Pulukuri S M, Rao J S
Program of Cancer Biology, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, IL 61656, USA.
Oncogene. 2006 Aug 3;25(33):4559-72. doi: 10.1038/sj.onc.1209462. Epub 2006 Jun 19.
14-3-3sigma proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3sigma expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we first examined the methylation status of the 14-3-3sigma promoter-associated CpG islands and 14-3-3sigma gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3sigma expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3sigma promoter in LNCaP and Tramp-C1 cells but not in 14-3-3sigma-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3sigma promoter region. The 14-3-3sigma gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3sigma gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3sigma promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3sigma expression in LNCaP cells. We also investigated whether the loss of 14-3-3sigma expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3sigma-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3sigma expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3sigma-active PC3 and DU145 cells, which normally express 14-3-3sigma. Finally, 14-3-3sigma knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3sigma gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.
14-3-3σ蛋白调控着许多对癌症发展至关重要的细胞过程。其生物学作用之一涉及DNA损伤后G2期细胞周期停滞。也有报道称，通过CpG甲基化导致14-3-3σ表达缺失可能会损害G2期细胞周期检查点功能，从而导致遗传缺陷积累，进而促进恶性转化。然而，与甲基化CpG结合蛋白（MeCPs）相关的启动子甲基化如何影响CpG甲基化依赖性沉默机制仍不清楚。为了更好地理解该机制，我们首先使用甲基化特异性PCR（MSP）、基于HhaI的DNA甲基化检测方法以及逆转录PCR（RT-PCR），检测了一部分前列腺癌细胞系中14-3-3σ启动子相关CpG岛的甲基化状态和14-3-3σ基因表达。我们发现，在LNCaP和Tramp-C1前列腺癌细胞系中14-3-3σ表达缺失，而用表观遗传沉默修饰剂5-氮杂-2'-脱氧胞苷（5-aza）和曲古抑菌素A（TSA）处理后，这种表达得以恢复。这些结果表明存在通过启动子相关CpG甲基化导致的转录沉默。染色质免疫沉淀分析显示，甲基化CpG结合蛋白2（MBD2）在LNCaP和Tramp-C1细胞中优先与14-3-3σ启动子中的甲基化CpG岛结合，而在表达14-3-3σ的PC3和DU145细胞中则不然，后者在14-3-3σ启动子区域含有未甲基化的CpG岛。由于CpG甲基化导致的14-3-3σ基因沉默与MBD2的结合相关。此外，5-aza和TSA联合处理激活14-3-3σ基因表达还涉及MBD2从LNCaP和Tramp-C1细胞中14-3-3σ启动子甲基化CpG岛的释放。此外，通过小干扰RNA（siRNA）敲低MBD2可刺激LNCaP细胞中14-3-3σ的表达。我们还通过MTT实验研究了LNCaP和Tramp-C1细胞中14-3-3σ表达缺失是否影响细胞增殖。有趣的是，我们观察到，在用5-aza和TSA处理恢复14-3-3σ表达后，14-3-3σ失活的LNCaP和Tramp-C1细胞的细胞增殖以及增殖细胞核抗原（PCNA）的蛋白表达明显降低。另一方面，相同处理对正常表达14-3-3σ的14-3-3σ活性PC3和DU145细胞没有显著影响。最后，通过siRNA敲低14-3-3σ导致PC3和DU145细胞增殖增加。这些发现表明，14-3-3σ基因的转录沉默是由与MBD2相关的启动子CpG岛甲基化引起的，这可能在前列腺癌侵袭和转移阶段的疾病进展中起重要作用。
