Akino Kimishige, Toyota Minoru, Suzuki Hiromu, Mita Hiroaki, Sasaki Yasushi, Ohe-Toyota Mutsumi, Issa Jean-Pierre J, Hinoda Yuji, Imai Kohzoh, Tokino Takashi
First Department of Internal Medicine, Cancer Research Institute, Sapporo Medical University, Japan.
Gastroenterology. 2005 Jul;129(1):156-69. doi: 10.1053/j.gastro.2005.03.051.
BACKGROUND & AIMS: Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells.
The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing.
Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas.
RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.
Ras信号通路的激活是结直肠癌（CRC）的一个标志，但Ras负调控因子的作用尚未完全明确。我们的目的是通过研究CRC细胞中Ras-Ras效应基因的遗传和表观遗传改变来解决这个问题。
采用RT-PCR和亚硫酸氢盐PCR检测6个RASSF家族基因在CRC细胞系、原发性CRC及结直肠腺瘤中的表达和甲基化状态。采用集落形成试验和流式细胞术评估RASSF1和RASSF2的肿瘤抑制活性。用免疫荧光显微镜确定RASSF2表达改变对细胞形态的影响。采用单链构象分析和直接测序鉴定K-ras、BRAF和p53的突变。
RASSF2的异常甲基化和组蛋白去乙酰化与CRC中该基因的沉默有关。RASSF2的活性与RASSF1不同，包括诱导形态改变和凋亡；此外，其预防细胞转化的能力表明RASSF2在CRC中作为肿瘤抑制因子发挥作用。显示K-ras/BRAF突变的原发性CRC也经常显示RASSF2甲基化，RASSF2失活增强K-ras诱导的致癌转化。RASSF2甲基化在结直肠腺瘤中也经常被发现。
RASSF2是一个新的肿瘤抑制基因，可调节Ras信号通路，在结直肠癌发生的早期阶段起关键作用。
