Malignant and nonmalignant T cell lines from human T cell lymphotropic virus type I-negative patients with Sézary syndrome.

The establishment of an in vitro model for cutaneous T cell lymphomas and Sézary syndrome has been difficult since T cells from individuals with these diseases do not proliferate in response to T cell mitogens. We found that conditioned media, collected from mitogen-activated PBMC from Sézary patients, contain an IL-2 receptor inducing factor. Despite their ostensible proliferative disorder, using a combination Sézary cell-conditioned media and rIL-2, we established IL-2 responsive, human T cell lymphotropic virus type I negative T cell lines from 23 patients, nine of which contain cells with the structural and/or genetic characteristics of neoplastic Sézary T cells.     

Transmembrane protein 106A is silenced by promoter region hypermethylation and suppresses gastric cancer growth by inducing apoptosis

Inactivation of tumour suppressor genes by promoter methylation plays an important role in the initiation and progression of gastric cancer (GC). Transmembrane 106A gene (TMEM106A) encodes a novel protein of previously unknown function. This study analysed the biological functions, epigenetic changes and the clinical significance of TMEM106A in GC. Data from experiments indicate that TMEM106A is a type II membrane protein, which is localized to mitochondria and the plasma membrane. TMEM106A was down‐regulated or silenced by promoter region hypermethylation in GC cell lines, but expressed in normal gastric tissues. Overexpression of TMEM106A suppressed cell growth and induced apoptosis in GC cell lines, and retarded the growth of xenografts in nude mice. These effects were associated with the activation of caspase‐2, caspase‐9, and caspase‐3, cleavage of BID and inactivation of poly (ADP‐ribose) polymerase (PARP). In primary GC samples, loss or reduction of TMEM106A expression was associated with promoter region hypermethylation. TMEM106A was methylated in 88.6% (93/105) of primary GC and 18.1% (2/11) in cancer adjacent normal tissue samples. Further analysis suggested that TMEM106A methylation in primary GCs was significantly correlated with smoking and tumour metastasis. In conclusion, TMEM106A is frequently methylated in human GC. The expression of TMEM106A is regulated by promoter hypermethylation. TMEM106A is a novel functional tumour suppressor in gastric carcinogenesis.     

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer

Chromosome region 3p12‐14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down‐regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation‐specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9‐transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E‐cadherin, VIM, SNAIL, VEGFA, NFκB‐p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFβ1/TβR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR‐ and TGFβ1/TβR(I/II)‐activated AKT signaling.     

SC5 mAb represents a unique tool for the detection of extracellular vimentin as a specific marker of Sezary cells

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4(+)CD45RO(+) T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.     

Subpopulations of human T lymphocytes. IV. Quantitation and distribution in patients with mycosis fungoides and Sézary syndrome.

T cell subpopulations (Tμ and Tγ cells) were examined in the peripheral blood from fourteen patients with mycosis fungoides and Sézary syndrome. One patient with Sézary syndrome having low lymphocyte count had higher proportions of Tγ cells when compared to controls while the other with high lymphocyte count (75% Sézary cells) lacked Tγ cells and had normal proportions of Tμ cells. T cells from a third patient with Sézary syndrome having high lymphocyte count (95% Sézary cells) lacked almost completely both Tμ and Tγ cells. Three of eleven patients with mycosis fungoides had a high proportion of Tγ cells and one had a high proportion of Tμ cells. Study of T cells in the peripheral blood, lymph nodes, and bone marrow from two patients with mycosis fungoides demonstrated that the quantitative abnormality of tμ and Tγ cells is shared by the peripheral blood and bone marrow and not by the lymph nodes. Heterogeneity of T cells subsets in mycosis fungoides appears to be in non-malignant T cells. However, in Sézary syndrome malignant Sézary T cells demonstrate heterogeneity with regard to receptors for IgM (Tμ) and IgG (Tγ).     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.     

miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.     

DNA甲基化通过锌指蛋白185调控慢性粒细胞白血病细胞的增殖

锌指蛋白185(ZNF185)属于LIM结构域蛋白,参与细胞的增殖和分化,在多种肿瘤细胞中具有抑癌基因的功能.ZNF185在正常人血液系统细胞中高表达,但目前对白血病细胞的作用未见研究.采用Western blot检测人外周血中性粒细胞、急性粒细胞白血病细胞系HL-60和慢性粒细胞白血病细胞系K562细胞中ZNF185的表达,发现ZNF185在HL-60和K562细胞中的表达水平显著低于外周血中性粒细胞.为了阐明ZNF185对慢性粒细胞白血病细胞增殖的影响,从人外周血中性粒细胞克隆ZNF185编码序列,转染K562细胞,MTT检测细胞增殖,发现过表达ZNF185显著抑制K562细胞的增殖.甲基化特异PCR分析表明:ZNF185启动子在HL-60和K562细胞中高甲基化,用5-氮杂-2′-脱氧胞苷处理K562细胞,促进ZNF185的表达,显著抑制细胞增殖.研究结果表明,ZNF185启动子高甲基化导致其在K562细胞中的表达降低和细胞增殖抑制作用减弱.可能是慢性粒细胞白血病发生或发展的原因之一.     

PRDM5 基因甲基化及在HTB-182、A549 中的表达

目的:本实验检测PRDM5在HTB-182、A549、HBE正常支气管上皮细胞系中甲基化状态及去甲基化处理对其表达的影响。方法:运用MSP甲基化特异性PCR检测PRDM5在A549、HTB-182和HBE正常支气管上皮细胞系的甲基化状态,再加入不同浓度的去甲基化药物检测PRDM5在A549、HTB-182和HBE正常支气管上皮细胞的甲基化状态,用RT-PCR检测PRDM5在加药前后的mRNA表达水平;用Western-Blot检测PRDM5在加药前后的蛋白表达水平。结果:在肺肿瘤细胞系中,PRDM5存在不同程度的高甲基化,加入不同浓度去甲基化药物后,甲基化表达水平逐渐减弱(P<0.05),mRNA表达水平逐渐增强(P<0.05),蛋白表达水平也逐渐增强(P<0.05)。结论:在肺癌细胞系中PRDM5启动子高甲基化是导致PRDM5表达水平降低的主要原因,PRD-M5启动子去甲基化可能成为肺癌治疗的新靶点。     

TMEM207 hinders the tumour suppressor function of WWOX in oral squamous cell carcinoma

The WW domain‐containing oxidoreductase (WWOX) functions as a tumour suppressor in oral carcinogenesis. As aberrant TMEM207 expression may lead to tumour progression by hampering the tumour suppressor function of WWOX in various cancers, we explored the expression and pathobiological properties of TMEM207, focusing on the WWOX‐mediated regulation of the HIF‐1α pathway in oral squamous cell carcinoma (OSCC). TMEM207 immunoreactivity was detected in 40 of 90 OSCC samples but not in neighbouring non‐tumorous epithelial tissues. Moreover, TMEM207 expression was significantly correlated with lymph node metastasis and poor prognosis. An in situ proximal ligation assay demonstrated the colocalization of TMEM207 and WWOX in invasive OSCC cells, especially glycogen‐rich ones. Enforced expression of TMEM207 abrogated the binding of WWOX to HIF‐1α, increased HIF‐1α and GLUT‐1 expression, even under normoxic conditions, and promoted tumour growth in a xenoplant assay using SAS tongue squamous cancer cells. In contrast, TMEM207 knockdown decreased GLUT‐1 expression in two OSCC cell lines. As a whole, our findings indicate that the aberrant expression of TMEM207 contributes to tumour progression in OSCC, possibly via promoting aerobic glycolysis.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.