Publications by authors named "Chunbo Shao"

28 Publications

  • Page 1 of 1

A novel siRNA-gemcitabine construct as a potential therapeutic for treatment of pancreatic cancer.

NAR Cancer 2020 Sep 11;2(3):zcaa016. Epub 2020 Aug 11.

Sirnaomics Inc., Suite 280, 401 Professional Drive, Gaithersburg, MD 20879, USA.

The non-nucleoside analog gemcitabine has been the standard of care for treating pancreatic cancer. The drug shows good potency in pancreatic cancer cells but, due to poor bioavailability, requires administration in large doses by infusion and this systemic exposure results in significant toxicity for the patient. Genes have been identified that, when silenced by siRNA, synergize with gemcitabine treatment and offer a means of reducing the gemcitabine dosage required for efficacy. However, benefiting from the synergism between the two agents requires that the gemcitabine and siRNA penetrate the same cells. To ensure co-delivery, we incorporated gemcitabine covalently within siRNAs against targets synergistic with gemcitabine (CHK1 or RAD17). We demonstrated that specific bases within an siRNA can be replaced with gemcitabine to increase efficacy. The result is a single drug molecule that simultaneously co-delivers gemcitabine and a synergistic siRNA. The siRNA-gemcitabine constructs demonstrate a 5-30-fold improvement in potency compared with gemcitabine alone. Co-delivering a CHK1 siRNA-gemcitabine construct together with a WEE1 siRNA resulted in a 10-fold improvement in IC compared with gemcitabine alone. These constructs demonstrate efficacy across a wide array of pancreatic tumor cells and may represent a novel therapeutic approach for treating pancreatic cancer.
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http://dx.doi.org/10.1093/narcan/zcaa016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209983PMC
September 2020

To "grow" or "go": TMEM16A expression as a switch between tumor growth and metastasis in SCCHN.

Clin Cancer Res 2014 Sep 11;20(17):4673-88. Epub 2014 Jun 11.

VA Pittsburgh Health System, Pittsburgh, Pennsylvania. Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsylvania.

Purpose: Tumor metastasis is the leading cause of death in patients with cancer. However, the mechanisms that underlie metastatic progression remain unclear. We examined TMEM16A (ANO1) expression as a key factor shifting tumors between growth and metastasis.

Experimental Design: We evaluated 26 pairs of primary and metastatic lymph node (LN) tissue from patients with squamous cell carcinoma of the head and neck (SCCHN) for differential expression of TMEM16A. In addition, we identified mechanisms by which TMEM16A expression influences tumor cell motility via proteomic screens of cell lines and in vivo mouse studies of metastasis.

Results: Compared with primary tumors, TMEM16A expression decreases in metastatic LNs of patients with SCCHN. Stable reduction of TMEM16A expression enhances cell motility and increases metastases while decreasing tumor proliferation in an orthotopic mouse model. Evaluation of human tumor tissues suggests an epigenetic mechanism for decreasing TMEM16A expression through promoter methylation that correlated with a transition between an epithelial and a mesenchymal phenotype. These effects of TMEM16A expression on tumor cell size and epithelial-to-mesenchymal transition (EMT) required the amino acid residue serine 970 (S970); however, mutation of S970 to alanine does not disrupt the proliferative advantages of TMEM16A overexpression. Furthermore, S970 mediates the association of TMEM16A with Radixin, an actin-scaffolding protein implicated in EMT.

Conclusions: Together, our results identify TMEM16A, an eight transmembrane domain Ca2+-activated Cl- channel, as a primary driver of the "Grow" or "Go" model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-0363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160843PMC
September 2014

Expression microarray analysis reveals alternative splicing of LAMA3 and DST genes in head and neck squamous cell carcinoma.

PLoS One 2014 27;9(3):e91263. Epub 2014 Mar 27.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States of America; Milton J. Dance Head and Neck Center, Greater Baltimore Medical Center, Baltimore, Maryland, United States of America.

Purpose: Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.

Experimental Design: We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score-the Maximum-Minimum Exon Score (MMES)--designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.

Results: After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).

Conclusion: Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091263PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967989PMC
December 2015

Aquaporin-1 promoter hypermethylation is associated with improved prognosis in salivary gland adenoid cystic carcinoma.

Otolaryngol Head Neck Surg 2014 May 3;150(5):801-7. Epub 2014 Feb 3.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Objectives: Aquaporin-1 (AQP1) is a candidate oncogene that is epigenetically modified in adenoid cystic carcinoma (ACC). We sought to (1) assess AQP1 promoter methylation and expression in an ACC cohort, (2) identify correlations between AQP1 and clinical outcomes, and (3) explore the role of AQP1 in tumor progression in vitro.

Study Design: Laboratory study, retrospective chart review.

Setting: Academic medical center.

Methods: DNA and RNA were isolated from ACC tumors and control salivary gland tissues. Quantitative methylation-specific polymerase chain reaction (PCR) was performed on bisulfite-treated DNA. Quantitative reverse transcription PCR was performed after cDNA synthesis. Cell lines stably overexpressing an AQP1 plasmid or empty vector were generated. Cell scratch and Matrigel invasion assays were performed. Retrospective chart review was performed for collection of clinical information.

Results: Methylation results from 77 tumors and 30 controls demonstrated that AQP1 was hypomethylated in tumors (P < .0001). Fifty-eight tumors (75.3%) displayed AQP1 hypomethylation compared with controls. AQP1 expression levels assessed in 58 tumors and 23 controls demonstrated a trend toward increased expression in tumors (P = .08). Univariate analysis revealed that AQP1 hypermethylation was associated with increased overall survival. No associations between AQP1 expression level and survival were found. AQP1 overexpression did not affect cell migratory or invasive capacities in vitro.

Conclusion: AQP1 promoter hypomethylation is common in ACC, and AQP1 tends to be overexpressed in these tumors. Increased AQP1 methylation is associated with improved prognosis on univariate analysis, but expression is not associated with outcomes. Further in vitro studies are necessary to clarify the role of AQP1 in ACC.
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http://dx.doi.org/10.1177/0194599814521569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318231PMC
May 2014

Clusterin is a gene-specific target of microRNA-21 in head and neck squamous cell carcinoma.

Clin Cancer Res 2014 Feb 10;20(4):868-77. Epub 2013 Dec 10.

Authors' Affiliations: Departments of Otolaryngology-Head and Neck Surgery and Pathology, Johns Hopkins Medical Institutions; The Milton J Dance, Jr. Head and Neck Center, Greater Baltimore Medical Center, Baltimore, Maryland; Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey; and Asuragen, Inc. Austin, Texas.

Purpose: MicroRNA-21 (miRNA-21) has proto-oncogenic properties, although no miRNA-21-specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology.

Experimental Design: miRNA expression profiles of 10 HNSCCs and 10 normal mucosa samples were investigated using a custom miRNA microarray. Thirteen HNSCCs and five normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of 16 HNSCCs and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets.

Results: miRNA-21 was upregulated in HNSCCs and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3'-untranslated region (UTR) of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCCs and correlated with miRNA-21 overexpression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCCs and showed growth suppression function that was reversed by miRNA-21 overexpression.

Conclusions: CLU is a specific, functional target of oncogenic miRNA-21 in HNSCCs. CLU-1 isoform is the predominant growth-suppressive variant targeted by miRNA-21.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-2675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970211PMC
February 2014

Targeting aberrant DNA double-strand break repair in triple-negative breast cancer with alpha-particle emitter radiolabeled anti-EGFR antibody.

Mol Cancer Ther 2013 Oct 19;12(10):2043-54. Epub 2013 Jul 19.

Corresponding Author: George Sgouros, The Johns Hopkins University School of Medicine, Rm 4M61 Cancer Research Building II, 1550 Orleans Street, Baltimore, MD 21231.

The higher potential efficacy of alpha-particle radiopharmaceutical therapy lies in the 3- to 8-fold greater relative biological effectiveness (RBE) of alpha particles relative to photon or beta-particle radiation. This greater RBE, however, also applies to normal tissue, thereby reducing the potential advantage of high RBE. As alpha particles typically cause DNA double-strand breaks (DSB), targeting tumors that are defective in DSB repair effectively increases the RBE, yielding a secondary, RBE-based differentiation between tumor and normal tissue that is complementary to conventional, receptor-mediated tumor targeting. In some triple-negative breast cancers (TNBC; ER(-)/PR(-)/HER-2(-)), germline mutation in BRCA-1, a key gene in homologous recombination DSB repair, predisposes patients to early onset of breast cancer. These patients have few treatment options once the cancer has metastasized. In this study, we investigated the efficacy of alpha-particle emitter, (213)Bi-labeled anti-EGF receptor antibody, cetuximab, in BRCA-1-defective TNBC. (213)Bi-cetuximab was found to be significantly more effective in the BRCA-1-mutated TNBC cell line HCC1937 than BRCA-1-competent TNBC cell MDA-MB-231. siRNA knockdown of BRCA-1 or DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), a key gene in non-homologous end-joining DSB repair pathway, also sensitized TNBC cells to (213)Bi-cetuximab. Furthermore, the small-molecule inhibitor of DNA-PKcs, NU7441, sensitized BRCA-1-competent TNBC cells to alpha-particle radiation. Immunofluorescent staining of γ-H2AX foci and comet assay confirmed that enhanced RBE is caused by impaired DSB repair. These data offer a novel strategy for enhancing conventional receptor-mediated targeting with an additional, potentially synergistic radiobiological targeting that could be applied to TNBC.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804319PMC
October 2013

Novel role of MDA-9/syntenin in regulating urothelial cell proliferation by modulating EGFR signaling.

Clin Cancer Res 2013 Sep 19;19(17):4621-33. Epub 2013 Jul 19.

Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA 23298, USA.

Purpose: Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression.

Experimental Design: Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knockdown was examined in multiple cells lines and key findings were validated in primary tumors.

Results: Significantly higher (P=0.002-0.003) expression of MDA-9/Syntenin was observed in 64% (28 of 44) of primary tumors and an association was evident with stage (P=0.01), grade (P=0.03), and invasion status (P=0.02). MDA-9/Syntenin overexpression in nontumorigenic HUC-1 cells increased proliferation (P=0.0012), invasion (P=0.0001), and EGF receptor (EGFR), AKT, phosphoinositide 3-kinase (PI3K), and c-Src expression. Alteration of β-catenin, E-cadherin, vimentin, claudin-1, ZO-1, and T-cell factor-4 (TCF4) expression was also observed. MDA-9/Syntenin knockdown in three UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and colocalization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin:AKT expressions with stage (P=0.04, EGFR; P=0.01, AKT). A correlation between MDA-9/Syntenin:β-catenin coexpression with stage (P=0.03) and invasion (P=0.04) was also evident.

Conclusions: Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring, and therapeutic strategies for managing UCC.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-0585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3872137PMC
September 2013

Hypermethylation of genes detected in urine from Ghanaian adults with bladder pathology associated with Schistosoma haematobium infection.

PLoS One 2013 18;8(3):e59089. Epub 2013 Mar 18.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

Purpose: Schistosoma haematobium is associated with chronic bladder damage and may subsequently induce bladder cancer in humans, thus posing a serious threat where the parasite is endemic. Here we evaluated aberrant promoter DNA methylation as a potential biomarker to detect severe bladder damage that is associated with schistosomiasis by analyzing urine specimens.

Materials And Methods: A quantitative methylation-specific PCR (QMSP) assay was used to examine the methylation status of seven genes (RASSF1A, RARβ2, RUNX3, TIMP3, MGMT, P16, ARF) in 57 urine samples obtained from volunteers that include infected and uninfected by S. haematobium from an endemic region. The Fishers Exact Test and Logistic Regression analysis were used to evaluate the methylation status with bladder damage (as assessed by ultrasound examination) in subjects with S. haematobium infection.

Results: RASSF1A and TIMP3 were significant to predict severe bladder damage both in univariate (p = 0.015 and 0.023 respectively) and in multivariate (p = 0.022 and 0.032 respectively) logistic regression analysis. Area under the receiver operator characteristic curves (AUC-ROC) for RASSF1A and TIMP3 to predict severe bladder damage were 67.84% and 63.73% respectively. The combined model, which used both RASSF1A and TIMP3 promoter methylation, resulted in significant increase in AUC-ROC compared to that of TIMP3 (77.55% vs. 63.73%.29; p = 0.023).

Conclusions: In this pilot study, we showed that aberrant promoter methylation of RASSF1A and TIMP3 are present in urine sediments of patients with severe bladder damage associated with S. haematobium infection and that may be used to develop non-invasive biomarker of S. haematobium exposure and early molecular risk assessmentof neoplastic transformation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059089PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601097PMC
September 2013

Suprabasin is hypomethylated and associated with metastasis in salivary adenoid cystic carcinoma.

PLoS One 2012 7;7(11):e48582. Epub 2012 Nov 7.

Department of Otolaryngology-Head and Neck Surgery, the Johns Hopkins Medical Institutions, Baltimore, MD, USA.

Background: Salivary gland adenoid cystic carcinoma (ACC) is a rare cancer, accounting for only 1% of all head and neck malignancies. ACC is well known for perineural invasion and distant metastasis, but its underlying molecular mechanisms of carcinogenesis are still unclear.

Principal Findings: Here, we show that a novel oncogenic candidate, suprabasin (SBSN), plays important roles in maintaining the anchorage-independent and anchorage-dependent cell proliferation in ACC by using SBSN shRNA stably transfected ACC cell line clones. SBSN is also important in maintaining the invasive/metastatic capability in ACC by Matrigel invasion assay. More interestingly, SBSN transcription is significantly upregulated by DNA demethylation induced by 5-aza-2'-deoxycytidine plus trichostatin A treatment and the DNA methylation levels of the SBSN CpG island located in the second intron were validated to be significantly hypomethylated in primary ACC samples versus normal salivary gland tissues.

Conclusions/significance: Taken together, these results support SBSN as novel oncogene candidate in ACC, and the methylation changes could be a promising biomarker for ACC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048582PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492451PMC
April 2013

SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling.

J Mol Med (Berl) 2013 Mar 12;91(3):381-93. Epub 2012 Sep 12.

Department of Human and Molecular Genetics, VCU Institute of Molecular Medicine, VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA.

The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p = 0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced overexpression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p = 0.0015-0.030) and in vivo (p = 0.016) cellular growth, invasion (p = 0.029-0.049), and colony formation (p = 0.023-0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser(473)), STAT3 (Tyr(705)), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated ß-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
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http://dx.doi.org/10.1007/s00109-012-0955-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691869PMC
March 2013

Dose-dependent activation of putative oncogene SBSN by BORIS.

PLoS One 2012 5;7(7):e40389. Epub 2012 Jul 5.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States of America.

Testis-specific transcription factor BORIS (Brother of the Regulator of Imprinted Sites), a paralog and proposed functional antagonist of the widely expressed CTCF, is abnormally expressed in multiple tumor types and has been implicated in the epigenetic activation of cancer-testis antigens (CTAs). We have reported previously that suprabasin (SBSN), whose expression is restricted to the epidermis, is epigenetically derepressed in lung cancer. In this work, we establish that SBSN is a novel non-CTA target of BORIS epigenetic regulation. With the use of a doxycycline-inducible BORIS expressing vector, we demonstrate that relative BORIS dosage is critical for SBSN activation. At lower concentrations, BORIS induces demethylation of the SBSN CpG island and disruption and activation of chromatin around the SBSN transcription start site (TSS), resulting in a 35-fold increase in SBSN expression in the H358 human lung cancer cell line. Interestingly, increasing BORIS concentrations leads to a subsequent reduction in SBSN expression via chromatin repression. In a similar manner, increase in BORIS concentrations leads to eventual decrease of cell growth and colony formation. This is the first report demonstrating that different amount of BORIS defines its varied effects on the expression of a target gene via chromatin structure reorganization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040389PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390376PMC
April 2013

Molecular biology of adenoid cystic carcinoma.

Head Neck 2012 Nov 17;34(11):1665-77. Epub 2011 Oct 17.

University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Background: Adenoid cystic carcinoma (ACC) is an unusual salivary gland malignancy that remains poorly understood. Standard treatment, including surgery with postoperative radiation therapy, has attained reasonable local control rates, but the propensity for distant metastases has limited any improvement in survival over time. Our understanding of the molecular mechanisms driving ACC is quite rudimentary, due to the infrequent nature of its occurrence.

Methods: An extensive literature review was performed on salivary gland ACCs and basic science research findings.

Results: This review highlights many findings that are emerging about the carcinogenesis of ACC including cytogenetics, tumor suppressor genes, oncogenes, epigenetic alterations, mitochondrial alterations, and biomarker studies.

Conclusion: Although there have been many discoveries, much still remains unknown about this rare malignancy. © 2011 Wiley Periodicals, Inc. Head Neck, 2011.
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http://dx.doi.org/10.1002/hed.21849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3262103PMC
November 2012

Mitochondrial DNA mutations in respiratory complex-I in never-smoker lung cancer patients contribute to lung cancer progression and associated with EGFR gene mutation.

J Cell Physiol 2012 Jun;227(6):2451-60

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, Maryland, USA.

Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with epidermal growth factor receptor (EGFR) and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients' samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P = 0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P = 0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients' population. MtDNA mutation was significantly (P = 0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P = 0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion, and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
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http://dx.doi.org/10.1002/jcp.22980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256258PMC
June 2012

Detection of mitochondrial deoxyribonucleic acid alterations in urine from urothelial cell carcinoma patients.

Int J Cancer 2012 Jul 30;131(1):158-64. Epub 2011 Aug 30.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, USA.

Our study aims at understanding the timing and nature of mitochondrial deoxyribonucleic acid (mtDNA) alterations in urothelial cell carcinoma (UCC) and their detection in urine sediments. The entire 16.5 kb mitochondrial genome was sequenced in matched normal lymphocytes, tumor and urine sediments from 31 UCC patients and compared to different clinical stages and histological grades. The mtDNA content index was examined in all the specimens. Sixty-five percent (20/31) of the patients harbored at least 1 somatic mtDNA mutation. A total of 25 somatic mtDNA mutations were detected, which were more frequent in the respiratory complex coding regions (Complex I, III, IV and V) of the mtDNA and significantly affected respiratory Complex III compared to the other complexes (p = 0.021-0.039). Compared to Stage Ta, mtDNA mutation was higher in Stage T1 and significantly higher in Stage T2 (p = 0.01) patients. MtDNA mutation was also significantly higher (p = 0.04) in Stage T2 compared to Stage T1 patients. Ninety percent (18/20) of the patients harboring mtDNA mutation in the tumor also had mutation in their urine sediments. Eighty percent (20/25) of the tumor-associated mtDNA mutations was detectable in the urine sediments. Compared to the normal lymphocytes, the mtDNA content increased significantly in the tumor (p = 0.0013) and corresponding urine sediments (p = 0.0025) in 19/25 (76%) patients analyzed. Our results indicate that mtDNA alterations occur frequently in progressive stages of UCC patients and are readily detectable in the urine sediments. MtDNA mutations appear to provide a promising tool for developing early detection and monitoring strategies for UCC patients.
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http://dx.doi.org/10.1002/ijc.26357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328657PMC
July 2012

Promoter methylation of leukemia inhibitory factor receptor gene in colorectal carcinoma.

Int J Oncol 2011 Aug 23;39(2):337-44. Epub 2011 May 23.

Department of Otolaryngology, Head and Neck Surgery, The Johns Hopkins University, 1550 Orleans Street, CRBII (Rm 574), Baltimore, MD 21231, USA.

Aberrant methylation of gene promoters and corresponding loss of gene expression plays a critical role in the initiation and progression of colorectal cancer. An IL-6-type cytokine receptor, leukemia inhibitory factor receptor (LIFR), is a component of cell-surface receptor complexes for multifunctional cytokines such as LIF. Herein, we report that LIFR is methylated in human colon cancer. LIFR promoter was methylated in primary tumor tissues with high frequency (65%, 52/80). Quantitative methylation-specific PCR (TaqMan-MSP) demonstrated differential promoter methylation of LIFR in primary colorectal cancer tissues as compared to normal colon tissues (5%, 4/80). LIFR methylation was not detectable in 13 normal colon mucosa samples obtained from patients without cancer. The mRNA expression of LIFR was significantly down-regulated in colon cancer tissues as compared to corresponding normal tissues. A strong expression of LIFR protein was observed in all non-malignant normal and adjacent normal colon mucosa tissues whereas down-regulated LIFR protein expression was observed in primary tumors. These results demonstrate that cancer-specific methylation and a specific decrease of LIFR expression are a common inactivation event in colon cancer development.
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http://dx.doi.org/10.3892/ijo.2011.1050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601031PMC
August 2011

BORIS binding to the promoters of cancer testis antigens, MAGEA2, MAGEA3, and MAGEA4, is associated with their transcriptional activation in lung cancer.

Clin Cancer Res 2011 Jul 10;17(13):4267-76. Epub 2011 May 10.

Department of Otolaryngology-Head, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Purpose: Aim of this study was to determine whether BORIS (Brother of the Regulator of Imprinted Sites) is a regulator of MAGEA2, MAGEA3, and MAGEA4 genes in lung cancer.

Experimental Design: Changes in expression of MAGEA genes upon BORIS induction/knockdown were studied. Recruitment of BORIS and changes in histone modifications at their promoters upon BORIS induction were analyzed. Luciferase assays were used to study their activation by BORIS. Changes in methylation at these promoters upon BORIS induction were evaluated.

Results: Alteration of BORIS expression by induction/knockdown directly correlated with expression of MAGEA genes. BORIS was enriched at their promoters in H1299 cells, which show high expression of these cancer testis antigens (CTA), compared with normal human bronchial epithelial (NHBE) cells which show low expression of the target CTAs. BORIS induction in A549 cells resulted in increased amounts of BORIS and activating histone modifications at their promoters along with a corresponding increase in their expression. Similarly, BORIS binding at these promoters in H1299 correlates with enrichment of activating modifications, whereas absence of BORIS binding in NHBE is associated with enrichment of repressive marks. BORIS induction of MAGEA3 was associated with promoter demethylation, but no methylation changes were noted with activation of MAGEA2 and MAGEA4.

Conclusions: These data suggest that BORIS positively regulates these CTAs by binding and inducing a shift to a more open chromatin conformation with promoter demethylation for MAGEA3 or independent of promoter demethylation in case of MAGEA2 and MAGEA4 and may be a key effector involved in their derepression in lung cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-0653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172963PMC
July 2011

Integrated, genome-wide screening for hypomethylated oncogenes in salivary gland adenoid cystic carcinoma.

Clin Cancer Res 2011 Jul 6;17(13):4320-30. Epub 2011 May 6.

Department of Otolaryngology-Head and Neck Surgery and Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Purpose: Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy that is poorly understood. To look for relevant oncogene candidates under the control of promoter methylation, an integrated, genome-wide screen was conducted.

Experimental Design: Global demethylation of normal salivary gland cell strains using 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA), followed by expression array analysis was conducted. ACC-specific expression profiling was generated using expression microarray analysis of primary ACC and normal samples. Next, the two profiles were integrated to identify a subset of genes for further validation of promoter demethylation in ACC versus normal. Finally, promising candidates were further validated for mRNA, protein, and promoter methylation levels in larger ACC cohorts. Functional validation was then conducted in cancer cell lines.

Results: We found 159 genes that were significantly re-expressed after 5-aza-dC/TSA treatment and overexpressed in ACC. After initial validation, eight candidates showed hypomethylation in ACC: AQP1, CECR1, C1QR1, CTAG2, P53AIP1, TDRD12, BEX1, and DYNLT3. Aquaporin 1 (AQP1) showed the most significant hypomethylation and was further validated. AQP1 hypomethylation in ACC was confirmed with two independent cohorts. Of note, there was significant overexpression of AQP1 in both mRNA and protein in the paraffin-embedded ACC cohort. Furthermore, AQP1 was upregulated in 5-aza-dC/TSA-treated SACC83. Finally, AQP1 promoted cell proliferation and colony formation in SACC83.

Conclusions: Our integrated, genome-wide screening method proved to be an effective strategy for detecting novel oncogenes in ACC. AQP1 is a promising oncogene candidate for ACC and is transcriptionally regulated by promoter hypomethylation.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131484PMC
July 2011

Evaluation of MYB promoter methylation in salivary adenoid cystic carcinoma.

Oral Oncol 2011 Apr 15;47(4):251-5. Epub 2011 Feb 15.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD, USA.

The transcription factor MYB was recently proposed to be a promising oncogene candidate in salivary gland adenoid cystic carcinoma (ACC). However, the up-regulation of MYB in ACC could not be explained solely by deletion of its 3' end. It is widely accepted that the promoter methylation status can regulate the transcription of genes, especially in human cancers. Therefore, it is important to know whether MYB promoter demethylation could explain the over-expression of MYB in ACC. By using the Methprimer program, we identified nine CpG islands in the promoter of MYB. All of these CpG islands were located within the -864 to +2082 nt region relative to the transcription start site of MYB. We then used bisulfite genomic sequencing to evaluate the methylation levels of the CpG islands of MYB in 18 primary ACC tumors, 13 normal salivary gland tissues and nine cancer cell lines. Using cell lines, we also determined the relative MYB expression levels and correlated these with the methylation levels. With bisulfite genomic sequencing, we found no detectable methylation in the CpG islands of MYB in either ACC or normal salivary gland tissues. There was a variable degree of MYB expression in the cell lines tested, but none of these cell lines demonstrated promoter methylation. Promoter hypomethylation does not appear to explain the differential expression of MYB in ACC. An alternative mechanism needs to be proposed for the transcriptional control of MYB in ACC.
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http://dx.doi.org/10.1016/j.oraloncology.2011.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065551PMC
April 2011

Value of chest CT in the diagnosis and management of tracheobronchial foreign bodies.

Pediatr Int 2011 Aug;53(4):515-8

Department of Otolaryngology, Shengjing Hospital, China Medical University, Shenyang, China.

Background: The aim of the present study was to investigate the value of chest multidetector computed tomography (CT) in the evaluation of children with suspected foreign body aspiration.

Methods: Chest CT was performed in 45 consecutive children with suspected foreign body aspiration, and plain chest X-ray was conducted at the same time. Multiplanar reformatted imaging was carried out after multidetector CT. Rigid bronchoscopy and removal of the foreign body was performed under general anesthesia.

Results: All 42 patients (100%) with tracheobronchial foreign bodies were identified on chest CT. Three patients avoided unnecessary operations due to negative CT scans. For the patients with tracheobronchial foreign bodies, the occurrence of unilateral hyperlucent lung and post-obstructive lobar or segmental infiltrates on plain chest X-ray was 42.9% (18/42) and 4.8% (2/42), respectively. Twenty-two of the 42 patients (52.4%) had no abnormalities on plain X-ray. The difference between multidetector CT and plain X-ray results was statistically significant (P < 0.001). Surgical plans were designed and appropriate foreign body forceps were selected based on the CT scans. All foreign bodies were removed successfully, and no severe complications were observed. The location, shape, and volume of the foreign bodies found at surgery were consistent with the CT images.

Conclusions: The diagnosis of foreign body aspiration of the airway in children can be accomplished by using chest multidetector CT. It is often useful in delineating the exact shape, location, volume and form of a bronchial foreign body and can help the surgeon plan for operative bronchoscopy and safe removal of the foreign body.
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http://dx.doi.org/10.1111/j.1442-200X.2010.03299.xDOI Listing
August 2011

Quantitative methylation profiles for multiple tumor suppressor gene promoters in salivary gland tumors.

PLoS One 2010 May 26;5(5):e10828. Epub 2010 May 26.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institution, Baltimore, Maryland, USA.

Background: Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected.

Methodology: DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results.

Principal Findings: The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-beta, and Timp3.

Conclusions/significance: Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877085PMC
May 2010

TKTL1 is activated by promoter hypomethylation and contributes to head and neck squamous cell carcinoma carcinogenesis through increased aerobic glycolysis and HIF1alpha stabilization.

Clin Cancer Res 2010 Feb 26;16(3):857-66. Epub 2010 Jan 26.

Department of Otolaryngology, Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA.

Purpose: This study aims to investigate the role of the aberrant expression of Transkelolase-like 1 (TKTL1) in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and to characterize TKTL1 contribution to HNSCC tumorigenesis through aerobic glycolysis and HIF1alpha stabilization.

Experimental Design: TKTL1 promoter hypomethylation and mRNA/protein aberrant expression were studied in human HNSCC tumor samples and normal mucosas. Oncogenic functions of TKTL1 were examined in HNSCC cell line panels and tumor xenograft models with TKTL1 expression construct. The metabolite levels of fructose-6-phosphate, glyceraldehydes-3-phosphate, pyruvate, lactate, and the levels of HIF1alpha protein and its downsteam glycolytic targets were compared between the TKTL1-expressing and vehicle-expressing HNSCC cells. Meanwhile, the effects of HIF1alpha/glycolytic inhibitors were evaluated on the TKTL1 transfectants.

Results: TKTL1 exhibits high frequency of promoter hypomethylation in HNSCC tumors compared with the normal mucosas, correlating with its overexpression in HNSCC. Overexpression of TKTL1 in HNSCC cells promoted cellular proliferation and enhanced tumor growth in vitro and in vivo. Overexpression of TKTL1 increased the production of fructose-6-phosphate and glyceraldehyde-3-phosphate, in turn elevating the production of pyruvate and lactate, resulting in the normoxic stabilization of the malignancy-promoting transcription factor HIF1alpha and the upregulation of downstream glycolytic enzymes. Notably, the reduction of TKTL1 expression decreased HIF1alpha accumulation and inhibition with HIF1alpha and/or the glycolysis inhibitor could abrogate the growth effects mediated by TKTL1 overexpression.

Conclusion: TKTL1 is a novel candidate oncogene that is epigenetically activated by aberrant hypomethlation and contributes to a malignant phenotype through altered glycolytic metabolism and HIF1alpha accumulation.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-2604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824550PMC
February 2010

Conservative management of transnasal intracranial injury.

Am J Otolaryngol 2011 Mar-Apr;32(2):165-7. Epub 2010 Jan 8.

Department of Otorhinolaryngology, Shengjing Hospital, China Medical University, Shenyang, China.

The purpose of this study was to explore the conservative management for an unusual case of transnasal intracranial injury. A 3-year-old female child presenting with transnasal injuries after a domestic accident whereby she apparently fell while holding a large pair of scissors, which then penetrated her left nasal cavity, piercing her nasal cavity, ethmoid sinus, and skull base. The scissors were removed from her nasal cavity. The patient had scant cerebrospinal rhinorrhea and no other additional neurologic deficits noted at the time, as well as no long-term developmental deficits. This report highlights the occurrence of this rare condition. The role of radiologic studies such as computed tomographic scans and plain films in diagnosis and management of this case is affirmed. The strategy of minimally invasive treatment of this injury can be a reasonable treatment option.
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http://dx.doi.org/10.1016/j.amjoto.2009.11.005DOI Listing
July 2011

Mitochondrial mutations in adenoid cystic carcinoma of the salivary glands.

PLoS One 2009 Dec 30;4(12):e8493. Epub 2009 Dec 30.

Department of Surgery, Division of Plastic and Reconstructive Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States of America.

Background: The MitoChip v2.0 resequencing array is an array-based technique allowing for accurate and complete sequencing of the mitochondrial genome. No studies have investigated mitochondrial mutation in salivary gland adenoid cystic carcinomas.

Methodology: The entire mitochondrial genome of 22 salivary gland adenoid cystic carcinomas (ACC) of salivary glands and matched leukocyte DNA was sequenced to determine the frequency and distribution of mitochondrial mutations in ACC tumors.

Principal Findings: Seventeen of 22 ACCs (77%) carried mitochondrial mutations, ranging in number from 1 to 37 mutations. A disproportionate number of mutations occurred in the D-loop. Twelve of 17 tumors (70.6%) carried mutations resulting in amino acid changes of translated proteins. Nine of 17 tumors (52.9%) with a mutation carried an amino acid changing mutation in the nicotinamide adenine dinucleotide dehydrogenase (NADH) complex.

Conclusions/significance: Mitochondrial mutation is frequent in salivary ACCs. The high incidence of amino acid changing mutations implicates alterations in aerobic respiration in ACC carcinogenesis. D-loop mutations are of unclear significance, but may be associated with alterations in transcription or replication.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008493PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795173PMC
December 2009

Hemimethylation footprints of DNA demethylation in cancer.

Epigenetics 2009 Apr 23;4(3):165-75. Epub 2009 Apr 23.

Hayward Human Genetics Center, Tulane Medical School, New Orleans, LA 70112, USA.

Hypomethylation of DNA repeats, including satellite 2 DNA (Sat2), is one of the most frequent epigenetic changes in cancer. We examined ovarian epithelial tumors and diverse control tissues for methylation on only one strand (hemimethylation), both strands (symmetrical methylation), or neither strand at Sat2 CpG dyads using hairpin genomic sequencing. Analysis of the resulting cloned DNA molecules indicated that although carcinomas displayed much symmetrical hypomethylation of CpG dyads, there was cancer-linked hypermethylation at one of the thirteen dyads in the examined 0.2 kb Sat2 region. Hemimethylated sites were seen in both carcinomas and controls but, importantly, in carcinoma DNA molecules, they were significantly more likely to occur in clusters displaying the same orientation (the same strand methylated). Our data suggest that hemimethylated CpG dyads are intermediates in active demethylation during carcinogenesis and not just due to a failure of maintenance methylation during replicative DNA synthesis. Constitutive heterochromatin may be especially suitable for providing a snapshot of demethylation intermediates because hemimethylation might be more long-lived in heterochromatin due to its highly condensed state.
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http://dx.doi.org/10.4161/epi.4.3.8277DOI Listing
April 2009

ICF, an immunodeficiency syndrome: DNA methyltransferase 3B involvement, chromosome anomalies, and gene dysregulation.

Autoimmunity 2008 May;41(4):253-71

Hayward Human Genetics Program, Tulane Medical School, New Orleans, LA 70112, USA.

The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) is the only disease known to result from a mutated DNA methyltransferase gene, namely, DNMT3B. Characteristic of this recessive disease are decreases in serum immunoglobulins despite the presence of B cells and, in the juxtacentromeric heterochromatin of chromosomes 1 and 16, chromatin decondensation, distinctive rearrangements, and satellite DNA hypomethylation. Although DNMT3B is involved in specific associations with histone deacetylases, HP1, other DNMTs, chromatin remodelling proteins, condensin, and other nuclear proteins, it is probably the partial loss of catalytic activity that is responsible for the disease. In microarray experiments and real-time RT-PCR assays, we observed significant differences in RNA levels from ICF vs. control lymphoblasts for pro- and anti-apoptotic genes (BCL2L10, CASP1, and PTPN13); nitrous oxide, carbon monoxide, NF-kappaB, and TNFalpha signalling pathway genes (PRKCH, GUCY1A3, GUCY1B3, MAPK13; HMOX1, and MAP4K4); and transcription control genes (NR2F2 and SMARCA2). This gene dysregulation could contribute to the immunodeficiency and other symptoms of ICF and might result from the limited losses of DNA methylation although ICF-related promoter hypomethylation was not observed for six of the above examined genes. We propose that hypomethylation of satellite 2 at 1qh and 16qh might provoke this dysregulation gene expression by trans effects from altered sequestration of transcription factors, changes in nuclear architecture, or expression of noncoding RNAs.
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http://dx.doi.org/10.1080/08916930802024202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430169PMC
May 2008

Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

Nucleic Acids Res 2008 Apr 16;36(7):2196-207. Epub 2008 Feb 16.

Human Genetics Program and Department of Biochemistry and Tulane Cancer Center, Tulane Medical School, Department of Mathematics, Tulane University, New Orleans, LA 70112, USA.

DNA methylation and chromatin DNaseI sensitivity were analyzed in and adjacent to D4Z4 repeat arrays, which consist of 1 to approximately 100 tandem 3.3-kb units at subtelomeric 4q and 10q. D4Z4 displayed hypomethylation in some cancers and hypermethylation in others relative to normal tissues. Surprisingly, in cancers with extensive D4Z4 methylation there was a barrier to hypermethylation spreading to the beginning of this disease-associated array (facioscapulohumeral muscular dystrophy, FSHD) despite sequence conservation in repeat units throughout the array. We infer a different chromatin structure at the proximal end of the array than at interior repeats, consistent with results from chromatin DNaseI sensitivity assays indicating a boundary element near the beginning of the array. The relative chromatin DNaseI sensitivity in FSHD and control myoblasts and lymphoblasts was as follows: a non-genic D4Z4-adjacent sequence (p13E-11, array-proximal)> untranscribed gene standards > D4Z4 arrays> constitutive heterochromatin (satellite 2; P < 10(-4) for all comparisons). Cancers displaying D4Z4 hypermethylation also exhibited a hypermethylation-resistant subregion within the 3.3-kb D4Z4 repeat units. This subregion contains runs of G that form G-quadruplexes in vitro. Unusual DNA structures might contribute to topological constraints that link short 4q D4Z4 arrays to FSHD and make long ones phenotypically neutral.
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http://dx.doi.org/10.1093/nar/gkn055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2367708PMC
April 2008

Interphase chromosomal abnormalities and mitotic missegregation of hypomethylated sequences in ICF syndrome cells.

Chromosoma 2005 Jul 27;114(2):118-26. Epub 2005 Apr 27.

Department of Clinical Genetics, University Hospital, 22185 Lund, Sweden.

The immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disease. Usually, it is caused by mutations in the DNA methyltransferase 3B gene, which result in decreased methylation of satellite DNA in the juxtacentromeric heterochromatin at 1qh, 16qh, and 9qh. Satellite II-rich 1qh and 16qh display high frequencies of abnormalities in mitogen-stimulated ICF lymphocytes without these cells being prone to aneuploidy. Here we show that in lymphoblastoid cell lines from four ICF patients, there was increased colocalization of the hypomethylated 1qh and 16qh sequences in interphase, abnormal looping of pericentromeric DNA sequences at metaphase, formation of bridges at anaphase, chromosome 1 and 16 fragmentation at the telophase-interphase transition, and, in apoptotic cells, micronuclei with overrepresentation of chromosome 1 and 16 material. Another source of anaphase bridging in the ICF cells was random telomeric associations between chromosomes. Our results elucidate the mechanism of formation of ICF chromosome anomalies and suggest that 1qh-16qh associations in interphase can lead to disturbances of mitotic segregation, resulting in micronucleus formation and sometimes apoptosis. This can help explain why specific types of 1qh and 16qh rearrangements are not present at high frequencies in ICF lymphoid cells despite diverse 1qh and 16qh aberrations continuously being generated.
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http://dx.doi.org/10.1007/s00412-005-0343-7DOI Listing
July 2005

Cytogenetic and immuno-FISH analysis of the 4q subtelomeric region, which is associated with facioscapulohumeral muscular dystrophy.

Chromosoma 2004 May 11;112(7):350-9. Epub 2004 May 11.

Human Genetics Program and Department of Biochemistry, Tulane Medical School, New Orleans, LA 70112, USA.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the shortening of a copy-number polymorphic array of 3.3 kb repeats (D4Z4) at one allelic 4q35.2 region. How this contraction of a subtelomeric tandem array causes FSHD is unknown but indirect evidence suggests that a short array has a cis effect on a distant gene or genes. It was hypothesized that the length of the D4Z4 array determines whether or not the array and a large proximal region are heterochromatic and thereby controls gene expression in cis. To test this, we used fluorescence in situ hybridization probes with FSHD and control myoblasts to characterize the distal portion of 4q35.2 with respect to the following: intense staining with the chromatin dye 4',6-diamidino-2-phenylindole; association with constitutively heterochromatic foci; extent of binding of heterochromatin protein 1alpha; histone H3 methylation at lysine 9 and lysine 4; histone H4 lysine 8 acetylation; and replication timing within S-phase. Our results indicate that 4q35.2 does not resemble constitutive heterochromatin in FSHD or control myoblasts. Furthermore, in these analyses, the allelic 4q35.2 regions of FSHD myoblasts did not behave differently than those of control myoblasts. Other models for how D4Z4 array contraction causes long-distance regulation of gene expression in cis need to be tested.
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http://dx.doi.org/10.1007/s00412-004-0280-xDOI Listing
May 2004
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