抑癌基因WWOX对人肝癌细胞SMMC-7721的作用及其机制的初步研究
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摘要
第一部分WWOX基因在肝细胞癌中的表达及其意义
目的：研究原发性肝细胞癌(HCC)中WWOX基因的表达及其与HCC临床病理特征之间的关系。
方法：采用免疫组织化学SP法和逆转录聚合酶链反应(RT-PCR)检测50例肝癌及其癌旁组织、15例肝外伤后正常肝组织标本中WWOX蛋白及mRNA的表达情况，并分析其与肝细胞癌的临床病理因素之间的关系。
结果：在50例HCC中WWOX蛋白阳性表达率为38．00％(19／50)，癌旁组织中的蛋白阳性表达率为72．00％(36／50)，正常肝组织中全部呈阳性表达，HCC组织中WWOX蛋白阳性表达率显著低于相应癌旁及正常肝组织(P＜0．05)；WWOXmRNA在HCC组织中阳性表达率为42．00％(19／50)，明显低于相应癌旁中的阳性表达84．00％(39／50)及正常肝组织的阳性表达100％(15／15)(P＜0．05)；WWOX蛋白的表达缺失与肿瘤的病理学分级、HbsAg是否阳性及有无门静脉癌栓有关(P＜0．05)。
结论：WWOX蛋白的缺失与肝细胞癌的发生、发展、侵袭、转移过程有关，WWOX功能失活是肝细胞癌发生、发展的机制之一，提示WWOX可能成为一种新的分子指标，可用于肝细胞癌的诊断、治疗和监测患者的预后。
第二部分WWOX基因真核表达载体的构建
目的：从正常人体肝组织提取总RNA，利用RT-PCR法反应获得目的基因WWOX的cDNA片段，构建具备可进行细胞转染和容易被识别筛选的pEGFP-N1-WWOX质粒载体。
方法：应用Trizol法从人体正常肝组织中提取mRNA，按照GeneBank公布的WWOX基因序列设计引物：5’端引物：5’-GGGAAGCTTTTGGAGCGGGAGTGAG-3’；3’端引物：5’-ATGGGATCCCAGCAGTTGTTGAAGTACA-3’。RT-PCR反应获得1 234bp大小的WWOX cDNA片段。WWOX和载体pEGFP-N1用HindⅢ、BamHⅠ进行双酶切，二者进行连接，构建包含目的基因WWOX的重组质粒pEGFP-N1-WWOX。
结果：使用Trizol法分离和提取得到正常人体的总RNA，通过RT-PCR法反应获得目的基因WWOX cDNA片段，构建完成了能够稳定表达和具有筛选特征的pEGFP-N1-WWOX质粒载体，供后续转染和表达实验使用。
结论：Trizol法简便快速提取到正常人体总RNA，RT-PCR法准确地获得目的基因WWOX cDNA片段，正确构建完成了pEGFP-N1-WWOX质粒载体。
第三部分重组表达载体的转染及对肝癌细胞SMMC-7721生长的影响
目的：对重组pEGFP-N1-WWOX质粒载体进行扩增、纯化，将重组载体转染入肝癌SMMC-7721细胞中，筛选出阳性细胞克隆；并研究转染WWOX对SMMC-7721细胞的生长和侵袭力是否会受到抑制，是否会有显著的细胞凋亡出现，探讨WWOX是否会影响SMMC-7721的细胞周期进展和分布比例，以判断WWOX在肝细胞癌的发生和进展方面是否扮演着关键性的作用，进而说明WWOX是否是肝细胞癌病因学中的抑癌基因。
方法：采用脂质体Li pofectami ne2000介导pEGFP-N1-WWOX在肝癌SMMC-7721细胞处于对数生长期进行转染，并采用含G418的培养基筛选一获得阳性克隆的整合了WWOX基因的SMMC-7721细胞，继续传代培养。检测转染前后WWOX mRNA和蛋白量，MTT法检测细胞增殖变化，Transwell小室测定细胞的体外侵袭能力。流式细胞仪检测细胞凋亡率和细胞周期变化情况。
结果：MTT法观察显示，转染WWOX基因后的SMMC-7721细胞生长抑制率为(32．1±4．2)％，对照组为(10．4±2．3)％，两组比较p＜0．01，转染后细胞生长增殖受到明显的抑制。Transwell小室测定转染WWOX基因后的SMMC-7721细胞侵袭能力受到抑制。流式细胞仪检测转染WWOX基因后SMMC-7721细胞的细胞周期中G_0／G_1期占(64．90±1．03)％，对照组(44．24±2．35)％，两组比较p＜0．01，细胞凋亡率(17．96±1．2)％明显高于对照组(2．84±0．64)％，两组比较p＜0．01，显示WWOX基因诱导肝癌细胞的凋亡和细胞周期阻滞。
结论：重组质粒pEGFP-N1-WWOX成功转染肝癌SMMC-7721细胞，筛选获得包含WWOX的阳性克隆SMMC-7721细胞。转染WWOX基因后SMMC-7721细胞生长增殖受到显著的抑制，WWOX通过阻滞肝癌SMMC-7721的细胞细胞周期进展诱导细胞的凋亡。
第四部分抑癌基因WWOX转染对人肝癌裸鼠移植瘤的作用
目的：构建裸鼠人肝癌动物模型，观察WWOX基因在体内对肿瘤的作用。
方法：将转染pEGFP-N1-WWOX和转染空载体pEGFP-N1的SMMC-7721细胞分别接种裸鼠皮下，建立裸鼠人肝癌荷瘤模型，测量肿瘤的体积，质量，计算肿瘤抑制瘤。免疫组织化学法测定Bcl-2、CyclinD_1蛋白的表达情况，TUNEL法检测肿瘤细胞的凋亡情况。
结果：接种转染pEGFP-N1-WWOX细胞的裸鼠成瘤能力明显降低，瘤结节的体积小，抑制率为59．42％，免疫组化测定转染pEGFP-N1-WWOX的裸鼠移植瘤Bcl-2、CyclinD_1的表达均降低，TUNEL法显示凋亡增加，凋亡指数(15．24±2．57)％。
结论：转染pEGFP-N1-WWOX的肝癌SMMC-7721细胞成瘤性差，WWOX基因能抑制肝癌裸鼠移植瘤的发生和生长，并诱导其凋亡。
PART ONE
Expression of WWOX gene in human hepatocellularcarcinoma and its significance
Objectives:To investigate the expressional and clinical meaning of WWOX inhepatocellular carcinoma(HCC).
Methods:Expression of WWOX protein in HCC and the paired para-carcinoma livertissues from 50 HCC patients was detected by immunohistochemistry(SP method),andWWOX mRNA expression was analyzed by reverse transcription polymerase chainreaction(RT-PCR).The relation ship between expression of WWOX gene andclinicopathologic parameters in patients with HCC was analysed.
Results:The positive rates of WWOX protein in HCC were respectively 38.00%(19/50) which were prominently lower than those in paired para-carcinoma liver tissues72.00%(36/50)(p＜0.05),The positive rates of WWOX mRNA were respectively 42.00%(19/50) which were prominently lower than those in paired para-carcinoma liver tissues84.00% (39/50) .Chisquare test analyses revealed that reduced WWOX gene expressionwas correlated with tumor clinical grade and stage,portal cancer embolus(p＜0.05).
Conclusions:The WWOX protein deletion may play an important role in thecarcinogenesis of HCC.It indicate that WWOX may be a new molocular biology parameterin evaluating diagnosis,therapy and prognosis for HCC.
PART TWO
Construction of eukaryotic expression vectorcontaining the WWOX gene
Objectives:To extract total RNA from the normal tissue of human liver,clone cDNAof anti-cancer gene WWOX by RT-PCR,design and achieve plasmid vectorpEGFP-N 1-WWOX which contains anti-cancer gene WWOX.
Methods:We extracted from normal human liver,designed the primer referring tosequence of WWOX mRNA from the GeneBank,We cloned cDNA of anti-cancer geneWWOX by RT-PCR.The cDNA of WWOX was 1234bp.The cDNA of WWOX andpEGFP-N1 were cut by endonuclease HindⅢand BamHⅠ,then they were joined together.We gained recombined plasmid vector pEGFP-N1-WWOX containing anti-cancer geneWWOX.
Results:We extracted mRNA from normal human liver successfully,gained thecDNA of WWOX by RT-PCR successfully.The cDNA of WWOX was 1234bp onelectrophoresis site.We gained recombined plasmid vector pEGFP-N1-WWOX .ThecDNA of WWOX was right.
Conclusions:We can gain the cDNA of WWOX by RT-PCR properly and recombinedplasmid vector pEGFP-N1-WWOX successfully.The recombined plasmid can betransfected into cell and be indentified and screened out.
PART THREE
Experimental studies of eukaryotic expression vectortransfecting hepatoma cells in vitro
Objectives:To copy,reproduce,purify and analyse the recombined plasmidpEGFP-N1-WWOX into SMMC-7721 cell screen out the SMMC-7721 cell with WWOX.To expiore the effect of WWOX on the growth,proliferation,apoptosis and cell growthcycle phase of SMMC-7721 cell,discuss the process mechanism of WWOX inhepatocellular carcinoma.
Methods:SMMC-7721 cell was cultured in RPIM-1640 with 10% FCS and glutamine,then mixed with liposome Lipofectamine2000 and plasmid vector pEGFP-N1-WWOX,totransfect recombined plasmid vector pEGFP-N1-WWOX into SMMC-7721 cell Weobserved the growth and proliferation of SMMC-7721 cell containing pEGFP-N1-WWOX,by MTT method,analysed the apoptosis and cell growth cycle phase of SMMC-7721 cellcontaining pEGFP-N1-WWOX,by Flow Cytometry method.
Results:We transfected the recombined plasmid vector pEGFP-N1-WWOX,intoSMMC-7721 cell successfully and screen out the signle clone SMMC-7721 cell withWWOX.After transfected WWOX into SMMC-7721 cell,WWOX enhanced the growthsuppression of SMMC-7721 [Suppression ratio=(32.1±4.2)%,p＜0.01],induced theapoptosis of SMMC-7721 cell [Apoptosis ratio=(17.96±1.2)%,p＜0.01],Anti-cancergene WWOX also increased the ratio of G_0/G_1 from (44.24±2.35)% to (64.90±1.03)%(p＜0.05),inhibited the advance of SMMC-7721 cell phase significatively,arrested andstopped SMMC-7721 cell at G_0/G_1 phase.
Conclusions:The recombined plasmid vector pEGFP-N1-WWOX can be transfectedinto SMMC-7721 cell with transfection vector liposome Lipofectamine2000.WWOX genecan enhanced the growth suppression and induce apoptosis of SMMC-7721 cell in vitrosignificatively.The mechanism of WWOX on hepatocellular carcinoma may correlate withthe growth suppression,inducement of apoptosis and inhibition and arrestment ofSMMC-7721 cell cycle.
PART FOUR
Inhibitory effects of WWOX gene transfection on hepatocellularcarcinoma xenograft in mude mice
Objectives:Construct nude mice bearing human hepatocellular carcinoma andobserve the effect of WWOX on tumorigenesis in nude mice.
Methods:Construct nude mice bearing human hepatocellular carcinoma.the lengthand short-diameter and weight of the transplantation tumor were measured,so did tumorinhabitation rate.The expressions of Bcl-2、CyclinD_1 protein in nude mice were detected byimmunohistochemsitry.Tumor cell apoptosis were monitored by use of TUNEL.
Results:The tumorigenesis capability in nude mice injected with transfectedpEGFP-N1-WWOX cells was lower than in nude mice injiected with nontransfected cells.The tumor volume in transfected nude mice was smaller than in nontransfected nude mice,the inhibition rate was 59.42%.The expressions of Bcl-2、CyclinD_1 protein in nude micewere inhibited by immunohistochemsitry method,and apoptosis was increased by TUNEL.
Conclusions:The tumorigenesis capability in SMMC-7721 cells transfected bypEGFP-N 1-WWOX reduced,the tumor growth was inhibited and apoptosis was induced.
目的：研究原发性肝细胞癌(HCC)中WWOX基因的表达及其与HCC临床病理特征之间的关系。
方法：采用免疫组织化学SP法和逆转录聚合酶链反应(RT-PCR)检测50例肝癌及其癌旁组织、15例肝外伤后正常肝组织标本中WWOX蛋白及mRNA的表达情况，并分析其与肝细胞癌的临床病理因素之间的关系。
结果：在50例HCC中WWOX蛋白阳性表达率为38．00％(19／50)，癌旁组织中的蛋白阳性表达率为72．00％(36／50)，正常肝组织中全部呈阳性表达，HCC组织中WWOX蛋白阳性表达率显著低于相应癌旁及正常肝组织(P＜0．05)；WWOXmRNA在HCC组织中阳性表达率为42．00％(19／50)，明显低于相应癌旁中的阳性表达84．00％(39／50)及正常肝组织的阳性表达100％(15／15)(P＜0．05)；WWOX蛋白的表达缺失与肿瘤的病理学分级、HbsAg是否阳性及有无门静脉癌栓有关(P＜0．05)。
结论：WWOX蛋白的缺失与肝细胞癌的发生、发展、侵袭、转移过程有关，WWOX功能失活是肝细胞癌发生、发展的机制之一，提示WWOX可能成为一种新的分子指标，可用于肝细胞癌的诊断、治疗和监测患者的预后。
第二部分WWOX基因真核表达载体的构建
目的：从正常人体肝组织提取总RNA，利用RT-PCR法反应获得目的基因WWOX的cDNA片段，构建具备可进行细胞转染和容易被识别筛选的pEGFP-N1-WWOX质粒载体。
方法：应用Trizol法从人体正常肝组织中提取mRNA，按照GeneBank公布的WWOX基因序列设计引物：5’端引物：5’-GGGAAGCTTTTGGAGCGGGAGTGAG-3’；3’端引物：5’-ATGGGATCCCAGCAGTTGTTGAAGTACA-3’。RT-PCR反应获得1 234bp大小的WWOX cDNA片段。WWOX和载体pEGFP-N1用HindⅢ、BamHⅠ进行双酶切，二者进行连接，构建包含目的基因WWOX的重组质粒pEGFP-N1-WWOX。
结果：使用Trizol法分离和提取得到正常人体的总RNA，通过RT-PCR法反应获得目的基因WWOX cDNA片段，构建完成了能够稳定表达和具有筛选特征的pEGFP-N1-WWOX质粒载体，供后续转染和表达实验使用。
结论：Trizol法简便快速提取到正常人体总RNA，RT-PCR法准确地获得目的基因WWOX cDNA片段，正确构建完成了pEGFP-N1-WWOX质粒载体。
第三部分重组表达载体的转染及对肝癌细胞SMMC-7721生长的影响
目的：对重组pEGFP-N1-WWOX质粒载体进行扩增、纯化，将重组载体转染入肝癌SMMC-7721细胞中，筛选出阳性细胞克隆；并研究转染WWOX对SMMC-7721细胞的生长和侵袭力是否会受到抑制，是否会有显著的细胞凋亡出现，探讨WWOX是否会影响SMMC-7721的细胞周期进展和分布比例，以判断WWOX在肝细胞癌的发生和进展方面是否扮演着关键性的作用，进而说明WWOX是否是肝细胞癌病因学中的抑癌基因。
方法：采用脂质体Li pofectami ne2000介导pEGFP-N1-WWOX在肝癌SMMC-7721细胞处于对数生长期进行转染，并采用含G418的培养基筛选一获得阳性克隆的整合了WWOX基因的SMMC-7721细胞，继续传代培养。检测转染前后WWOX mRNA和蛋白量，MTT法检测细胞增殖变化，Transwell小室测定细胞的体外侵袭能力。流式细胞仪检测细胞凋亡率和细胞周期变化情况。
结果：MTT法观察显示，转染WWOX基因后的SMMC-7721细胞生长抑制率为(32．1±4．2)％，对照组为(10．4±2．3)％，两组比较p＜0．01，转染后细胞生长增殖受到明显的抑制。Transwell小室测定转染WWOX基因后的SMMC-7721细胞侵袭能力受到抑制。流式细胞仪检测转染WWOX基因后SMMC-7721细胞的细胞周期中G_0／G_1期占(64．90±1．03)％，对照组(44．24±2．35)％，两组比较p＜0．01，细胞凋亡率(17．96±1．2)％明显高于对照组(2．84±0．64)％，两组比较p＜0．01，显示WWOX基因诱导肝癌细胞的凋亡和细胞周期阻滞。
结论：重组质粒pEGFP-N1-WWOX成功转染肝癌SMMC-7721细胞，筛选获得包含WWOX的阳性克隆SMMC-7721细胞。转染WWOX基因后SMMC-7721细胞生长增殖受到显著的抑制，WWOX通过阻滞肝癌SMMC-7721的细胞细胞周期进展诱导细胞的凋亡。
第四部分抑癌基因WWOX转染对人肝癌裸鼠移植瘤的作用
目的：构建裸鼠人肝癌动物模型，观察WWOX基因在体内对肿瘤的作用。
方法：将转染pEGFP-N1-WWOX和转染空载体pEGFP-N1的SMMC-7721细胞分别接种裸鼠皮下，建立裸鼠人肝癌荷瘤模型，测量肿瘤的体积，质量，计算肿瘤抑制瘤。免疫组织化学法测定Bcl-2、CyclinD_1蛋白的表达情况，TUNEL法检测肿瘤细胞的凋亡情况。
结果：接种转染pEGFP-N1-WWOX细胞的裸鼠成瘤能力明显降低，瘤结节的体积小，抑制率为59．42％，免疫组化测定转染pEGFP-N1-WWOX的裸鼠移植瘤Bcl-2、CyclinD_1的表达均降低，TUNEL法显示凋亡增加，凋亡指数(15．24±2．57)％。
结论：转染pEGFP-N1-WWOX的肝癌SMMC-7721细胞成瘤性差，WWOX基因能抑制肝癌裸鼠移植瘤的发生和生长，并诱导其凋亡。
PART ONE
Expression of WWOX gene in human hepatocellularcarcinoma and its significance
Objectives:To investigate the expressional and clinical meaning of WWOX inhepatocellular carcinoma(HCC).
Methods:Expression of WWOX protein in HCC and the paired para-carcinoma livertissues from 50 HCC patients was detected by immunohistochemistry(SP method),andWWOX mRNA expression was analyzed by reverse transcription polymerase chainreaction(RT-PCR).The relation ship between expression of WWOX gene andclinicopathologic parameters in patients with HCC was analysed.
Results:The positive rates of WWOX protein in HCC were respectively 38.00%(19/50) which were prominently lower than those in paired para-carcinoma liver tissues72.00%(36/50)(p＜0.05),The positive rates of WWOX mRNA were respectively 42.00%(19/50) which were prominently lower than those in paired para-carcinoma liver tissues84.00% (39/50) .Chisquare test analyses revealed that reduced WWOX gene expressionwas correlated with tumor clinical grade and stage,portal cancer embolus(p＜0.05).
Conclusions:The WWOX protein deletion may play an important role in thecarcinogenesis of HCC.It indicate that WWOX may be a new molocular biology parameterin evaluating diagnosis,therapy and prognosis for HCC.
PART TWO
Construction of eukaryotic expression vectorcontaining the WWOX gene
Objectives:To extract total RNA from the normal tissue of human liver,clone cDNAof anti-cancer gene WWOX by RT-PCR,design and achieve plasmid vectorpEGFP-N 1-WWOX which contains anti-cancer gene WWOX.
Methods:We extracted from normal human liver,designed the primer referring tosequence of WWOX mRNA from the GeneBank,We cloned cDNA of anti-cancer geneWWOX by RT-PCR.The cDNA of WWOX was 1234bp.The cDNA of WWOX andpEGFP-N1 were cut by endonuclease HindⅢand BamHⅠ,then they were joined together.We gained recombined plasmid vector pEGFP-N1-WWOX containing anti-cancer geneWWOX.
Results:We extracted mRNA from normal human liver successfully,gained thecDNA of WWOX by RT-PCR successfully.The cDNA of WWOX was 1234bp onelectrophoresis site.We gained recombined plasmid vector pEGFP-N1-WWOX .ThecDNA of WWOX was right.
Conclusions:We can gain the cDNA of WWOX by RT-PCR properly and recombinedplasmid vector pEGFP-N1-WWOX successfully.The recombined plasmid can betransfected into cell and be indentified and screened out.
PART THREE
Experimental studies of eukaryotic expression vectortransfecting hepatoma cells in vitro
Objectives:To copy,reproduce,purify and analyse the recombined plasmidpEGFP-N1-WWOX into SMMC-7721 cell screen out the SMMC-7721 cell with WWOX.To expiore the effect of WWOX on the growth,proliferation,apoptosis and cell growthcycle phase of SMMC-7721 cell,discuss the process mechanism of WWOX inhepatocellular carcinoma.
Methods:SMMC-7721 cell was cultured in RPIM-1640 with 10% FCS and glutamine,then mixed with liposome Lipofectamine2000 and plasmid vector pEGFP-N1-WWOX,totransfect recombined plasmid vector pEGFP-N1-WWOX into SMMC-7721 cell Weobserved the growth and proliferation of SMMC-7721 cell containing pEGFP-N1-WWOX,by MTT method,analysed the apoptosis and cell growth cycle phase of SMMC-7721 cellcontaining pEGFP-N1-WWOX,by Flow Cytometry method.
Results:We transfected the recombined plasmid vector pEGFP-N1-WWOX,intoSMMC-7721 cell successfully and screen out the signle clone SMMC-7721 cell withWWOX.After transfected WWOX into SMMC-7721 cell,WWOX enhanced the growthsuppression of SMMC-7721 [Suppression ratio=(32.1±4.2)%,p＜0.01],induced theapoptosis of SMMC-7721 cell [Apoptosis ratio=(17.96±1.2)%,p＜0.01],Anti-cancergene WWOX also increased the ratio of G_0/G_1 from (44.24±2.35)% to (64.90±1.03)%(p＜0.05),inhibited the advance of SMMC-7721 cell phase significatively,arrested andstopped SMMC-7721 cell at G_0/G_1 phase.
Conclusions:The recombined plasmid vector pEGFP-N1-WWOX can be transfectedinto SMMC-7721 cell with transfection vector liposome Lipofectamine2000.WWOX genecan enhanced the growth suppression and induce apoptosis of SMMC-7721 cell in vitrosignificatively.The mechanism of WWOX on hepatocellular carcinoma may correlate withthe growth suppression,inducement of apoptosis and inhibition and arrestment ofSMMC-7721 cell cycle.
PART FOUR
Inhibitory effects of WWOX gene transfection on hepatocellularcarcinoma xenograft in mude mice
Objectives:Construct nude mice bearing human hepatocellular carcinoma andobserve the effect of WWOX on tumorigenesis in nude mice.
Methods:Construct nude mice bearing human hepatocellular carcinoma.the lengthand short-diameter and weight of the transplantation tumor were measured,so did tumorinhabitation rate.The expressions of Bcl-2、CyclinD_1 protein in nude mice were detected byimmunohistochemsitry.Tumor cell apoptosis were monitored by use of TUNEL.
Results:The tumorigenesis capability in nude mice injected with transfectedpEGFP-N1-WWOX cells was lower than in nude mice injiected with nontransfected cells.The tumor volume in transfected nude mice was smaller than in nontransfected nude mice,the inhibition rate was 59.42%.The expressions of Bcl-2、CyclinD_1 protein in nude micewere inhibited by immunohistochemsitry method,and apoptosis was increased by TUNEL.
Conclusions:The tumorigenesis capability in SMMC-7721 cells transfected bypEGFP-N 1-WWOX reduced,the tumor growth was inhibited and apoptosis was induced.
引文
[1] Bednarek AK, Laflin KJ, Daniel RL, et al. WWOX, a novel WW domain- containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res,2000, 60(8):2140-2145.
[2] Paris PL, Witte JS, Kupelian PA, et al.Identification and fine mapping of a region showing a high frequency of all elic imbalance on chromosome 16q23.2 that corresponds to a prostate cancer susceptibility focus. Cancer Res, 2000, 60(13)3645-3649.
[3] Aqeilan R I, Pekarsky Y, Herrero J J, et al. Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. Proc Natl Acad Sci USA, 2004, 101(13):4401-4406.
[4] Ludes-Meyers JH, Kil H, Bednarek AK, et al. WWOX binds the specific praline-rich ligand PPXY: identification of candidate interacting proteins. Oncogene, 2004, 23(29):5049-5055.
[5] Chang NS, Doherty I, Ensign A, et al. Molecular mechanisms underlying WOX1 activation during apoptotic and stress responses. Biochem Pharmacol, 2003, 66(8):1347-1354.
[6] Chang NS, Pratt N, Heath I, et al. Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity. J Biol Chem, 2001, 276(5):3361-3370.
[7] Chang NS, The non-ankyrin C terminus of physically interacts with p53 in vivo and dissociates in response to apoptotic stress hypoxia DNA damage, and transforming growth factor-beta 1-mediated growth suppression. J Biol Chem, 2002, 277(12):10323-10331.
[8] Chang NS, Doherty I, Ensign A. c-Jun N-terminal kinase 1 physically interacts with WW domaining oxidoreductase(WOXl) and inhibits WOX1-mediated apoptosis. J Biol Chem, 2003, 278(11):9195-9202.
[9] Yendamuri S, Trapasso E, Kuroki T, et al. WW domain containing oxidoreductase gene expression is altered in nonsmall cell lung cancer. Cancer Res,2003, 63(4):878-881.
[10]Aqeilan R I, Pekarsky Y, Kuroki T, et al. Loss of WWOX expression in gastric carcinoma. Clin Cancer Res, 2004, 10(9):3053-3058.
[11]Nunez M I, Ludes-Meyers JH, Rosen DG, et al. WWOX protein expression varies among carcinoma histotypes and correlates with less favorable outcome. BMC Cancer, 2005, 5(1):64-68.
[12]Ried K, Hobson L, Finnis M, et al. Common chromosomal fragile site FRA16D sequence identification of the gene spanning FRA16D and homozygous deletions and translocation break points in cancer cells. Hum Mol Genet, 2000, 9(11):1651-1663.
[13]Pluciennik E, Potemski P, Kusinska R, et al. WWOX the FRA16D cancer gene expression correlation with breast cancer progression and prognosis. Eur J Surg Oncol, 2006, 32(2): 153-157.
[14]Qin HR, Semba S, Hiopoulos D, et al. A role for the WWOX gene in prostate cancer. Cancer Res, 2006, 66(13):6477-6481.
[15] Park SW, Zimonjic DB, Ludes-Meyers J, et al. Frequent down regulation and loss of WWOX gene expression in human hepatocellular carcinoma. Br J Cancer, 2004, 91(4):753-759.
[1] Bednarek AK, Laflin KJ, Daniel RL, et al. WWOX, a novel WW domaincontaining protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res, 2000, 60(8):2140-2145.
[2] Ried K, Finnis M, Hobson L, et al. Common chromosomal fragile site FRA16D sequence identification of the FOR gene spanning FRA16D and homozygous deletions and translocation break points in cancer cells. Hum Mol Genet, 2000, 9(11):1651-1663.
[3] Bednarek AK, Daniel RL, Keck-Waggoner CL, et al. WWOX, the FRA16D gene, as a suppressor of tumor growth. Cancer Res, 2001, 61(22):8068-8073.
[4] Chang NS, Hsu LJ, Lin YS, et al. WW domain-containing oxidoreductase a candidate tumor suppressor. Trends Mol Med, 2007, 13(l):12-22.
[5] Ingham RI, Colwill K, Howard C, et al. WW domains provide a platform for the assembly of multiprotein in networks. Mol Cell Biol, 2005, 25(16):7092-7106.
[6] Thavathiru E, Ludes-Meyers JH, MacLeod MC, et al. Expression of common chromosomal site genes. WWOX/FRA16D and is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR.Mol Carcinog, 2005, 44(3): 174182.
[7] Paige A I, Taylor C, Taylor K I, et al. WWOX: a candidate tumor suppressor gene involved in multiple tumor types. Proc Natl Acad SciUSA, 2001, 98(20):11417-11422.
[8] Bednarek AK, Keck-Waggoner CI,Daniel RI, et al. WWOX, the FRA16D gene, as a suppressor of tumor growth. Cancer Res, 2001, 61(22):8068-8073.
[9] Park SW, Zimonjic DB, Ludes-Meyers J, et al. Frequent down regulation and loss of WWOX gene expression in human hepatocellular carcinoma. Br J Cancer, 2004, 91(4):753-759.
[10]Aqeilan RI, Hussain S,Trapasso F, et al.Targeted deletion of WWOX reveals a tumor suppressor function. PNAS, 2007, 104(13):3949-3954.
[11] Fabbri M, Trapasso F, Iliopoulos D, et al. WWOX gene restoration prevents lung cancer growth in vitro and in vivo. PNAS, 2005, 102(43): 15611-15616.
[12] Iliopoulos D, Druck T,Fabbri M, et al.Inhibition of breast cancer cell growth in vitro and in vivo effect of restoration of WWOX expression. Clin Cancer Res, 2007, 13(1)268-274.
[1] Bednarek AK, Laflin KJ, Daniel RL, et al. WWOX, a novel WW domaincontaining protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res, 2000, 60(8):2140-2145.
[2] Chang NS. A potential role of P53 and W0X1 in mitochondrial apoptosis. Int J MolMed, 2002,9(0:19-24.
[3] Sze CI, Cleton-Jansen A., Moerland E., Kuipers-Dijkshoorn N, Callen D, Sutherland G, Hansen B, Devilee P, Cornelisse C. At least two different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Genes Chromosomes Cancer 1994;9:101-107.
[4] Chen ST, Chunang JI, Cheng CL. et al. Light-induced retinal danmange involves tyrosine 33 phosphorylation, mitochondrial and nucler translocation of WW domain-containing oxidoreductase in vivo. Neuroscience, 2005,130(1):397-407.
[5] Chang NS, Doherty J, Ensign A, et al. In vivo p53 function is indispensable for DNA damageinduced apoptotic signaling in Drosophila. FEBS Lett 2003; 550:5-10.
[6] Yendamuri S, Kuroki T, Trapasso E, et al. WW domain containing oxidoreductase gene expression is altered in nonsmall cell lung cancer. Cancer Res,2003, 63(4):878-881.
[7] Aqeilan R I, Sudol M. The WW domain: a signalling site in dystrophin. Trends Biochem. Sci. 1994; 19:531-533.
[8] Nunez M I,Rosen DG, Ludes-Meyers JH, et al. WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome. BMC Cancer, 2005, 5(1):64-68.
[9] Ried K, Finnis M, Hobson L, et al. Common chromosomal fragile site FRA16D sequence identification of the FOR gene spanning FRA16D and homozygous deletions and translocation break points in cancer cells. Hum Mol Genet, 2000, 9(11):1651-1663.
[10]Pluciennik E, Kusinska R, Potemski P, et al. WWOX the FRA16D cancer gene expression correlation with breast cancer progression and prognosis. Eur J Surg Oncol, 2006, 32(2): 153-157.
[11]Qin HR, Hiopoulos D, Semba S, et al. The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem. Sci. 1995; 20:347-349.
[12]Park SW, Ludes-Meyers J, Zimonjic DB, et al. Frequent down regulation and loss of WWOX gene expression in human hepatocellular carcinoma. Br J Cancer, 2004, 91(4):753-759.
[13]Fabbri M, Hiopoulos D, Trapasso F, et al. WWOX gene restoration prevents lung cancer growth in vitro and in vivo. PNAS, 2005, 102(43):15611-15616.
[14]Thavathiru E, Ludes-Meyers JH, MacLeod MC, et al. 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.Mol Carcinog, 2005, 44(3): 174182.
[15] Hiopoulos D, Fabbri M, Druck T, et al.Inhibition of breast cancer cell growth in vitro and in vivo effect of restoration of WWOX expression. Clin Cancer Res,2007, 13(1)268-274.
[1] Yu K, Toral BL, Discafani C, et al. mTOR, a novel target in breast cancer: the effect of CC12779, an mTOR inhibitor, in preclinical models of breast cancer. Endocl Relate Cancer, 2001, 8(3):249-258.
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[2] Paris PL, Witte JS, Kupelian PA, et al.Identification and fine mapping of a region showing a high frequency of all elic imbalance on chromosome 16q23.2 that corresponds to a prostate cancer susceptibility focus. Cancer Res, 2000, 60(13)3645-3649.
[3] Aqeilan R I, Pekarsky Y, Herrero J J, et al. Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. Proc Natl Acad Sci USA, 2004, 101(13):4401-4406.
[4] Ludes-Meyers JH, Kil H, Bednarek AK, et al. WWOX binds the specific praline-rich ligand PPXY: identification of candidate interacting proteins. Oncogene, 2004, 23(29):5049-5055.
[5] Chang NS, Doherty I, Ensign A, et al. Molecular mechanisms underlying WOX1 activation during apoptotic and stress responses. Biochem Pharmacol, 2003, 66(8):1347-1354.
[6] Chang NS, Pratt N, Heath I, et al. Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity. J Biol Chem, 2001, 276(5):3361-3370.
[7] Chang NS, The non-ankyrin C terminus of physically interacts with p53 in vivo and dissociates in response to apoptotic stress hypoxia DNA damage, and transforming growth factor-beta 1-mediated growth suppression. J Biol Chem, 2002, 277(12):10323-10331.
[8] Chang NS, Doherty I, Ensign A. c-Jun N-terminal kinase 1 physically interacts with WW domaining oxidoreductase(WOXl) and inhibits WOX1-mediated apoptosis. J Biol Chem, 2003, 278(11):9195-9202.
[9] Yendamuri S, Trapasso E, Kuroki T, et al. WW domain containing oxidoreductase gene expression is altered in nonsmall cell lung cancer. Cancer Res,2003, 63(4):878-881.
[10]Aqeilan R I, Pekarsky Y, Kuroki T, et al. Loss of WWOX expression in gastric carcinoma. Clin Cancer Res, 2004, 10(9):3053-3058.
[11]Nunez M I, Ludes-Meyers JH, Rosen DG, et al. WWOX protein expression varies among carcinoma histotypes and correlates with less favorable outcome. BMC Cancer, 2005, 5(1):64-68.
[12]Ried K, Hobson L, Finnis M, et al. Common chromosomal fragile site FRA16D sequence identification of the gene spanning FRA16D and homozygous deletions and translocation break points in cancer cells. Hum Mol Genet, 2000, 9(11):1651-1663.
[13]Pluciennik E, Potemski P, Kusinska R, et al. WWOX the FRA16D cancer gene expression correlation with breast cancer progression and prognosis. Eur J Surg Oncol, 2006, 32(2): 153-157.
[14]Qin HR, Semba S, Hiopoulos D, et al. A role for the WWOX gene in prostate cancer. Cancer Res, 2006, 66(13):6477-6481.
[15] Park SW, Zimonjic DB, Ludes-Meyers J, et al. Frequent down regulation and loss of WWOX gene expression in human hepatocellular carcinoma. Br J Cancer, 2004, 91(4):753-759.
[1] Bednarek AK, Laflin KJ, Daniel RL, et al. WWOX, a novel WW domaincontaining protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res, 2000, 60(8):2140-2145.
[2] Ried K, Finnis M, Hobson L, et al. Common chromosomal fragile site FRA16D sequence identification of the FOR gene spanning FRA16D and homozygous deletions and translocation break points in cancer cells. Hum Mol Genet, 2000, 9(11):1651-1663.
[3] Bednarek AK, Daniel RL, Keck-Waggoner CL, et al. WWOX, the FRA16D gene, as a suppressor of tumor growth. Cancer Res, 2001, 61(22):8068-8073.
[4] Chang NS, Hsu LJ, Lin YS, et al. WW domain-containing oxidoreductase a candidate tumor suppressor. Trends Mol Med, 2007, 13(l):12-22.
[5] Ingham RI, Colwill K, Howard C, et al. WW domains provide a platform for the assembly of multiprotein in networks. Mol Cell Biol, 2005, 25(16):7092-7106.
[6] Thavathiru E, Ludes-Meyers JH, MacLeod MC, et al. Expression of common chromosomal site genes. WWOX/FRA16D and is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR.Mol Carcinog, 2005, 44(3): 174182.
[7] Paige A I, Taylor C, Taylor K I, et al. WWOX: a candidate tumor suppressor gene involved in multiple tumor types. Proc Natl Acad SciUSA, 2001, 98(20):11417-11422.
[8] Bednarek AK, Keck-Waggoner CI,Daniel RI, et al. WWOX, the FRA16D gene, as a suppressor of tumor growth. Cancer Res, 2001, 61(22):8068-8073.
[9] Park SW, Zimonjic DB, Ludes-Meyers J, et al. Frequent down regulation and loss of WWOX gene expression in human hepatocellular carcinoma. Br J Cancer, 2004, 91(4):753-759.
[10]Aqeilan RI, Hussain S,Trapasso F, et al.Targeted deletion of WWOX reveals a tumor suppressor function. PNAS, 2007, 104(13):3949-3954.
[11] Fabbri M, Trapasso F, Iliopoulos D, et al. WWOX gene restoration prevents lung cancer growth in vitro and in vivo. PNAS, 2005, 102(43): 15611-15616.
[12] Iliopoulos D, Druck T,Fabbri M, et al.Inhibition of breast cancer cell growth in vitro and in vivo effect of restoration of WWOX expression. Clin Cancer Res, 2007, 13(1)268-274.
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