Xu Shanhai, Furukawa Toru, Kanai Naomi, Sunamura Makoto, Horii Akira
Department of Molecular Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan.
Gastroenterological Surgery, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan.
J Hum Genet. 2005;50(4):159-167. doi: 10.1007/s10038-005-0235-y. Epub 2005 Apr 12.
Our previous study indicated that DUSP6/MKP-3/PYST1 could act as a tumor suppressor in human pancreatic cancer. DUSP6 was frequently underexpressed in primary pancreatic cancer tissues by an unknown mechanism. In this study, we demonstrated that hypermethylation of the expressional control region of DUSP6 could account for its abrogation in cultured human pancreatic cancer cells and in primary pancreatic cancer tissues. First, we checked intrinsic transcriptional expression levels of DUSP6 by a quantitative real time PCR assay in 16 cultured pancreatic cancer cell lines and found that the cells could be classified into four groups: very-low-level expression, low-level expression, high-level expression, and very-high-level expression. We observed restored expression of DUSP6 after treatment with 5-azacytidine and trichostatin A, a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, respectively, in cells with intrinsically very-low-level and low-level expression of DUSP6. Using a sodium-bisulfite-modification assay, we found that CpG sequences in intron 1 of DUSP6 were heavily methylated in MIA PaCa-2 and PAN07JCK, both showing the very low level of intrinsic expression of the gene. On the other hand, no methylation in this region was detected in 14 other cell lines. We checked the methylation state of this region by a methylation-specific PCR method in 12 primary pancreatic cancer tissues and compared it with the expression state of DUSP6 investigated by immunohistochemistry. Methylation was detected in five of eight cases with abolished expressions of DUSP6, four of which were poorly differentiated adenocarcinoma. On the other hand, none of the four cases with preserved expression of DUSP6 showed methylation. The methylation state significantly correlated with both the abolishment of protein expression (p = 0.038) and the histological subtype of adenocarcinoma (p = 0.023) by chi-square test. These results indicate that hypermethylation of the CpG islands in intron 1 may account for the strong suppression of DUSP6 expression. Other mechanism(s) and/or other CpG sites outside of our investigation may have some influence upon expressional suppression. Our combined results suggest that hypermethylation with modification of histone deacetylation play an important role in transcriptional suppression of DUSP6 in human pancreatic cancer.
我们之前的研究表明，双特异性磷酸酶6（DUSP6）/丝裂原活化蛋白激酶磷酸酶3（MKP-3）/含SH2结构域的蛋白酪氨酸磷酸酶1（PYST1）可作为人类胰腺癌的肿瘤抑制因子。DUSP6在原发性胰腺癌组织中经常以未知机制低表达。在本研究中，我们证明DUSP6表达调控区域的高甲基化可解释其在培养的人类胰腺癌细胞和原发性胰腺癌组织中的缺失。首先，我们通过定量实时PCR检测法检测了16种培养的胰腺癌细胞系中DUSP6的内在转录表达水平，发现这些细胞可分为四组：极低水平表达、低水平表达、高水平表达和极高水平表达。我们观察到，在用5-氮杂胞苷和曲古抑菌素A（分别为DNA甲基转移酶抑制剂和组蛋白脱乙酰酶抑制剂）处理后，DUSP6内在表达水平极低和低水平的细胞中DUSP6表达得以恢复。使用亚硫酸氢钠修饰检测法，我们发现DUSP6第1内含子中的CpG序列在MIA PaCa-2和PAN07JCK中高度甲基化，这两种细胞系中该基因的内在表达水平都非常低。另一方面，在其他14种细胞系中未检测到该区域的甲基化。我们通过甲基化特异性PCR方法检测了12例原发性胰腺癌组织中该区域的甲基化状态，并将其与通过免疫组织化学研究的DUSP6表达状态进行比较。在8例DUSP6表达缺失的病例中，有5例检测到甲基化，其中4例为低分化腺癌。另一方面，4例DUSP6表达保留的病例均未显示甲基化。通过卡方检验，甲基化状态与蛋白表达缺失（p = 0.038）和腺癌组织学亚型（p = 0.023）均显著相关。这些结果表明，第1内含子中CpG岛的高甲基化可能是DUSP6表达受到强烈抑制的原因。我们研究范围之外的其他机制和/或其他CpG位点可能对表达抑制有一定影响。我们的综合结果表明，高甲基化与组蛋白去乙酰化修饰在人类胰腺癌中DUSP6的转录抑制中起重要作用。
