The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells.

J Exp Med 2016 10 26;213(11):2249-2257. Epub 2016 Sep 26.

Division of Head and Neck Surgery, Department of Otolaryngology, Program in Immunology, Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305

A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1CD3) cells, defined as CD49aTRAILCXCR6DX5 cells in the mouse liver, constitutively express AhR. In AhR mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49aTRAILCXCR6DX5 liver-resident NK cells and their hapten memory function.
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http://dx.doi.org/10.1084/jem.20151998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068230PMC
October 2016

Transcription factor Dlx3 induces aryl hydrocarbon receptor promoter activity.

Biochem Biophys Rep 2016 Sep 30;7:353-360. Epub 2016 Jun 30.

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA 94305, USA; Program in Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford CA 94305, USA.

The Distal-less (Dlx) homeobox transcription factors (TFs) play a prominent role in regulating multiple facets of vertebrate biology. Though widely studied as mediators of tissue development, recent work has uncovered a role for this TF family in modulating the vertebrate hematopoietic compartment. Pertinent to our study, murine are expressed in an innate lymphocyte population known as natural killer (NK) cells, and they are implicated to assume a functional role in the NK cell maturation pathway. However, Dlx target genes are poorly understood. In , the invertebrate ortholog () regulates another transcription factor called (), which is critical for specifying distal antennal segments. Importantly, the vertebrate ortholog of is the aryl hydrocarbon receptor (AhR), a transcription factor recently shown to be important in the regulation of a number of immune cell subsets, including NK cells. Given these findings, we investigated whether Dlx TF family members might analogously regulate in an NK cell context. Our results demonstrate that is constitutively co-expressed with in murine and human CD127 NK cells. Critically, we show that Dlx3 induces promoter activity by binding to a regulatory region that resides ~5.5 kb upstream of the transcriptional start site. This mechanism is functionally relevant, as expression in human NK cells significantly enhances TF activity at AhR DNA-binding elements (Xenobiotic Responsive Elements, XREs). Thus, our study defines Dlx3 as a positive regulator of the aryl hydrocarbon receptor.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074085PMC
http://dx.doi.org/10.1016/j.bbrep.2016.06.023DOI Listing
September 2016

ESM1 mediates NGFR-induced invasion and metastasis in murine oral squamous cell carcinoma.

Oncotarget 2016 Oct;7(43):70738-70749

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Oral squamous cell carcinoma (OSCC) is a highly invasive and metastatic malignancy. The nerve growth factor receptor (NGFR) has been observed to be expressed on a subset of cells in OSCC, and NGFR+ cells have greater tumor-initiating capacity in vivo. Further, inhibition of NGFR reduces tumor growth, indicating a functional role of this receptor; however, the mechanisms by which NGFR confers enhanced tumor formation are not known. Here, we used an established murine model of OSCC and gene expression array analysis to identify ESM1 as a downstream target gene of NGFR, critical for tumor invasion and metastasis. ESM1 encodes a protein called endocan, which has the property of regulating proliferation, differentiation, migration, and adhesion of different cell types. Incubation of NGFR+ murine OSCC cells with nerve growth factor resulted in increased expression of ESM1. Importantly, ESM1 overexpression conferred an enhanced migratory, invasive, and metastatic phenotype, similar to what has been correlated with NGFR expression. Conversely, shRNA knockdown of ESM1 in NGFR overexpressing OSCC cells abrogated the tumor growth kinetics and the invasive and metastatic properties associated with NGFR. Together, our data indicate that NGFR plays an important role in the pathogenesis and progression of OSCC via regulation of ESM1.
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http://dx.doi.org/10.18632/oncotarget.12210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342586PMC
October 2016

Accelerated hydration reaction of an unsymmetrical tolan evidenced by a Hg(ii)-trapped macrocycle and its application as a Hg(ii)-selective indicator.

Chem Commun (Camb) 2016 Sep 11;52(71):10759-62. Epub 2016 Aug 11.

Department of Chemistry, Inha University, Incheon 402-751, Korea.

Hg(ii)-mediated hydration reactions of unsymmetrical quinoline type tolans were studied. The observed accelerated reactions of the tolans rely on the additional binding motifs of the tolan, as supported by the X-ray structure of the macrocycle (2b). The analyte-specific reaction allows us to detect Hg(ii) in buffered media.
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http://dx.doi.org/10.1039/c6cc06012aDOI Listing
September 2016

CD44+ Cells in Head and Neck Squamous Cell Carcinoma Suppress T-Cell-Mediated Immunity by Selective Constitutive and Inducible Expression of PD-L1.

Clin Cancer Res 2016 07 10;22(14):3571-81. Epub 2016 Feb 10.

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford Cancer Institute, Stanford, California. Program in Immunology, Stanford University School of Medicine, Stanford, California. Stanford Cancer Institute, Stanford, California. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California.

Purpose: Human tumors consist of heterogeneous populations of cells with distinct marker expression and functional properties. In squamous cell carcinoma of the head and neck (SCCHN), CD44 is a well-characterized marker of a resilient subpopulation of cells associated with increased tumorigenesis, radioresistance, and chemoresistance. Evidence indicates that these cells have an immunosuppressive phenotype; however, mechanisms have been elusive.

Experimental Design: Using primary human SCCHN tumor samples and patient-derived xenografts, we examined the phenotypes of subsets of tumor cells and investigated mechanisms regulating their immunogenicity.

Results: CD44(+) cells in primary human SCCHN were found to have an epithelial-to-mesenchymal (EMT) phenotype and were less immunogenic than CD44(-) cells when cultured with autologous CD8(+) tumor-infiltrating T cells. Selective expression of the programmed death-ligand 1 (PD-L1) was observed on CD44(+) cells compared with CD44(-) cells and was associated with constitutive phosphorylation of STAT3 on CD44(+) cells. Importantly, inhibition of STAT3 decreased expression of PD-L1 on CD44(+) cells. IFNγ treatment preferentially induced even further PD-L1 expression on CD44(+) cells and was associated with enhanced IFNγ receptor expression and phosphorylation of STAT1. Finally, the decreased immunogenicity of CD44(+) cells was partially reversed by antibody blockade of the programmed death 1 (PD-1) receptor, indicating that the differences in PD-L1 expression between CD44(+) and CD44(-) cells are biologically and clinically relevant.

Conclusions: Our findings provide a mechanism by which long-lived CD44(+) tumor-initiating cells can selectively evade host immune responses and provide rationale for targeting the PD-1 pathway in the adjuvant therapy setting of SCCHN. Clin Cancer Res; 22(14); 3571-81. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623594PMC
July 2016

Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Oncotarget 2015 Apr;6(12):9897-907

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA, USA.

Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496405PMC
http://dx.doi.org/10.18632/oncotarget.3393DOI Listing
April 2015

CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma.

Oncotarget 2014 Aug;5(16):6854-66

Division of Head and Neck Surgery, Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA. Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA.

Tumor-initiating cells (TICs) in squamous cell carcinoma of the head and neck (SCCHN) are best characterized by their surface expression of CD44. Although there is great interest in identifying strategies to target this population, no marker of these cells has been found to be functionally active. Here, we examined the expression of the purported marker of normal human oral epithelial stem cells, CD271. We show that CD271 expression is restricted to a subset of the CD44+ cells. Using xenograft assays, we show that the CD44+CD271+ subpopulation contains the most tumorigenic cells. Loss of CD271 function results in a block in the G2-M phase of the cell cycle and a profound negative impact on the capacity of these cells to initiate tumor formation in vivo. Incubation with recombinant NGF results in enhanced phosphorylation of Erk, providing additional evidence that CD271 is functionally active. Finally, incubation of SCCHN cells with antibody to CD271 results in decreased Erk phosphorylation and decreased tumor formation in vivo. Thus, our data are the first to demonstrate that CD271 more specifically identifies the TIC subpopulation within the CD44+ compartment in SCCHN and that this receptor is a functionally active and targetable molecule.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196168PMC
http://dx.doi.org/10.18632/oncotarget.2269DOI Listing
August 2014

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon receptor.

Proc Natl Acad Sci U S A 2013 Jul 8;110(30):12391-6. Epub 2013 Jul 8.

Department of Otolaryngology-Head, and Neck Surgery, Stanford Cancer Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA.

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.
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http://dx.doi.org/10.1073/pnas.1302856110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725066PMC
July 2013

Reprogramming of single-cell-derived mesenchymal stem cells into hair cell-like cells.

Otol Neurotol 2012 Dec;33(9):1648-55

Model Animal Research Center of Nanjing University, Nanjing, People's Republic of China.

Hypothesis: Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination.

Background: Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells.

Methods: Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer, and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed using reverse transcriptase-polymerase chain reaction, and cell structures were examined using immunocytochemistry.

Results: Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells before overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model.

Conclusion: We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells.
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http://dx.doi.org/10.1097/MAO.0b013e3182713680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498597PMC
December 2012

CD44(+) cells have cancer stem cell-like properties in nasopharyngeal carcinoma.

Int Forum Allergy Rhinol 2012 Nov 7;2(6):465-70. Epub 2012 Aug 7.

Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.

Background: A subpopulation of cells within a tumor appears to have the exclusive ability to initiate tumors, self-renew, and differentiate. These "cancer stem cells" (CSCs) are CD44(+) in several epithelial malignancies. We examined the potential of CD44 to identify the CSC population in nasopharyngeal carcinoma (NPC).

Methods: C666, an Epstein-Barr virus-positive (EBV(+) ) human NPC cell line, was stained for CD44 and sorted by fluorescence-activated cell sorting (FACS). CD44(+) and CD44(-) subpopulations were evaluated for (1) proliferative potential, (2) ability to differentiate, (3) expression of markers of epithelial-to-mesenchymal transition (EMT) and EBV genes, and (4) the ability to initiate tumors in vivo. Immunocompromised mice were injected with CD44(+) and CD44(-) populations to assess the tumor-initiating capacity. Immunohistochemistry for CD44 was performed on an 87-patient tissue microarray (TMA), and clinical correlations were examined.

Results: Heterogeneous expression of CD44 was seen among C666 cells. CD44(+) cells differentiated into CD44(-) cells, indicating a hierarchical relationship. Further, CD44(+) cells exhibited a more robust tumor-initiating capacity in the xenograft model. However, no differences were seen in proliferation rates in vitro, EBV gene expression, or expression of EMT markers between CD44(+) and CD44(-) subsets. Patient tumors were heterogeneous for CD44 staining, and a trend toward an association between CD44 expression and clinical outcome was observed.

Conclusion: NPC contains a CD44(+) subpopulation with features consistent with CSCs. There was a trend toward an association between CD44 expression within NPC tumors and decreased time to local failure/relapse in patients.
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http://dx.doi.org/10.1002/alr.21068DOI Listing
November 2012

The manipulation of natural killer cells to target tumor sites using magnetic nanoparticles.

Biomaterials 2012 Aug 8;33(22):5584-92. Epub 2012 May 8.

Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, Department of Radiology, Stanford University, 1201 Welch Rd, Stanford, CA 94305, USA.

The present work demonstrates that Cy5.5 conjugated Fe(3)O(4)/SiO(2) core/shell nanoparticles could allow us to control movement of human natural killer cells (NK-92MI) by an external magnetic field. Required concentration of the nanoparticles for the cell manipulation is as low as ~20 μg Fe/mL. However, the relative ratio of the nanoparticles loaded NK-92MI cells infiltrated into the target tumor site is enhanced by 17-fold by applying magnetic field and their killing activity is still maintained as same as the NK-92MI cells without the nanoparticles. This approach allows us to open alternative clinical treatment with reduced toxicity of the nanoparticles and enhanced infiltration of immunology to the target site.
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http://dx.doi.org/10.1016/j.biomaterials.2012.04.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571620PMC
August 2012

Corepressor MMTR/DMAP1 is an intrinsic negative regulator of CAK kinase to regulate cell cycle progression.

Biochem Biophys Res Commun 2010 Nov;402(1):110-5

Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.

We have previously reported that MMTR (MAT1-mediated transcriptional repressor) is a co-repressor that inhibits TFIIH-mediated transcriptional activity via interaction with MAT1 (Kang et al., 2007). Since MAT1 is a member of the CAK kinase complex that is crucial for cell cycle progression and that regulates CDK phosphorylation as well as the general transcription factor TFIIH, we investigated MMTR function in cell cycle progression. We found that MMTR over-expression delayed G1/S and G2/M transitions, whereas co-expression of MAT1 and MMTR rescued the cell growth and proliferation rate. Moreover, MMTR was required for inhibition of CAK kinase-mediated CDK1 phosphorylation. We also showed that the expression level of MMTR was modulated during cell cycle progression. Our data support the notion that MMTR is an intrinsic negative cell cycle regulator that modulates the CAK kinase activity via interaction with MAT1.
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http://dx.doi.org/10.1016/j.bbrc.2010.09.126DOI Listing
November 2010

PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific alpha-globin expression.

Nucleic Acids Res 2010 Sep 26;38(16):5456-71. Epub 2010 Apr 26.

Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul, 133-791, Korea.

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.
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http://dx.doi.org/10.1093/nar/gkq286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938217PMC
September 2010

Corepressor MMTR/DMAP1 is involved in both histone deacetylase 1- and TFIIH-mediated transcriptional repression.

Mol Cell Biol 2007 May 19;27(10):3578-88. Epub 2007 Mar 19.

Department of Life Science, College of Natural Sciences, Hanyang University, Haengdang 17, Sungdong-gu, Seoul 133-791, South Korea.

A transcription corepressor, MAT1-mediated transcriptional repressor (MMTR), was found in mouse embryonic stem cell lines. MMTR orthologs (DMAP1) are found in a wide variety of life forms from yeasts to humans. MMTR down-regulation in differentiating mouse embryonic stem cells in vitro resulted in activation of many unrelated genes, suggesting its role as a general transcriptional repressor. In luciferase reporter assays, the transcriptional repression activity resided at amino acids 221 to 468. Histone deacetylase 1 (HDAC1) interacts with MMTR both in vitro and in vivo and also interacts with MMTR in the nucleus. Interestingly, MMTR activity was only partially rescued by competition with dominant-negative HDAC1(H141A) or by treatment with an HDAC inhibitor, trichostatin A (TSA). To identify the protein responsible for HDAC1-independent MMTR activity, we performed a yeast two-hybrid screen with the full-length MMTR coding sequence as bait and found MAT1. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase which constitutes a subcomplex of TFIIH. The coiled-coil domain in the middle of MAT1 was confirmed to interact with the C-terminal half of MMTR, and the MMTR-mediated transcriptional repression activity was completely restored by MAT1 in the presence of TSA. Moreover, intact MMTR was required to inhibit phosphorylation of the C-terminal domain in the RNA polymerase II largest subunit by TFIIH kinase in vitro. Taken together, these data strongly suggest that MMTR is part of the basic cellular machinery for a wide range of transcriptional regulation via interaction with TFIIH and HDAC.
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http://dx.doi.org/10.1128/MCB.01808-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899998PMC
May 2007

Prophylactic and therapeutic functions of T-type calcium blockers against noise-induced hearing loss.

Hear Res 2007 Apr 31;226(1-2):52-60. Epub 2006 Dec 31.

Department of Otolaryngology, Center for Aging, Washington University, 4560 Clayton Avenue, St. Louis, MO 63110, USA.

Cochlear noise injury is the second most frequent cause of sensorineural hearing loss, after aging. Because calcium dysregulation is a widely recognized contributor to noise injury, we examined the potential of calcium channel blockers to reduce noise-induced hearing loss (NIHL) in mice. We focused on two T-type calcium blockers, trimethadione and ethosuximide, which are anti-epileptics approved by the Food and Drug Administration. Young C57BL/6 mice of either gender were divided into three groups: a 'prevention' group receiving the blocker via drinking water before noise exposure; a 'treatment' group receiving the blocker via drinking water after noise exposure; and controls receiving noise alone. Trimethadione significantly reduced NIHL when applied before noise exposure, as determined by auditory brainstem recording. Both ethosuximide and trimethadione were effective in reducing NIHL when applied after noise exposure. Results were influenced by gender, with males generally receiving greater benefit than females. Quantitation of hair cell and neuronal density suggested that preservation of outer hair cells could account for the observed protection. Immunocytochemistry and RT-PCR suggested that this protection involves direct action of T-type blockers on alpha1 subunits comprising one or more Ca(v)3 calcium channel types in the cochlea. Our findings provide a basis for clinical studies testing T-type calcium blockers both to prevent and treat NIHL.
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http://dx.doi.org/10.1016/j.heares.2006.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1903349PMC
April 2007

Preferentially enhanced gene expression from a synthetic human telomerase reverse transcriptase promoter in human cancer cells.

Oncol Rep 2006 Nov;16(5):975-9

Department of Microbiology, University of Ulsan College of Medicine, 138-736 Seoul, Korea.

Although the human telomerase reverse transcriptase (hTERT) promoter can regulate cancer-specific genes, it is generally too weak to be effective. We therefore attempted to improve the potency of synthetic hTERT promoters by fusing the core element (E) of the hTERT promoter (H) and the tripartite leader sequence (T) from human adenovirus 5 in a combinatorial manner. To determine the potential as cancer-specific promoters, we measured luciferase activity driven by the chimeric hTERT promoters in human cancer cells. Among various constructs, the E3-H-T promoter induced the strongest luciferase activity in all the tested cancer cells. SK-Hep1 and Hela cells experienced 1000- and 11-fold higher expression than the basic hTERT promoter, respectively. Relative to the SV40 universal promoter, the E3-H-T promoter led to higher levels of gene expression. Using EMSA, we found that the hTERT enhancer region was specifically bound to c-Myc and Sp1. Thus, the data suggest that the E3-H-T promoter with up-regulated cancer-specific gene expression could be useful in cancer gene therapy.
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November 2006

Profiling of differentially expressed genes in human stem cells by cDNA microarray.

Mol Cells 2006 Jun;21(3):343-55

Department of Life Science, Hanyang University, Seoul 133-791, Korea.

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation, although a wide variety of adult stem cells as well as embryonic stem cells have been identified and stem cell plasticity has recently been reported. To identify genes implicated in the control of the stem cell state as well as the characteristics of each stem cell line, we analyzed the expression profiles of genes in human embryonic, hematopoietic (CD34+ and CD133+), and mesenchymal stem cells using cDNA microarrays, and identified genes that were differentially expressed in specific stem cell populations. In particular we were able to identify potential hESC signature-like genes that encode transcription factors (TFAP2C and MYCN), an RNA binding protein (IMP-3), and a functionally uncharacterized protein (MAGEA4). The overlapping sets of 22 up-regulated and 141 down-regulated genes identified in this study of three human stem cell types may also provide insight into the developmental mechanisms common to all human stem cells. Furthermore, our comprehensive analyses of gene expression profiles in various adult stem cells may help to identify the genetic pathways involved in self-renewal as well as in multi-lineage specific differentiation.
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June 2006

Stem cell therapy for hearing loss: Math1 overexpression in VOT-E36 cells.

Otol Neurotol 2006 Apr;27(3):414-21

Department of Otolaryngology, Center for Aging, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Hypothesis: VOT-E36 cells acquire mechanosensitivity after mammalian atonal homolog 1 (Math1) overexpression.

Background: VOT-E36 cells are derived from a population of epithelial cells in the ventral region of the otocyst at embryonic Day 10.5, before hair cell differentiation. These cells express a number of specific molecular markers for hair cells under both proliferation and differentiation states. Overexpression of Math1 can convert nonsensory epithelial cells into hair cells in the cochlea. Based on this information, we tested whether VOT-E36 cells can be converted into hair cells by Math1 overexpression.

Methods: Using reverse transcriptase-polymerase chain reaction-based analysis, we first compared the expression patterns of various molecular markers for hair cell development in VOT-E36 cells between proliferation and differentiation states, and also before and after overexpression of Math1. Subsequently, with a standard calcium imaging method, we examined whether VOT-E36 cells overexpressing Math1 could detect mechanical vibrations and activate spiral ganglion neurons in a coculture model. In addition, using confocal and scanning electron microscopes, we examined morphologic changes of VOT-E36 cells after Math1 overexpression.

Results: Consistent with previous reports, this study has shown that VOT-E36 cells express a number of specific molecular markers for hair cells in both proliferation and differentiation states. Under appropriate culture conditions, Math1 is transiently expressed in this cell line during conditional differentiation. In VOT-E36 cells overexpressing Math1, the normal expression pattern of certain molecular markers for mature hair cells is partially restored. Interestingly, after coculture with spiral ganglion neurons, VOT-E36 cells overexpressing Math1 are able to respond to mechanical vibrations and activate spiral ganglion neurons. Possible molecular mechanisms underlying this novel finding have been explored.

Conclusion: Math1 overexpression can partially restore presumably downstream signaling cascades for normal hair cell differentiation in VOT-E36 cells, which are able to detect mechanical vibrations after being cocultured with spiral ganglion neurons.
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http://dx.doi.org/10.1097/00129492-200604000-00020DOI Listing
April 2006

Erythroid cell-specific alpha-globin gene regulation by the CP2 transcription factor family.

Mol Cell Biol 2005 Jul;25(14):6005-20

Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Haengdang 17, Sungdong-gu, Seoul 133-791, South Korea.

We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of alpha-globin through an unknown mechanism. This mechanism is reported here to involve specific CP2 splice variants and protein inhibitor of activated STAT1 (PIAS1). We identify a novel murine splice isoform of CP2, CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon. CP2b has an erythroid cell-specific transcriptional activation domain, which requires the extra exon and can form heteromeric complexes with other CP2 isoforms, but lacks the DNA binding activity found in CP2a and CP2c. Transcriptional activation of alpha-globin occurred following dimerization between CP2b and CP2c in erythroid K562 and MEL cells, but this dimerization did not activate the alpha-globin promoter in nonerythroid 293T cells, indicating that an additional erythroid factor is missing in 293T cells. PIAS1 was confirmed as a CP2 binding protein by the yeast two-hybrid screen, and expression of CP2b, CP2c, and PIAS1 in 293T cell induced alpha-globin promoter activation. These results show that ubiquitously expressed CP2b exerts potent erythroid cell-specific alpha-globin gene expression by complexing with CP2c and PIAS1.
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http://dx.doi.org/10.1128/MCB.25.14.6005-6020.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1168829PMC
July 2005

Development of artificial chimerical gene regulatory elements specific for cancer gene therapy.

Oncol Rep 2003 Nov-Dec;10(6):2063-9

Department of Life Science and Research Institute of Natural Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791, Korea.

To achieve satisfactory outcome by the expression of therapeutic genes, it is of great importance to obtain efficient and high level of gene expression as well as minimizing inappropriate gene expression in non-target cells. To accomplish this goal for cancer gene therapy, we have evaluated the potential of cancer specific gene expression of functional promoter/enhancer elements in six putative cancer-specific genes (Tcf1alpha, C-Ha-Ras, CyclinE, Cdc25A, HK II, and hTert) using a luciferase reporter assay. Most of the reporter constructs showed higher activity in HepG2 cells than in non-transformed or stem cells, and, in particular, the hTert (E) or Tcf1alpha (T) regulatory element showed significantly higher activity. We have also constructed a series of artificial chimerical regulatory elements by combinatorial linking of E promoter and T enhancer. A dramatic decrease of activity was observed as the copy number of concatenated T/E regulatory elements increased. In contrast, in chimerical constructs containing two or three copies of regulatory elements of T/E, cell type preferential expression profiles were changed. Thus, both pGL3-TE and -TEE showed higher activity specifically in MCF7 breast cancer cells, whereas pGL3-TET showed moderate activity in several cancer cell lines of different origins. Our results demonstrate that although the transcriptional activities of synthetic promoters are weak, some cancer-specific regulatory elements are useful in developing optimized and systemic cancer-specific regulatory regions with potential application in targeted cancer cell therapy.
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June 2004