Han Youqi, San-Marina Serban, Yang Lin, Khoury Haytham, Minden Mark D
Department of Cellular and Molecular Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 2M9, Canada.
Breast Cancer Res. 2007;9(4):R43. doi: 10.1186/bcr1743.
There is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein.
Using full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture.
Transfection with WT1-ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1-ZF form lacking the lysine-threonine-serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1-ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1-ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells.
Our observations strongly argue that the WT1-ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1-ZF - KTS provides a potential candidate of gene therapy for breast cancer.
越来越多的证据表明，威尔姆斯瘤1抑癌基因（WT1）在某些形式的乳腺癌中表现为癌基因。先前的研究表明，WT1的N端结构域可通过自我结合发挥显性负性作用。在本研究中，我们探讨了WT1的锌指结构域（ZF）是否也具有显性负性效应，从而进一步加深我们对该蛋白的理解。
使用全长和仅含ZF的WT1形式，通过荧光素酶和染色质免疫沉淀试验评估它们对WT1和c-myc启动子的影响。通过定量实时RT-PCR、Northern印迹和Western印迹测定基因表达水平。我们还评估了仅含ZF的形式对培养的乳腺癌细胞系生长的影响。
在乳腺癌细胞中，用WT1-ZF质粒转染对WT1启动子的抑制作用比全长WT1更强。缺乏赖氨酸-苏氨酸-丝氨酸（KTS）插入片段的WT1-ZF形式（ZF - KTS）可结合WT1启动子区域内的大多数WT1共有位点，而含有插入片段的ZF形式（ZF + KTS）仅结合近端启动子中的位点。在乳腺癌细胞中稳定表达ZF - KTS后，内源性WT1 mRNA和蛋白的丰度显著降低。WT1靶基因，包括c-myc、Bcl-2、双调蛋白和端粒酶逆转录酶的表达也同样受到ZF - KTS的抑制。此外，WT1-ZF - KTS消除了由WT1的所有四种主要异构体（包括含有或缺乏可变剪接外显子5和9）介导的c-myc的转录激活。最后，WT1-ZF - KTS抑制MCF-7细胞的集落形成和细胞分裂，但诱导其凋亡。
我们的观察结果有力地表明，WT1-ZF质粒在乳腺癌细胞中作为内源性WT1的显性负性调节因子发挥作用。WT1-ZF - KTS对乳腺癌细胞增殖的抑制作用为乳腺癌基因治疗提供了一个潜在的候选方案。
