WWOX基因对人急性淋巴细胞性白血病细胞CCRF-CEM细胞凋亡及细胞周期的影响

目的:研究WWOX基因对人急性淋巴细胞性白血病细胞CCRF-CEM细胞凋亡及细胞周期的影响及分子机制。方法:体外培养CCRF-CRM细胞系，将WWOX基因转染入CCRF-CEM细胞，建立高表达WWOX的CCRF-CEM细胞，流式细胞仪法测定CCRF-CEM细胞的细胞周期和凋亡，Western blot法测定WWOX基因、细胞周期和凋亡相关因子的蛋白表达，qRT-PCR法测定caspase-3的mRNA表达。结果:与对照组和空白质粒组相比，转染入WWOX的CCRF-CEM细胞的WWOX蛋白表达显著增加，G0/G1期的细胞比例显著增加，凋亡细胞比例显著增加；CCRF-CEM细胞中cyclin D1、cyclin E、CDK2的蛋白表达显著降低而Wnt-5α和JNK的蛋白表达显著增加，caspase-3的mRNA表达促进CCRF-CEM细胞的凋亡。结论:WWOX基因在急性淋巴细胞性白血病CCRF-CEM细胞中发挥抑癌基因作用，可以有效促进细胞凋亡并抑制细胞周期进程。     

Expression of common chromosomal fragile site genes, WWOX/FRA16D and FHIT/FRA3B is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR

Common chromosomal fragile sites are unstable genomic loci susceptible to breakage, rearrangement, and are highly recombinogenic. Frequent alterations at these loci in tumor cells led to the hypothesis that they may contribute to cancer development. The two most common chromosomal fragile sites FRA16D and FRA3B which harbor WWOX and FHIT genes, respectively, are frequently altered in human cancers. Here we report that environmental carcinogens, ultraviolet (UV) light, and Benzo[a]pyrene diol epoxide (BPDE), significantly downregulate expression of both genes. On the other hand, we observe that ionizing radiation (IR) does not affect expression of these genes, suggesting that the effect of repression exerted by UV and BPDE is not just a consequence of DNA damage but may be a result of different signaling pathways triggered by specific DNA lesions. Such downregulation correlates with an induction of an S-phase delay in the cell cycle. Treatment of UV-irradiated cells with caffeine abrogates the S-phase delay while concomitantly overcoming the repression phenomenon. This suggests the involvement of unique cell cycle checkpoint mechanisms in the observed repression. Therefore, it is hypothesized that protracted downregulation of the putative tumor suppressor genes WWOX and FHIT by environmental carcinogens may constitute an additional mechanism of relevance in the initiation of tumorigenesis.     

WWOX基因杂合性丢失在恶性肿瘤发生中的作用

目的：探讨WWOX基因的改变对肿瘤发生的影响。方法：采用RT—PCR方法检测Hct116／4coildtype（野生型）、Hct116 p53 null（p53^-）、Hct116 Bax null（Bax^-）细胞株的WWOX基因表达。从琼脂糖凝胶中分离和提取DNA，进行DNA纯化，测序，测序结果与GeneBank上公布序列AF211943进行比较。结果：测序结果显示，第一对引物9F1与9R1，在T1497G有杂合性丢失，证实细胞株有二种类型的WWOX表达，全长的WWOX mRNA的转录，和外显子6～8丢失的WWOXmRNA的异常转录。第二对引物8F1与9R2，仅表达全长的WWOX mRNA的染色体才有PCR扩增，其产物在T1497G为G。第三对引物5F1与9R3，扩增产物为929bp和389bp。在389bp的T1497G，只有T，来自于WWOX mRNA的异常转录的染色体。结论：HCT116细胞株有二种类型的WWOX表达，全长的WWOX mRNA的转录，外显子6～8丢失的WWOX mRNA的异常转录，这二种转录来自两个不同的等位基因。     

WWOX基因真核表达载体的构建及其在SMMC-7721中的表达

目的克隆人WWOX基因及构建WWOX基因真核表达载体并研究其表达。方法取人新鲜正常肝组织，提取总RNA，采用逆转录-聚合酶联链反应（RT—PCR）扩增WWOX基因，将该基因定向克隆到真核表达载体pEGFP—N1中，构建真核细胞表达载体pEGFP—N1-WWOX，用限制性内切酶酶切分析，DNA序列分析鉴定重组质粒；将测序正确的WWOX基因通过脂质体介导下转染肝癌细胞SMMC-7721。结果重组质粒pEGFP—N1-WWOX经HindⅢ和BamHI双酶切后，获得4700bp片断和1234bp插入片断，序列分析表明插入的片段与Gene Bank发布的序列一致；荧光显微镜下可见转染的SMMC-7721细胞有绿色荧光蛋白的表达。结论成功构建了真核表达载体pEGFP—N1-WWOX，WWOX基因在SMMC-7721细胞内成功表达。     

脆性位点基因FHIT和WWOX在肝细胞癌中的表达及其意义

目的 探讨脆性位点基因脆性组氨酸三聚体基因(FHIT)和含有WW结构域的氧化还原酶基因(WWOX)在肝细胞癌(HCC)中的表达及其与临床病理特征的关系. 方法采用免疫组织化学染色方法评价142例HCC及其癌旁肝组织和15例正常肝组织中FHIT和WWOX的表达情况,结合临床病理资料进行分析.统计学分析采用Kruskal-Wallis H检验对2或多个独立样本的计数资料或等级资料进行分析,Wilcoxon符号秩检验对2个配对样本的等级资料进行分析,Spearman秩相关进行指标间的相关性分析.结果 68.3%和77.5%的HCC患者中出现FHIT和WWOX表达缺失或下降.肝癌与其相应癌旁组织及正常肝组织表达强度和总水平比较,u值为-9.952～-7.564,P值均＜0.01;且二者的表达具有明显的相关性(P＜0.01).随HCC分化程度的降低,FHIT和WWOX的表达也减弱(P＜0.05),并且与血管侵犯、包膜破坏、卫星病灶出现等提示肿瘤侵袭转移的指标有关(P＜0.05),在术后复发与未复发组中,二者的表达亦有差别(P＜0.05).结论 FHIT与WWOX在HCC中的缺失或低表达与HCC的恶性表型有关,预示HCC患者的不良预后,二者可能起肿瘤抑制作用.     

WWOX基因在肝细胞癌及手术切缘中的表达意义

目的 探讨WWOX基因在肝细胞癌(HCC)和切缘中的表达状况和术后复发及预后的关系及其临床意义.方法 用免疫组化方法检测76例肝癌组织及其切缘组织、20例正常肝组织中WWOX基因蛋白产物表达水平,用TUNEL法检测76例肝细胞癌的细胞凋亡水平,结合临床病理指标和术后生存率进行分析.结果 WWOX基因蛋白产物在HCC中定位于细胞核,在癌旁组织中定位于细胞核与细胞浆.肝癌组织中WWOX基因蛋白表达指数为(35.2±20.1)%,低于癌旁组织和正常肝组织(P<0.05).并与肝癌的细胞凋亡率下降、PCNA增高、肿瘤较大、门脉分支浸润、低分化高恶性度以及术后复发等指标有关(P<0.05).肝癌切缘ww0X基因蛋白表达水平与术后复发率和生存率有关(P<0.05),而与1 cm以上切缘距离无关.结论 肝癌组织和手术切缘存在低WWOX表达和定位异常,并可能与影响肿瘤进展、预后的生物学行为有关.肝癌组织和手术切缘WWOX表达水平测定有望成为评估肝癌进展等生物学行为和预后的有用指标之一,并有助于确定较为安全的根治切除分子切缘.     

胃癌组织中FHIT和WWOX的表达与临床病理特征的关系

目的探讨脆性位点抑癌基因组氨酸三联体（FHIT）和包含WW域的氧化还原酶（WWOX）的蛋白在胃癌组织中的表达及其与临床病理特征的关系。方法采用免疫组化PV-9000二步法检测70例胃癌组织、30例癌旁正常组织中FHIT和WWOX基因的蛋白表达情况。结果胃癌组织中FHIT和WWOX蛋白阳性表达率明显低于癌旁正常组织（P〈0.05）,与胃癌临床分期、组织学分级及淋巴结转移密切相关（P〈0.05）,与年龄、性别、肿瘤大小无关（P〉0.05）。FHIT和WWOX蛋白表达之间有相关性（P〈0.05）。结论 FHIT和WWOX基因可能参与了胃癌的发生发展,两者在胃癌的发生发展过程中可能存在协同作用。     

MPP+-induced neuronal death in rats involves tyrosine 33 phosphorylation of WW domain-containing oxidoreductase WOX1

WW domain-containing oxidoreductase (named WWOX, FOR or WOX1) is a pro-apoptotic protein and tumor suppressor. Animals treated with dopaminergic neurotoxin 1-methyl-4-phenyl-pyridinium (MPP⁺) develop Parkinson's disease (PD)-like symptoms. Here we investigated whether WOX1 is involved in MPP⁺-induced neurodegeneration. Upon insult with MPP⁺ in rat brains, WOX1 protein was upregulated and phosphorylated at Tyr33 (or activated) in the injured neurons in the striatum and cortex ipsilaterally to intoxication, as determined by immunohistochemistry and Western blotting. Also, WOX1 was present in the condensed nuclei and damaged mitochondria of degenerative neurons, as revealed by transmission immunoelectron microscopy. Time-lapse microscopy revealed that MPP⁺ induced membrane blebbing and shrinkage of neuroblastoma SK-N-SH cells. Dominant-negative WOX1, a potent inhibitor of Tyr33 phosphorylation, abolished this event, indicating a critical role of the phosphorylation in apoptosis. c-Jun N -terminal kinase (JNK1) is known to bind and counteract the apoptotic function of WOX1. Suppression of JNK1 function by a dominant-negative spontaneously induced WOX1 activation. WOX1 physically interacted with JNK1 in SK-N-SH cells and rat brain extracts. MPP⁺ rapidly increased the binding, followed by dissociation, which is probably needed for WOX1 to exert apoptosis. We synthesized a short Tyr33-phosphorylated WOX1 peptide (11 amino acid residues). Interestingly, this peptide blocked MPP⁺-induced neuronal death in the rat brains, whereas non-phospho-WOX1 peptide had no effect. Together, activated WOX1 plays an essential role in the MPP⁺-induced neuronal death. Our synthetic phospho-WOX1 peptide prevents neuronal death, suggestive of its therapeutic potential in mitigating the symptoms of PD.     

Concurrent deletion of 16q23 and PTEN is an independent prognostic feature in prostate cancer

The deletion of 16q23‐q24 belongs to the most frequent chromosomal changes in prostate cancer, but the clinical consequences of this alteration have not been studied in detail. We performed fluorescence in situ hybridization analysis using a 16q23 probe in more than 7,400 prostate cancers with clinical follow‐up data assembled in a tissue microarray format. Chromosome 16q deletion was found in 21% of cancers, and was linked to advanced tumor stage, high Gleason grade, accelerated cell proliferation, the presence of lymph node metastases (p < 0.0001 each) and positive surgical margin (p = 0.0004). 16q Deletion was more frequent in ERG fusion‐positive (27%) as compared to ERG fusion‐negative cancers (16%, p < 0.0001), and was linked to other ERG‐associated deletions including phosphatase and tensin homolog (PTEN) (p < 0.0001) and 3p13 (p = 0.0303). In univariate analysis, the deletion of 16q was linked to early biochemical recurrence independently from the ERG status (p < 0.0001). Tumors with codeletions of 16q and PTEN had a worse prognosis (p = 0.0199) than those with PTEN or the deletion of 16q alone. Multivariate modeling revealed that the prognostic value of 16q/PTEN deletion patterns was independent from the established prognostic factors. In summary, the results of our study demonstrate that the deletion of 16q and PTEN cooperatively drives prostate cancer progression, and suggests that deletion analysis of 16q and PTEN could be of important clinical value particularly for preoperative risk assessment of the clinically most challenging group of low‐ and intermediated grade prostate cancers.     

Construction of eukaryotic expression plasmid pEGFP-N1-WWOX and its transient expression in SMMC-7721 cells

Objective： To construct eukaryotic expression plasmid pEGFP-NI-WWOX and transiently express it in SMMC-7721 cells. Methods： Total mRNA was extracted from normal human liver tissue. RT-PCR was used to amplify the aimed segments WWOX cDNA which was then digested with Hindlll and BamHI and inserted into a eukaryotic expression plasmid pEGFP-N 1 to construct pEGFP-N 1-WMVOX. The constructed plasmid was transfected into SMMC-7721 cells by lipofectamine 2000 - mediated transfer method. The expression of WWOX in transfected SMMC-7721 cells was detected 24, 36 and 48 h post-transfection with fluorescence microscope and the expression level of WWOX mRNA in transfected SMMC-7721 cells was assay by using RT-PCR. The change of MMWOX expression and cell proliferation rates were detected by immunocyto- chemistry and MTT methods respectively. Results： The results showed pEGFP-N1-WWOX was successfully constructed and expressed transiently in SMMC-7721 cells. At 48th hour post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected, while no green fluorescence was detected in the control group. In SMMC-7721 cells transfected with pEGFP-NI-WWOX a high level of porcine WWOX was detected. WWOX ex- pressed by transfected cells could significantly inhibit the proliferation of SMMC-7721 cells. Conclusion： pEGFP-N1-WWOX was expressed successfully in SMMC-7721 cells, which suggested that might be used as a new therapeutic method for liver cancer.