WWOX Research Articles

Effects of 5-Aza-2′-deoxycytidine on the methylation state and function of the WWOX gene in the HO-8910 ovarian cancer cell line

The aim of this study was to explore the effects of 5-Aza-2′-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor, on the methylation state and function of the WWOX gene in the HO-8910 ovarian cancer cell line. The HO-8910 cells were divided into two groups, a control group and a 5-Aza-CdR-treated group. The methylation state of the WWOX gene was evaluated using a methylation-specific PCR assay. The effect of 5-Aza-CdR on the HO-8910 cells was analyzed using MTT and cell invasion assays, as well as flow cytometry. The animal models were established by intraperitoneal transplantation of the cells into nude mice. Following treatment with 5-Aza-CdR, a demethylation state was detected in the HO-8910 cells. WWOX protein expression was significantly higher in the 5-Aza-CdR-treated group compared with that in the control group. The cell growth rate at each tested time point and the number of invasive cells were lower in the 5-Aza-CdR-treated group compared with that in the control group. Flow cytometry revealed that 67.13% of the cells were arrested at the G0/G1 stage in the 5-Aza-CdR-treated group. The tumorigenic ability of the 5-Aza-CdR-treated group was lower compared with that of the control group. In conclusion, the methylation state of the WWOX gene in HO-8910 cells may be reversed using 5-Aza-CdR, which may also inhibit the growth of these cells.

New molecular insights into osteosarcoma targeted therapy

Recent translational studies in osteosarcoma are discussed with the purpose to shed light on the new molecular therapeutic targets. The genetic aberrations of vascular endothelial growth factor (VEGF), mammalian target of rapamycin, Wnt signaling pathway, the inactivation of p53, Rb, WWOX genes, and amplification of APEX1, c-myc, RECQL4, RPL8, MDM2, VEGFA might be involved in the pathogenesis of osteosarcoma. The promising therapeutic targets for osteosarcoma patients include: integrin, ezrin, statin, NOTCH/HES1, matrix metalloproteinases (MMPs), m-calpain, and Src, which are involved in tumor cell invasion and metastasis; aldolase A, fructose-bisphosphate, sulfotransferase family 3A, member 1, BCL2-associated athanogene 3, heat shock protein 70 (HSP70), B-cell lymphoma 2-interacting mediator (BIM), polo-like kinase 1, hypoxia inducible factor 1, alpha subunit, minibrain-related kinase, Bcl-xl, caspase-3, midkine, high mobility group box 1 protein (HMGB1), and Beclin1, which are involved in tumor proliferation and apoptosis; met proto-oncogene (hepatocyte growth factor receptor), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, insulin-like growth factor (IGF)-1R, fms-related tyrosine kinase 4, platelet-derived growth factor receptor, beta polypeptide, IGF-I/II, and c-kit, which are involved in tumor growth; endosialin, VEGF, thrombin, and MMPs, which are involved in tumor angiogenesis; transforming growth factor-α/β, parathyroid hormone-like hormone, interleukin-6, interleukin-11, receptor activator of nuclear factor-κB ligand, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1, and cathepsin, which are involved in osteoclast function; Myc, HSP90, p-Met, p-Akt, p-STAT3, and cyclin D1, which are transcriptional factors; p-GP, hydroxysteroid (17-beta) dehydrogenase 10, HMGB1, BIM, inorganic phosphate, Bcl-2, PARP, mdm2, p21, Bax, and mitogen-activated protein kinase 1, which are involved in drug sensitivity. Furthermore, microRNAs such as miR-215 are also therapeutic targets. These translational studies in osteosarcoma have identified new molecular targets for osteosarcoma.

Common chromosomal fragile site FRA16D tumor suppressor WWOX gene expression and metabolic reprograming in cells

The WWOX gene spans the FRA16D common chromosomal fragile site and is able to suppress tumor growth. FRA16D is a frequent site of DNA instability in cancer resulting in reduced levels of WWOX expression. Altered levels of WWOX have been shown to affect metabolism. Whereas metabolic reprograming of cells from oxidative phosphorylation to aerobic glycolysis is a major hallmark of tumors, the relationship between common chromosomal fragile site genes and altered metabolism has been unclear. Here we report that altering metabolism from glycolysis to oxidative phosphorylation causes stable increase in steady-state levels of transcripts of the WWOX gene. Consistent with this, exposure to hypoxic conditions, in which cells rely on glycolysis, causes a downregulation of WWOX mRNA. The function of WWOX is therefore intimately integrated with metabolism, as WWOX not only contributes to the metabolic state of cells, its transcript levels are also linked to intracellular metabolic state.

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose-dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogen-induced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p=0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.

Abstract 1217: Chromosomal imbalances detected by microarray CGH in malignant pleural mesothelioma.

Abstract Background CDKN2A (9p21 locus) and NF2 (22q locus) genes are inactivated in most Malignant Pleural Mesotheliomas (MPM). Few other recurrent molecular abnormalities have been described as well. A better understanding of the molecular pathways is needed to identify potential additional driver genes. We did an integrated analysis of chromosomal copy number variations from MPM specimens and cells lines. Patients and Methods In order to describe genomic profiles, we performed microarray Comparative Genomic Hybridization (CGH) (Agilent 400K). Analyses were carried out using genomic DNA samples extracted from 26 MPM primary tumours (frozen sections) and 5 MPM cell lines (Four ATCC cell lines and one cell line established in our laboratory from a pleural effusion). Subsequently the results were correlated with the baseline characteristics of the patients. Results The mean age of the patients was 60 years (30-81). The sex ratio (M/F) and the histologic epithelioid type were 1.6 and 69% respectively. Our results showed that MPM are characterized by a complex pattern of genetic changes where losses of genomic regions are more frequent than gains. Common losses (30-70%) were clustered at chromosomal regions: 1p36.13, 1p13.3, 1q21.3-1q24.2, 1p22.3-1p12 (41%) (NRAS gene), 1p36.33-1q21.2 (30%) (JUN gene) 3p22.1-3p14.2 (59%) (BAP1, RASSF1 genes), 4q13.2, 6p21.32, 6q14.1-6q27 (63%) (ROS1 gene), 7q36.1, 8p23.1, 9p24.1, 9p22.1-9p21 (52%) (CDKN2A gene), 10p11.1-10q26.3 (30%) (RET, PTEN genes), 11q25, 14q11.2, 14q32.33, 15q11.2, 15q14, 22q11.22-22q13.33 (63%) (NF2 gene). Common gains (30-50%) were located at chromosomal regions 1q44, 8p11.22, 11q11, 12p13.31, 15q11.2, 16p11, 20p13, 22q11.23, Xq22.2. The most common deletions (D) and amplifications (A) (>20%) defined as having a log2(ratio) ≤-1.0 and ≥1.0 respectively were located at chromosomal regions: 1p31.1 (A, D), 1q21, 1q24.2 (D), 6p21.32, 6q16.3, 6q27 (D), 7q22.1 (D), 8p11.22 (A), 9p21.3, 9q34.2 (D), 12p13.2 (D), 14q32.33 (D), 16q23.1, 16q22.1 (WWOX gene)(D), 20p13 (A, D), 22q11.23 (D). Higher frequency of genetic alterations was noted in cell lines than tumour specimens. Different patterns of genetic changes were observed according to gender, stage, histology, smoking history, asbestos exposure and performance status. Conclusion Our study confirmed the complexity pattern of genetic changes in MPM through high-resolution microarray CGH analysis. We found high number of chromosomal imbalances not yet well identified. Some correspond to known genes and others could be the areas of unknown genes involved in its development that we will try to identify thereafter. Moreover, identifying patient subgroups seems essential for having a more individualized therapy and personalized medicine in MPM (e.g. MOSCATO trial, ongoing in our cancer center). Citation Format: Jacques Raphael, Nicolas Dorvault, Cedric Orear, Bastien Job, Vincent Thomas de Montpreville, Francois Leroy-Ladurie, Philippe Girard, Pierre Validire, Gwenael Le Teuff, David Planchard. Chromosomal imbalances detected by microarray CGH in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1217. doi:10.1158/1538-7445.AM2013-1217

P73 participates in WWOX-mediated apoptosis in leukemia cells

The WWOX gene is considered to be a tumor-suppressor gene which encodes a protein (Wwox) implicated in various types of solid human cancers. It has been shown that overexpression of WWOX in human tumors promotes apoptosis in vitro and suppresses tumor growth in vivo. Recently, we investigated the effects of WWOX overexpression in vitro and observed marked growth arrest in human leukemia cells; however, the underlying mechanism(s) for this effect is unknown. The present study aimed to elucidate the primary mechanism(s) underlying WWOX-mediated apoptosis in human leukemia. We traced the interactions between WWOX and its associated factors p73 and p53 after WWOX overexpression was induced in Jurkat and K562 cells. Our data revealed that p73 participates in WWOX-mediated apoptosis in Jurkat and K562 cells through binding with Wwox in the cytoplasm without a nuclear-cytoplasmic translocation.

Tumor Suppressor WWOX and p53 Alterations and Drug Resistance in Glioblastomas

Tumor suppressor p53 are frequently mutated in glioblastomas (GBMs) and appears to contribute, in part, to resistance to temozolomide (TMZ) and therapeutic drugs. WW domain-containing oxidoreductase WWOX (FOR or WOX1) is a proapoptotic protein and is considered as a tumor suppressor. Loss of WWOX gene expression is frequently seen in malignant cancer cells due to promoter hypermethylation, genetic alterations, and translational blockade. Intriguingly, ectopic expression of wild type WWOX preferentially induces apoptosis in human glioblastoma cells harboring mutant p53. WWOX is known to physically bind and stabilize wild type p53. Here, we provide an overview for the updated knowledge in p53 and WWOX, and postulate potential scenarios that wild type and mutant p53, or isoforms, modulate the apoptotic function of WWOX. We propose that triggering WWOX activation by therapeutic drugs under p53 functional deficiency is needed to overcome TMZ resistance and induce GBM cell death.

Role of WWOX WOX1 in Alzheimer s disease pathology and in cell death signaling

Alzheimer's disease (AD) is the most common form of dementia with a progressive course. AD pathology is a manifestation of the underlying severity and neuroanatomic involvement of specific vulnerable brain regions and circuits that are responsible for neuronal dysfunction and death. The etiology of AD is largely unknown. It has been hypothesized that multiple factors, including genetic components, oxidative stress, intracellular or extracellular accumulation of amyloid, dysfunction of cystoskeletal and synapse components, neuronal loss by apoptosis, neuronal excitotoxicity, inflammation, mitochondria dysfunction, etc., may play important roles in the onset of the disease. WWOX/WOX1 is a candidate tumor suppressor. HumanWWOXgene, encoding the WW domain-containing oxidoreductase (designated WWOX, FOR, or WOX1) protein, has been mapped to a fragile site on the chromosome ch16q23.3-24.1. Functionally, the WW domain is not only a tumor suppressor, but also a participant in molecular interactions, signaling, and apoptosis in many diseases. In this article, we review the potential mechanism by which WWOX/WOX1 may participate in the pathogenesis of AD with a focus on cell death signaling pathways in neurons.

Methylation State of WWOX Gene Promoter CpG Islands in Epithelial Ovarian Cancer and Its Clinical Significance

Methylation State of WWOX Gene Promoter CpG Islands in Epithelial Ovarian Cancer and Its Clinical Significance

Genetic alterations of WWOX in Wilms’ tumor are involved in its carcinogenesis

Loss of heterozygosity (LOH) in 16q appears in ~20-30% cases of Wilms' tumor. Within this region, known as common fragile site FRA16D, the WWOX tumor suppressor gene is located. Abnormalities of WWOX gene expression levels were observed in many tumor types and were associated with worse prognosis. The purpose of this study was to investigate the role of the WWOX tumor suppressor gene in Wilms' tumor samples. We evaluated the correlation between expression of WWOX and genes involved in proliferation (Ki67), apoptosis (BCL2, BAX), signal transduction (ERBB4, ERBB2, EGFR), cell cycle (CCNE1, CCND1), cell adhesion (CDH1) and transcription (TP73) using real-time RT-PCR in 23 tumor samples. We also analyzed the potential causes of WWOX gene expression reduction i.e., promoter methylation status (MethylScreen method) and loss of heterozygosity (LOH) status. We revealed a positive correlation between WWOX expression and BCL2, BCL2/BAX ratio, EGFR, ERBB4 isoform JM-a, TP73 and negative correlation with both cyclins. Loss of heterozygosity of the WWOX gene was observed only at intron 8, however, it had no influence on the reduction of its expression levels. Contrary to LOH, methylation of the region covering the 3' end of the promoter and part of exon 1 was associated with statistically significant reduction of WWOX gene expression levels. In the present study we reveal that in Wilms' tumors the WWOX expression levels are positively associated with the process of apoptosis, signal transduction through the ErbB4 pathway and EGFR and negatively with the regulation of the cell cycle (by cyclin E1 and D1). Moreover, our analysis indicates that in this type of tumor the expression of the WWOX gene can be regulated by an epigenetic mechanism--its promoter methylation.

WWOX-mediated apoptosis in A549 cells mainly involves the mitochondrial pathway

The human WWOX gene, known as WW domain-containing oxidoreductase, is located on 16q23.3-24.1, a chromosome region that spans the common fragile site, FRA16D. Abnormal transcripts or even loss of expression are frequently found in a number of cancer cell types, including breast, ovarian, prostate and lung cancer cells. It has therefore been proposed that the WWOX gene encodes a candidate tumor suppressor, possibly a pro-apoptotic protein. However, the mechanism behind this is not entirely clear. In the present study, we examined the pro-apoptotic action of WWOX using transient expression in A549 cells. We observed that the ectopic expression of WWOX caused apoptosis in A549 cells. We further observed procaspase-3 and procaspase-9 activation and the release of cytochrome C from the mitochondria in A549 cells transfected with pcDNA3.0-WWOX. These data indicate that WWOX induces apoptosis in A549 cells via the mitochondrial pathway.

WWOX/WOX1 is essential in UV irradiation/frostbite‐induced membrane bubbling

Overexposure to extreme cold, along with incidental UV irradiation, causes tissue damage and death ‐ the so called frostbite. Time lapse imaging analysis showed that post UV irradiation, COS7 fibroblasts undergo tiny, fluctuating membrane blebbing and then explode with a big bubble from the nucleus. Cold shock at 4oC significantly enhances the UV‐induced bubbling event. UV energy appears to be absorbed by the nucleus, so as to punch off a tiny nuclear pore that allows pressure release from the nucleus and subsequent plasma membrane exploding, as evidenced by using ECFP‐tagged nuclear protein. Unlike apoptosis, these COS7 cells exhibit little or no DNA fragmentation, cell shrinkage and phosphatidylserine expsoure. Blebbistatin, a blebs formation inhibitor, failed to prevent cells from bubbling up. Intriguingly, bubbling of Wwox gene knock out MEF (Wwox−/−) cells are significantly reduced, suggesting a crucial role of tumor suppressor WWOX/WOX1 in the bubbling event. Mechanistically, WWOX/WOX1 dissociates from TRAF2 for mitochondrial and nuclear relocation that contributes, in part, bubbling and death. [Supported by NSC and NHRI, Taiwan, and DoD, USA]

Overexpression of WW domain-containing oxidoreductase WOX1 preferentially induces apoptosis in human glioblastoma cells harboring mutant p53

PurposeHuman WWOX gene encoding WW domain-containing oxidoreductase, named WWOX, FOR, or WOX1, has been studied in various types of cancer cells and shown to be a tumor suppressor with pro-apoptotic properties. Mutation or gain-of-function of p53 in glioma cells is associated with resistance to radiation therapy and poor prognosis. In this study, we overexpressed WOX1 to examine the pro-apoptotic activity against human glioblastoma cells harboring mutant p53. MethodsOverexpression of WOX1 in glioblastoma cell lines and apoptosis-related assays were performed. ResultsOur results showed that overexpressed WOX1 induced apoptosis of glioblastoma U373MG harboring mutant p53 by causing hypoploidy and DNA fragmentation. However, ectopic WOX1 had no effect with U87MG possessing wild type p53. Unlike temozolomide, WOX1 induced apoptosis of U373MG cells via a mitochondria-independent and caspase-3-independent pathway. ConclusionsOverexpression of WOX1 preferentially inhibited viability and induced apoptosis in human glioblastoma cells expressing mutant p53 via a mechanism independent of the intrinsic apoptotic pathway. Conceivably, the survival of human glioblastoma cells depends upon interactions between the gain-of-function of p53 and WOX1. This suggests that modulation of WOX1 expression may be a novel strategy for treating human glioblastoma cells with mutant p53.

Natural antioxidants in prevention and management of Alzheimer's disease

Alzheimer’s disease (AD) is the most common form of dementia with a progressive course. AD pathology is a manifestation of the underlying severity and neuroanatomic involvement of specific vulnerable brain regions and circuits that are responsible for neuronal dysfunction and death. The etiology of AD is largely unknown. It has been hypothesized that multiple factors, including genetic components, oxidative stress, intracellular or extracellular accumulation of amyloid, dysfunction of cystoskeletal and synapse components, neuronal loss by apoptosis, neuronal excitotoxicity, inflammation, mitochondria dysfunction, etc., may play important roles in the onset of the disease. WWOX/WOX1 is a candidate tumor suppressor. Human WWOX gene, encoding the WW domain-containing oxidoreductase (designated WWOX, FOR, or WOX1) protein, has been mapped to a fragile site on the chromosome ch16q23.3-24.1. Functionally, the WW domain is not only a tumor suppressor, but also a participant in molecular interactions, signaling, and apoptosis in many diseases. In this article, we review the potential mechanism by which WWOX/WOX1 may participate in the pathogenesis of AD with a focus on cell death signaling pathways in neurons.

Role of WWOX WOX1 in Alzheimer s disease pathology and in cell death signaling

Alzheimer's disease (AD) is the most common form of dementia with a progressive course. AD pathology is a manifestation of the underlying severity and neuroanatomic involvement of specific vulnerable brain regions and circuits that are responsible for neuronal dysfunction and death. The etiology of AD is largely unknown. It has been hypothesized that multiple factors, including genetic components, oxidative stress, intracellular or extracellular accumulation of amyloid, dysfunction of cystoskeletal and synapse components, neuronal loss by apoptosis, neuronal excitotoxicity, inflammation, mitochondria dysfunction, etc., may play important roles in the onset of the disease. WWOX/WOX1 is a candidate tumor suppressor. Human WWOX gene, encoding the WW domain-containing oxidoreductase (designated WWOX, FOR, or WOX1) protein, has been mapped to a fragile site on the chromosome ch16q23.3-24.1. Functionally, the WW domain is not only a tumor suppressor, but also a participant in molecular interactions, signaling, and apoptosis in many diseases. In this article, we review the potential mechanism by which WWOX/WOX1 may participate in the pathogenesis of AD with a focus on cell death signaling pathways in neurons.

WWOXinduces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721. Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis. WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis. Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.

A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development.

Effect of transfection of WWOX gene on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism

Objective To investigate the effect of transfection of WWOX gene on apoptosis of human cholangiocarcinoma QBC939 cells in vitro andin vivoand its possible mechanism.Methods The recombinant WWOX eukaryotic expression plasmid was introduced into QBC939 cells by liposome-mediated transfection.The mRNA and protein expression levels in QBC939 cells stably transfected with WWOX were detected by using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting before and after transfection.Cell apoptosis was assessed by flow cytometry (FCM).The alteration of mitochondria membrane potential ( △ Ψm) was assayed by JC-l staining method.The expression change of bcl-2 mRNA and protein was examined by using quantitative RT-PCR and Western blotting.All the experimental subjects were divided into three groups:QBC939 cells were cultured in natural status (nature control group) ; QBC939 cells were transfected with blank plasmid (blank control group),and QBC939 cells were transfected with WWOX/pmCherry-N1 (experimental group).The three groups were subcutaneously inoculated into nude mice and the growth of xenografted tumor was observed.TUNEL was used to evaluate the apoptosis.Results QBC939 cells with stable transfection of WWOX were established.QuantitativeRT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western blotting demonstrated that WWOX protein expression was markedly increased.FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.24 ±0.35)％ vs (1.73 ±0.48)％ vs (21.40 ±2.35)％,P＜0.01].JC-1 staining method indicated that the experimental group was loss of △Ψm [ (4.27 ± 0.64) ％ vs (4.96 ± 0.52 ) ％ vs (28.60 ± 3.94 ) ％,P ＜ 0.01 ].Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased (P ＜O.05 ).The growth of implanted tumor of WWOX/pmCherry-N1 cells was significantly slowed down (P ＜0.05 ).Number of apoptotic cells was significantly increased in the experimental group [ (4.1 ± 0.6) ％ vs (4.4±0.5)％ vs (13.6 ± 1.5)％,P＜0.01].Conclusion WWOX gene can promote apoptosis of cholangiocarcinoma cells,which may be associated with downregulation of bcl-2 expression,and activation of the mitochondrial apoptosis pathway. Key words: Cholangiocarcinoma; Gene transfection; Apoptosis

Genetic alterations of the WWOX gene in breast cancer

FRA3B and FRA16D are the most sensitive common chromosomal fragile site loci in the human genome and two tumor suppressor genes FHIT (Fragile Histidine Triad) and WWOX (WW domain-containing oxidoreductase gene) map to this sites. The WWOX gene is composed of 9 exons and encodes a 46-kD protein that contains 414 amino acids. Loss of heterozygosity, homozygous deletions, and chromosomal translocations affecting WWOX has been reported in several types of cancer, including ovarian, esophageal, lung and stomach carcinoma, and multiple myeloma. The aim of this study was to determine the role of WWOX as a tumor suppressor gene in patients with breast cancer. Tumor and adjacent non-cancerous tissue samples were obtained from 81 patients with breast cancer. DNA was isolated from all tissue samples, and all exons and flanking intronic sequences of the WWOX gene were analyzed by PCR amplification and direct sequencing. We detected 14 different alterations in the coding sequence and one base substitution at the intron 6 splice site (+1 G-A). In addition to exonic and splice-site alterations, we detected 23 different alterations in the non-coding region of the gene. All coding region mutations identified in this study were in the exons between 4 and 9. We did not observe any alterations in exons 1-3. We conclude that mutations in critical region of the WWOX gene are frequent and may have an important role in breast carcinogenesis.

Tumor suppressor genes FHIT and WWOX are deleted in primary effusion lymphoma (PEL) cell lines

AbstractPrimary effusion lymphoma (PEL) is a diffuse-large B-cell lymphoma with poor prognosis. One hundred percent of PELs carry the genome of Kaposi sarcoma–associated herpesvirus and a majority are coinfected with Epstein-Barr virus (EBV). We profiled genomic aberrations in PEL cells using the Affymetrix 6.0 SNP array. This identified for the first time individual genes that are altered in PEL cells. Eleven of 13 samples (85%) were deleted for the fragile site tumor suppressors WWOX and FHIT. Alterations were also observed in the DERL1, ETV1, RASA4, TPK1, TRIM56, and VPS41 genes, which are yet to be characterized for their roles in cancer. Coinfection with EBV was associated with significantly fewer gross genomic aberrations, and PEL could be segregated into EBV-positive and EBV-negative clusters on the basis of host chromosome alterations. This suggests a model in which both host genetic aberrations and the 2 viruses contribute to the PEL phenotype.