Alleman Wade G, Tabios Ray L, Chandramouli Gadisetti V R, Aprelikova Olga N, Torres-Cabala Carlos, Mendoza Arnulfo, Rogers Craig, Sopko Nikolai A, Linehan W Marston, Vasselli James R
Howard Hughes Medical Institute, Chevy Chase, MD, USA.
Clin Cancer Res. 2004 Oct 15;10(20):7011-21. doi: 10.1158/1078-0432.CCR-04-0516.
Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models.
Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis.
5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment.
Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.
透明细胞肾细胞癌（ccRCC）与冯·希佩尔-林道（VHL）肿瘤抑制基因的缺失密切相关。在高达19%的散发性ccRCC病例中，VHL基因通过高甲基化在功能上丧失。我们推测，使用去甲基化剂使ccRCC细胞中因甲基化而沉默的VHL重新表达，可能是治疗这类癌症患者的一种方法。我们测试了两种去甲基化剂在细胞培养和携带人ccRCC的小鼠中重新表达VHL的能力，并评估了重新表达的VHL在这些模型中的作用。
使用实时逆转录PCR评估zebularine和5-氮杂-2'-脱氧胞苷（5-aza-dCyd）在四种通过高甲基化导致VHL基因沉默的ccRCC细胞系中重新表达VHL的能力。我们评估了去甲基化剂处理后重新表达的VHL是否能在ccRCC细胞中重现当VHL基因通过细胞培养转染和异种移植小鼠模型重新表达时所观察到的类似表型变化。最后，我们使用微阵列分析评估我们的细胞中发生的全局基因表达变化。
5-aza-dCyd能够在我们的细胞系的培养物和异种移植的鼠肿瘤中重新表达VHL。在处理后的细胞系中观察到了VHL表达的典型表型变化，包括对基质胶侵袭性的降低、血管内皮生长因子和葡萄糖转运蛋白-1表达的降低。用5-aza-dCyd处理的小鼠中，VHL甲基化的ccRCC异种移植肿瘤的大小显著减小。携带未甲基化但VHL突变肿瘤的小鼠在5-aza-dCyd处理后未出现肿瘤缩小。
去甲基化剂可能对治疗由VHL甲基化细胞组成的ccRCC肿瘤患者有用。
