PDF(Pubmed)
Abstract：
BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved.
METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment.
RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation.
CONCLUSIONS: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.
METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment.
RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation.
CONCLUSIONS: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.
摘要：
背景：我们先前已经确定了GJB3的一个意外作用，表明这种连接蛋白蛋白的缺乏在人和鼠细胞中诱导非整倍性，并在异种移植模型中加速细胞转化以及肿瘤形成。GJB3丢失导致非整倍性和癌症发生和进展的分子机制仍未解决。
方法:通过RT-qPCR和Western印迹测定GJB3表达水平。通过中期染色体计数评估GJB3敲低对基因组不稳定性的影响，多核细胞，通过微核形成和纺锤体取向的确定。通过免疫沉淀和免疫细胞化学分析GJB3与α-微管蛋白和F-肌动蛋白的相互作用。光漂白实验后，通过活细胞成像和荧光恢复测量GJB3缺乏对微管和肌动蛋白动力学的影响，分别。免疫组织化学用于确定人和鼠膀胱癌组织切片上的GJB3水平。通过BBN处理化学诱导小鼠的膀胱癌。
结果：我们发现GJB3在输尿管和膀胱上皮中高表达，但它在浸润性膀胱癌细胞系中以及在人和小鼠膀胱癌的肿瘤进展过程中下调。GJB3表达的下调导致核型稳定的尿路上皮细胞的非整倍性和基因组不稳定性，并且GJB3水平的实验调节改变了膀胱癌细胞系的迁移和侵袭能力。重要的是,GJB3与α-微管蛋白和F-肌动蛋白相互作用。这些相互作用的损害改变了这些细胞骨架成分的动力学，并导致纺锤体取向缺陷。
结论：我们得出结论，微管和肌动蛋白动力学失调对适当的染色体分离和肿瘤细胞侵袭和迁移有影响。因此,这些观察结果表明GJB3在膀胱癌的发病和扩散中可能发挥作用,并证明在肿瘤细胞播散过程中增强的非整倍性和侵袭能力之间存在分子联系.
方法:通过RT-qPCR和Western印迹测定GJB3表达水平。通过中期染色体计数评估GJB3敲低对基因组不稳定性的影响，多核细胞，通过微核形成和纺锤体取向的确定。通过免疫沉淀和免疫细胞化学分析GJB3与α-微管蛋白和F-肌动蛋白的相互作用。光漂白实验后，通过活细胞成像和荧光恢复测量GJB3缺乏对微管和肌动蛋白动力学的影响，分别。免疫组织化学用于确定人和鼠膀胱癌组织切片上的GJB3水平。通过BBN处理化学诱导小鼠的膀胱癌。
结果：我们发现GJB3在输尿管和膀胱上皮中高表达，但它在浸润性膀胱癌细胞系中以及在人和小鼠膀胱癌的肿瘤进展过程中下调。GJB3表达的下调导致核型稳定的尿路上皮细胞的非整倍性和基因组不稳定性，并且GJB3水平的实验调节改变了膀胱癌细胞系的迁移和侵袭能力。重要的是,GJB3与α-微管蛋白和F-肌动蛋白相互作用。这些相互作用的损害改变了这些细胞骨架成分的动力学，并导致纺锤体取向缺陷。
结论：我们得出结论，微管和肌动蛋白动力学失调对适当的染色体分离和肿瘤细胞侵袭和迁移有影响。因此,这些观察结果表明GJB3在膀胱癌的发病和扩散中可能发挥作用,并证明在肿瘤细胞播散过程中增强的非整倍性和侵袭能力之间存在分子联系.
