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1.
Int J Gen Med ; 15: 1131-1146, 2022.
Article in English | MEDLINE | ID: mdl-35153505

ABSTRACT

PURPOSE: This study aimed to determine the potential application of the protein phosphatase 1 regulatory subunit 3 (PPP1R3B) gene as a prognostic marker in stomach adenocarcinoma (STAD), as well as its potential mediating biological processes and pathways. MATERIALS AND METHODS: Differential expression analyses were performed using the TIMER2.0 and UALCAN databases. Complete RNA-seq data and other relevant clinical and survival data were acquired from The Cancer Genome Atlas (TCGA). Univariate survival analyses, Cox regression modelling, and Kaplan-Meier curves were implemented to investigate the associations between PPP1R3B gene expression and clinical pathologic features. A genome wide gene set enrichment analysis (GSEA) was conducted to define the underlying molecular mechanisms mediating the observed associations between the PPP1R3B gene and STAD development. RESULTS: We found that PPP1R3B was overexpressed in STAD tissues, and that higher PPP1R3B expression correlated with worse prognoses in patients with STAD. Comprehensive survival analyses suggested that PPP1R3B might be an independent predictive factor for survival time in patients with STAD. The prognostic relationship between PPP1R3B and STAD was also verified using Kaplan-Meier curves. Patients with higher PPP1R3B levels had a shorter clinical survival time on average. Additionally, a GSEA demonstrated that PPP1R3B might be involved in multiple biological processes and pathways. CONCLUSION: Our findings demonstrate that the PPP1R3B gene has utility as a potential molecular marker for STAD prognoses.

2.
Biosci Rep ; 41(4)2021 04 30.
Article in English | MEDLINE | ID: mdl-33851708

ABSTRACT

OBJECTIVE: We aimed to explore the prognostic value of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes in gastric cancer (GC). METHODS: The RNA-sequencing (RNA-seq) expression data for 351 GC patients and other relevant clinical data were acquired from The Cancer Genome Atlas (TCGA). Survival analysis and a genome-wide gene set enrichment analysis (GSEA) were performed to define the underlying molecular value of the ADAMTS genes in GC development. Besides, qRT-PCR and immunohistochemistry were all employed to validate the relationship between the expression of these genes and GC patient prognosis. RESULTS: The Log rank test with both Cox regression and Kaplan-Meier survival analyses showed that ADAMTS6 expression profile correlated with the GC patients clinical outcome. Patients with a high expression of ADAMTS6 were associated with poor overall survival (OS). Comprehensive survival analysis of the ADAMTS genes suggests that ADAMTS6 might be an independent predictive factor for the OS in patients with GC. Besides, GSEA demonstrated that ADAMTS6 might be involved in multiple biological processes and pathways, such as the vascular endothelial growth factor A (VEGFA), kirsten rat sarcoma viral oncogene (KRAS), tumor protein P53, c-Jun N-terminal kinase (JNK), cadherin (CDH1) or tumor necrosis factor (TNF) pathways. It was also confirmed by immunohistochemistry and qRT-PCR that ADAMTS6 is highly expressed in GC, which may be related to the prognosis of GC patients. CONCLUSION: In summary, our study demonstrated that ADAMTS6 gene could be used as a potential molecular marker for GC prognosis.


Subject(s)
ADAMTS Proteins/metabolism , Biomarkers, Tumor/metabolism , Stomach Neoplasms/metabolism , ADAMTS Proteins/genetics , Biomarkers, Tumor/genetics , Female , Gene Regulatory Networks , Humans , Male , Middle Aged , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Survival Analysis
3.
Adv Healthc Mater ; 7(3)2018 02.
Article in English | MEDLINE | ID: mdl-28892262

ABSTRACT

A glycan-stimulated and poly(3,4-ethylene-dioxythiophene)s (PEDOT)-based nanomaterial platform is fabricated to purify circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This new platform, phenylboronic acid (PBA)-grafted PEDOT NanoVelcro, combines the 3D PEDOT nanosubstrate, which greatly enhances CTC capturing efficiency, with a poly(EDOT-PBA-co-EDOT-EG3) interfacial layer, which not only provides high specificity for CTC capture upon antibody conjugation but also enables competitive binding of sorbitol to gently release the captured cells. CTCs purified by this PEDOT NanoVelcro chip provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which may provide clinical insights into the disease.


Subject(s)
Biomarkers/analysis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Nanostructures/chemistry , Neoplastic Cells, Circulating/pathology , Polymers/chemistry , Prostatic Neoplasms/pathology , RNA/analysis , Cell Line, Tumor , Humans , Male , Neoplastic Cells, Circulating/metabolism , Prostatic Neoplasms/metabolism
4.
Oncol Lett ; 14(2): 2573-2579, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28789464

ABSTRACT

The present study aimed to determine the diagnostic concordance of plasma epidermal growth factor receptor (EGFR) mutation using droplet digital polymerase chain reaction (ddPCR) with tumor tissue samples and the predictive clinical significance of plasma EGFR mutation concentration. Plasma DNA samples from patients with non-small cell lung cancer (NSCLC) were analyzed for EGFR exon 21 codon 858 (L858R) mutation, deletion of exon 19 (ex19del) and exon 20 codon 790 (T790M) mutation using ddPCR. Firstly, the mutations in the plasma samples were compared with the matched tumor samples to determine the concordance. Secondly, image examination follow-ups were analyzed to assess the association between plasma EGFR mutation concentration and patients' response to EGFR-tyrosine kinase inhibitors (TKIs). A total of 51 patients with NSCLC were enrolled, including 48 newly diagnosed patients. Compared with tumor tissue samples, the sensitivity and specificity of ddPCR were 76.19% (16/21) and 96.55% (28/29) for mutant L858R, and 88.89% (8/9) and 100% (41/41) for ex19del, respectively. No patient exhibited the T790M mutation in the tumor tissue or plasma samples. Furthermore, 5 patients with the L858R mutation and 4 patients with ex19del in plasma and tumor tissue samples had been followed up with image examination for ≥3 months following EGFR-TKI treatment. The baseline mutant EGFR concentrations were positively correlated with a reduction in tumor burden (Spearman's r=0.7000, P=0.0358). When analyzed separately, ex19del concentrations (Spearman's r=1.0000, P<0.0001) were also positively correlated with the reduction, while mutant L858R concentrations were not (Spearman's r=0.7000, P=0.1881). In the present study, detection of plasma EGFR mutations using ddPCR exhibited sufficient concordance with tumor tissue sample results. Baseline plasma mutant EGFR and ex19del concentrations were significantly and positively correlated with response to EGFR-TKIs.

5.
Lung Cancer ; 109: 124-127, 2017 07.
Article in English | MEDLINE | ID: mdl-28577941

ABSTRACT

OBJECTIVES: Droplet digital polymerase chain reaction (ddPCR) has shown sufficient concordance in detecting plasma epidermal growth factor receptor (EGFR) status in non-small cell lung cancer (NSCLC), compared to tumor tissues. However, the clinical significance of the quantitative plasma mutated EGFR concentration remains unknown. The purpose of this study was to explore the relationship of plasma mutated EGFR concentration with tumor burden in advanced NSCLC patients. MATERIALS AND METHODS: Using ddPCR, plasma DNA samples prior to administration of therapies from 113 consecutive NSCLC patients were analyzed for EGFR L858R substitution and deletion of exon19 (ex19del). Plasma EGFR status was compared to tumor EGFR status to determine concordance. Then, we assessed the correlation of plasma mutated EGFR concentrations with tumor burden and other tumor characteristics. RESULTS AND CONCLUSION: Compared to tumor EGFR, the concordance rate of plasma and tissue EGFR status was 86.73%. Of the 64 patients who harbored tumor EGFR mutation, plasma mutated EGFR concentrations significantly correlated with number of metastatic sites (Spearman's r=0.4954, p<0.0001), number of lesions (Spearman's r=0.4484, p=0.0002), and sum of measurable lesions' diameters (Spearman's r=0.3539, p=0.0048). Number of metastatic sites was independently associated with mutated EGFR concentration in multiple linear regression. Besides, plasma mutated EGFR concentrations were significantly higher in those with extensive tumor burden (median concentration, 386.9 vs. 13.4copies/mL; p<0.0001) and stage IV disease (median concentration, 244.2 vs. 0copies/mL; p=0.0252). In conclusion, mutated plasma EGFR concentration determined by ddPCR analysis significantly correlated with tumor burden.


Subject(s)
Carcinoma, Non-Small-Cell Lung/diagnosis , ErbB Receptors/genetics , Lung Neoplasms/diagnosis , Mutation/genetics , Tumor Burden , Carcinoma, Non-Small-Cell Lung/genetics , DNA Mutational Analysis , ErbB Receptors/blood , Female , Humans , Lung Neoplasms/genetics , Male , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Prospective Studies , Sensitivity and Specificity
6.
Oncotarget ; 8(8): 13195-13205, 2017 Feb 21.
Article in English | MEDLINE | ID: mdl-28061461

ABSTRACT

Detection of circulating tumor DNA using droplet digital polymerase chain reaction (ddPCR) is a highly-sensitive, minimally invasive alternative to serial biopsies for assessment and management of cancer. We used ddPCR to assess the utility of measuring plasma concentrations of common epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, and T790M) in 57 non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs). High baseline plasma EGFR mutation (pEGFRmut) concentrations were associated with shorter progression-free survival (8.43 months) than low baseline pEGFRmut (16.23 months; p = 0.0019). By contrast, there were no differences in tumor shrinkage or overall survival between groups. During EGFR-TKI treatment, pEGFRmut levels decreased to zero in 89.58% of patients. Twenty-five of the 27 patients who progressed had basal pEGFRmut, and 18 also had circulating T790M. All 20 patients with dramatic progression (according to a categorization system for EGFR-TKIs failure) had basal pEGFRmut, and 13 had T790M mutation at progression. These results support the use of ddPCR for analysis of plasma EGFR mutations for prediction of PFS and to monitor clinical responses to EGFR-TKIs in NSCLC patients.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Mutation , Protein Kinase Inhibitors/therapeutic use , Aged , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/genetics , Disease-Free Survival , ErbB Receptors/blood , ErbB Receptors/genetics , Erlotinib Hydrochloride/therapeutic use , Female , Gefitinib , Humans , Kaplan-Meier Estimate , Lung Neoplasms/blood , Lung Neoplasms/genetics , Male , Middle Aged , Prognosis , Quinazolines/therapeutic use , Treatment Outcome
7.
Zhong Yao Cai ; 38(7): 1460-5, 2015 Jul.
Article in Chinese | MEDLINE | ID: mdl-26946844

ABSTRACT

OBJECTIVE: To study the effect of basil polysaccharide on the expression of histone methyltransferase G9a, demethylase JMJD1A and histone H3K9me2 methylation level in hepatoma cells MHCC97H and MHCC97L under hypoxic conditions, in order to explore the regulatory effect of basil polysaccharide on the epigenetics of hepatoma cells. METHODS: Cobalt chloride(CoCl2) was used to simulate hypoxic, MHCC97H and MHCC97L hepatoma cells hypoxia model was established in vitro,and then intervened with different concantration of basil polysaccharide intervened 24 h. The expression of HIF-1α, G9a and JMJD1A mRNA in hepatoma cells were detected by real time fluorescent quantitative PCR. The expression of HIF-lα, G9a, JMJD1A protein and histone H3K9me2 methylation level was detected by Western-blot method. RESULTS: Basil polysaccharide down-regulated the expression of HIF-lα, G9a, JMJD1A mRNA and protein and histone H3K9me2 methylation level in MHCC97H cells under hypoxic condition,and down-regulated the expression of HIF-lα mRNA and protein and histone H3K9me2 methylation level in MHCC97L cells under hypoxic condition(P <0. 05). CONCLUSION: Basil polysaccharide can regulate histone H3K9me2 methylation levels in hepatoma cells MHCC97H and MHCC97L which have different metastatic potential under hypoxic conditions. On hepatoma cell MHCC97H, the regulation of histone H3K9me2 methylation is associated with histone methyltransferase G9a and demethylase JMJDlA. In hepatoma cell MHCC97L, the regulation of histone H3K9me2 methylation was probably through other pathways.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Histones/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Ocimum basilicum/chemistry , Polysaccharides/pharmacology , Carcinoma, Hepatocellular/metabolism , Cell Hypoxia , Cell Line, Tumor/drug effects , Down-Regulation , Epigenesis, Genetic , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms , Methylation , RNA, Messenger
8.
Zhonghua Zhong Liu Za Zhi ; 35(4): 282-7, 2013 Apr.
Article in Chinese | MEDLINE | ID: mdl-23985257

ABSTRACT

OBJECTIVE: To investigate the expression of androgen receptor (AR) and hepatitis B virus X protein (HBx) in hepatocellular carcinoma (HCC), and analyze the relationship between AR and HBx expressions. METHODS: Tumor tissues and peritumoral tissues of 83 HBV-associated HCC cases were investigated in this study. Fourteen cases of HBV-negative HCC and 13 cases of hemangioma peritumoral tissues were considered as control. AR and HBx mRNA levels were determined by quantitative fluorescence real-time RT-PCR and their protein levels were assayed by Western blot. The expression of AR and HBx proteins in tissues were examined with EnVision immunohistochemical staining. The methylation status of AR promoter was determined using methylation-specific PCR (MSP). RESULTS: Both expression levels of AR mRNA and protein of the peritumoral tissues were significantly higher (0.17) than that of tumor tissues (0.09) in HBV-associated HCC (P < 0.01), but such a difference was not found in HBV-negative HCC (0.06 vs. 0.07, P > 0.05). The level of AR expression in peritumoral tissues was associated with tumor differentiation in HBV-associated HCC. AR mRNA and protein levels of peritumoral tissues in HBV-associated HCC were significantly higher than that in HBV-negative HCC and hemangioma (all P < 0.05). In the tumor tissues, HBV-associated HCC had significantly higher AR expression than HBV-negative HCC at mRNA level (P < 0.05), but not at protein level. Spearman rank correlation analysis showed that the AR mRNA or AR protein levels were positively correlated with HBx in both tumor and peritumoral tissues in HBV-associated HCC, but the expressions of AR and HBx were not associated with AR promoter methylation status. The relative expression levels of AR mRNA and protein in the HBV-associated peritumoral tissues were negatively correlated with tumor differentiation (r = -0.213, P < 0.05; r = -0.313, P < 0.05), the higher the AR expression, the poorer differentiation. But this correlation of AR mRNA and protein was not shown in the hepatocellular carcinoma tissues. CONCLUSIONS: HBx may enhance AR expression in HBV-associated HCC, but AR promoter demethylation maybe not been involved in its main mechanism. An increased AR expression is probably an early event during the development and progression of HBV-associated HCC, and AR expression in the peritumoral tissue is correlated with HBV-associated HCC differentiation. AR may play different roles in HBV-associated HCC and HBV-negative HCC.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Hepatitis B virus/isolation & purification , Liver Neoplasms/metabolism , Receptors, Androgen/metabolism , Trans-Activators/metabolism , Adult , Aged , Blotting, Western , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Cell Differentiation , DNA Methylation , Female , Hemangioma/metabolism , Humans , Immunohistochemistry , Liver/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/virology , Male , Middle Aged , Promoter Regions, Genetic , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Androgen/genetics , Viral Regulatory and Accessory Proteins
9.
Cell Rep ; 4(2): 362-75, 2013 Jul 25.
Article in English | MEDLINE | ID: mdl-23871671

ABSTRACT

A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.


Subject(s)
Huntington Disease/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Inhibitors of Activated STAT/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Aged , Aged, 80 and over , Amino Acid Sequence , Animals , Catalytic Domain , Drosophila , Female , HeLa Cells , Humans , Huntingtin Protein , Huntington Disease/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Molecular Sequence Data , Mutation , Protein Inhibitors of Activated STAT/genetics , Protein Processing, Post-Translational , Small Ubiquitin-Related Modifier Proteins/genetics , Transfection , Ubiquitin-Protein Ligases/metabolism
10.
Exp Mol Pathol ; 95(2): 144-50, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23797005

ABSTRACT

The aim of the present study was to optimize the pregelatinized starch technique for cell block preparation and apply this approach in cultured cells of all types of growing forms, suspension and adherent. In order to evenly mix the starch powder and the cell suspension, we crafted a special plastic dropper. To prove the effectiveness of this optimized technique we used different cell lines, NCI-H69, NCI-H345, HCT-116, SKBR3 and MDA-MB-231. The morphology features, immunocytochemistry (ICC) and fluorescent/chromogenic in-situ hybridization (FISH/CISH) on the cell block sections were evaluated. The morphology features, the ICC and ISH results of cell block sections prepared by the new method were satisfactory comparing with the results obtained in biopsies, the gold standard test for this kind of analysis. The most attractive advantage of our optimized pregelatinized starch technique is that this new method is based on cell suspensions instead of cell sediment, so with our technique every section will contain cells due to the even distribution of the starch powder and the cells forming a homogeneous cell block. To the authors' knowledge, this is the first description on cell block preparation based on cell suspension.


Subject(s)
Histocytological Preparation Techniques , Cell Line , Gelatin , Humans , Starch
11.
J Cardiothorac Surg ; 8: 146, 2013 Jun 07.
Article in English | MEDLINE | ID: mdl-23758949

ABSTRACT

Glomus tumor is usually a small, benign tumor and typically occurs in the dermis or subcutis or soft tissue of the extremities and rarely in the visceral locations. Its occurrence in the main bronchus is extremely rare. The current case reported a 30-year-old woman with dyspnea on exertion and hemoptysis, she had a glomus tumor which has large size, deep location and exhibits an infiltrative margin as well as increased atypical mitotic figures. These characteristics suggest malignant behavior. However, there is little data regarding glomus tumors arising in the bronchus, the need for caution in diagnosing this case as a malignant glomus tumor must be highlighted. Therefore, the diagnosis of bronchial glomus tumor of uncertain malignant potential was favored. To the best of our knowledge, both the type and the location of this glomus tumor are extremely rare. Accumulation of more cases are needed to clarify their diagnosis and significance since there is little data regarding glomus tumors arising in the bronchus.


Subject(s)
Bronchial Neoplasms/diagnosis , Bronchial Neoplasms/surgery , Glomus Tumor/diagnosis , Glomus Tumor/surgery , Adult , Biopsy , Bronchial Neoplasms/pathology , Bronchoscopy , Diagnosis, Differential , Diagnostic Imaging , Female , Glomus Tumor/pathology , Humans , Lymph Node Excision , Neoplasm Invasiveness
12.
J Cardiothorac Surg ; 8: 131, 2013 May 17.
Article in English | MEDLINE | ID: mdl-23684153

ABSTRACT

We herein reported a primary pulmonary papillary carcinoma with colloid-like luminal content in the glandular cavity and classic nuclear features such as pseudo-inclusions, intranuclear grooves in the tumor cell nuclei and ground glass nuclei which closely mimics papillary thyroid carcinoma. Meanwhile, lymph node in the left pulmonary hilum was involved and showed similar features to the primary pulmonary papillary carcinoma. This specific histopathological presentation caused a diagnostic dilemma.The patient didn't show previous concomitant or subsequent evidence of a thyroid tumor. Immunohistochemistry further confirmed pulmonary origin and excluded a metastasis from the thyroid, as it was thyroglobulin negative, thyroid transcription factor 1 and surfactant apoprotein A positive, which was consistent with the imageology and history.Based on the above features, the diagnosis of primary pulmonary papillary carcinoma was confirmed. Understanding the existence of papillary thyroid carcinoma-like pulmonary papillary carcinoma will avoid misdiagnosis or unnecessary clinical and radiologic investigations in future.


Subject(s)
Adenocarcinoma/diagnostic imaging , Adenocarcinoma/surgery , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/surgery , Adenocarcinoma/pathology , Aged, 80 and over , Carcinoma/diagnostic imaging , Carcinoma, Papillary , Diagnosis, Differential , Fatal Outcome , Hemorrhoidectomy , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Lymph Node Excision , Male , Pneumonectomy , Radiography , Thyroid Cancer, Papillary , Thyroid Neoplasms/diagnostic imaging , Ultrasonography
13.
Cell Tissue Res ; 350(2): 395-400, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22895623

ABSTRACT

Cell block sections serve as an important diagnostic annex for cytological smears, liquid-based SurePath cytology and the Liquid-based Thin-prep Cytology Test (TCT). A variety of methods for the preparation of cell blocks are described in the literature and the techniques in cell blocks are in continuous improvement. A new technique for making cell blocks was introduced in the present study. We first used pregelatinized starch as the frame for the cell block, which is a really simple and economic method, because it can be carried out at room temperature without additional special instruments. We have performed hematoxylin and eosin (HE) staining, immunohistochemistry analysis and fluorescence in situ hybridization (FISH) in the cell block sections in 122 cytological specimens. The results demonstrated in this article show that pregelatinized starch is a useful frame for cell blocks. The pregelatinized starch can effectively collect even a few cells with powerful adhesiveness. Therefore, this new technique for making cell blocks is especially useful for cytologic samples with low cellularity, such as cerebrospinal fluid specimens.


Subject(s)
Cytological Techniques/instrumentation , Cytological Techniques/methods , Adult , Aged , Aged, 80 and over , Female , Hematoxylin/chemistry , Humans , Immunohistochemistry , Male , Middle Aged , Young Adult
14.
Acta Virol ; 56(2): 101-10, 2012.
Article in English | MEDLINE | ID: mdl-22720699

ABSTRACT

UNLABELLED: Previously, we have found a new mutation at nt 1726-1730 that is associated with lower hepatitis B virus (HBV) DNA levels in the liver, and mutations at nt 1762/1764 that are correlated with higher HBV DNA levels. To confirm the effects of these mutations on the virus replication efficiency, substitutions nt 1726-1730 CTGAG and A1762T/G1764A in the HBV X (HBX) gene region were investigated alone or in combination. Cells Huh-7 or HepG2 were transfected with these constructs. The effects of these mutations on HBV were investigated at the gene and protein levels. The double mutation A1762T/G1764A increased whereas the nt 1726-1730 CTGAG mutations decreased the levels of released virion-associated and intracellular HBV DNA. The combined mutations had no appreciable effect on the replication capacity of the virus. Cells bearing the constructs with double mutations A1762T/G1764A contained the lowest levels of hepatitis B e antigen (HBeAg). Lowest expression of HBV X protein was in constructs that had both A1762T/G1764A and 1726-1730 CTGAG mutations. We think that changes in secondary RNA structure that were caused by these mutations might have been responsible for those results. KEYWORDS: hepatitis B virus; X gene; mutants; replication.


Subject(s)
Hepatitis B virus , Promoter Regions, Genetic , Hepatitis B e Antigens , Hepatitis B virus/genetics , Humans , Mutation , Virus Replication
15.
Histol Histopathol ; 26(1): 23-35, 2011 01.
Article in English | MEDLINE | ID: mdl-21117024

ABSTRACT

A remarkable feature of HBV-associated HCC is male predominance. The cooperation of hepatitis B virus X protein (HBx) with androgen receptor (AR) signaling pathway has been documented to contribute to this dominance. HBx, a multifunctional viral regulator, has been documented to induce promoter hypermethylation and low expression of tumor suppressor genes via activation of DNA methyl-transferase (DNMT) in hepatocarcinogenesis. In prostate cancer, hypermethylation of AR promoter is associated with loss of AR expression. However, the relationship among HBx, DNMTs, the methylation status of AR and AR expression in HBV-associated HCC is still unknown. In this report, we found that HBx correlated with high levels of AR in HCC cases and induced AR expression by stimulating its transcription in liver cell lines. HBx correlated with high expression of DNMTs in HCC cases too. Both in vivo and in vitro, however, the expression of AR was not associated with its promoter methylation status, and the methylation status of AR was not regulated by DNMTs. AR expression is higher in peritumoral tissues than in tumors, as well as being higher in HBV-associated HCC than in HBV-negative cases. Therefore, HBx-induced high expression of AR plays a role during hepatocarcinogenesis, but is not involved with its promoter methylation or DNMTs. HBx-mediated DNMT deregulation is gene-specific, and the expression and methylated regulation of AR is tissue-specific.


Subject(s)
Carcinoma, Hepatocellular/etiology , Hepatitis B/complications , Liver Neoplasms/etiology , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Trans-Activators/metabolism , Adult , Aged , Base Sequence , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , DNA Primers/genetics , Female , Hepatitis B/genetics , Hepatitis B/metabolism , Hepatitis B/virology , Hepatitis B virus/metabolism , Hepatitis B virus/pathogenicity , Humans , In Vitro Techniques , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/virology , Male , Middle Aged , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Signal Transduction , Viral Regulatory and Accessory Proteins
16.
Exp Mol Pathol ; 89(3): 268-75, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20620135

ABSTRACT

The hepatitis B virus×protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanism remains unknown. The aim of this study is to identify underlying mechanisms involved in HBx-mediated epigenetic modification in the process of HBx induced p16(INK4A) promoter hypermethylation. Liver cell lines were stably transfected with HBx-expressing vector. The methylation status of p16(INK4A) was examined by methyl-specific polymerase chain reaction (MSP) and bisulfite sequencing. Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR), Western blot and immunohistochemistry were used to analyze the expression of HBx, HBx-mediated DNA methylation abnormalities and p16(INK4A). Some cases of HCC and corresponding noncancerous liver tissues were studied. HBx up-regulates DNMT1 and DNMT3A expression in both mRNA level and protein level, and HBx represses p16(INK4A) expression through inducing hypermethylation of p16(INK4A) promoter. Moreover, HBx induces hypermethylation of p16(INK4A) promoter through DNMT1 and DNMT3A. Regulation of DNMT1 and DNMT3A by HBx promoted hypermethylation of p16(INK4A) promoter region. HBx-DNMTs-p16(INK4A) promoter hypermethylation may suggest a mechanism for tumorigenesis during hepatocarcinogenesis.


Subject(s)
Carcinoma, Hepatocellular/virology , DNA (Cytosine-5-)-Methyltransferases/biosynthesis , Gene Expression Regulation, Neoplastic , Genes, p16 , Liver Neoplasms/virology , Trans-Activators/metabolism , Adult , Aged , Blotting, Western , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1 , DNA Methylation/genetics , DNA Methyltransferase 3A , Female , Humans , Immunohistochemistry , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Male , Middle Aged , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Up-Regulation , Viral Regulatory and Accessory Proteins
17.
J Cell Biol ; 187(7): 1083-99, 2009 Dec 28.
Article in English | MEDLINE | ID: mdl-20026656

ABSTRACT

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species.


Subject(s)
I-kappa B Kinase/physiology , Lysosomes/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Animals , Brain/metabolism , Cell Line , Humans , Huntingtin Protein , Mice , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/chemistry , Nuclear Proteins/analysis , Nuclear Proteins/chemistry , Phosphorylation , Protein Structure, Tertiary , Rats , Rats, Sprague-Dawley , Solubility , Ubiquitination
18.
J Biol Chem ; 284(43): 29427-36, 2009 Oct 23.
Article in English | MEDLINE | ID: mdl-19710014

ABSTRACT

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH(2)-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.


Subject(s)
Huntington Disease/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Threonine/metabolism , Acetylation , HeLa Cells , Humans , Huntingtin Protein , Huntington Disease/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Phosphorylation , Protein Structure, Tertiary , Threonine/chemistry , Threonine/genetics
19.
Science ; 304(5667): 100-4, 2004 Apr 02.
Article in English | MEDLINE | ID: mdl-15064418

ABSTRACT

Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation.


Subject(s)
Huntington Disease/pathology , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , SUMO-1 Protein/metabolism , Animals , Animals, Genetically Modified , Cell Line , Cell Nucleus/metabolism , Corpus Striatum/cytology , Cytoplasm/metabolism , Drosophila , Genes, MDR , HeLa Cells , Humans , Huntingtin Protein , Huntington Disease/metabolism , Lysine/genetics , Lysine/metabolism , Mutation , Nerve Degeneration , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neurons/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Proline/genetics , Proline/metabolism , Promoter Regions, Genetic , Rats , Recombinant Fusion Proteins/metabolism , SUMO-1 Protein/genetics , Transcription, Genetic , Transfection , Ubiquitin/metabolism
20.
Nat Genet ; 30(4): 367-76, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11925563

ABSTRACT

Huntington disease is caused by the expansion of a polyglutamine repeat in the Huntingtin protein (Htt) that leads to degeneration of neurons in the central nervous system and the appearance of visible aggregates within neurons. We have developed and tested suppressor polypeptides that bind mutant Htt and interfere with the process of aggregation in cell culture. In a Drosophila model, the most potent suppressor inhibits both adult lethality and photoreceptor neuron degeneration. The appearance of aggregates in photoreceptor neurons correlates strongly with the occurrence of pathology, and expression of suppressor polypeptides delays and limits the appearance of aggregates and protects photoreceptor neurons. These results suggest that targeting the protein interactions leading to aggregate formation may be beneficial for the design and development of therapeutic agents for Huntington disease.


Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Peptides/chemistry , Animals , Blotting, Western , COS Cells , Cell Line , Cells, Cultured , DNA, Complementary/metabolism , Drosophila , Epitopes , Glutathione Transferase/metabolism , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Microscopy, Video , Molecular Sequence Data , Mutation , Neurons/metabolism , Photoreceptor Cells, Invertebrate/metabolism , Plasmids , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Amino Acid , Suppression, Genetic , Time Factors , Transfection
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