抑癌基因 AT motif 结合因子 1（ATBF1）在 TGF-β信号转导过程中与 runt 结构域转录因子 3（RUNX3）一起转移到细胞核。

Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-beta signal transduction.

机构信息

Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

出版信息

Biochem Biophys Res Commun. 2010 Jul 23;398(2):321-5. doi: 10.1016/j.bbrc.2010.06.090. Epub 2010 Jun 25.

DOI:10.1016/j.bbrc.2010.06.090

PMID:20599712

Abstract

BACKGROUND AND AIMS

AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of p21Waf1/Cip1. We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-beta signal transduction.

MATERIALS AND METHODS

To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21Waf1/Cip1 reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-beta signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-beta1 using confocal laser scanning microscopy.

RESULTS

Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear localization (rs=0.433, p<0.001). Co-IP revealed a physical association between ATBF1 and RUNX3. ATBF1 and RUNX3 up-regulated p21Waf1/Cip1 promoter activity synergistically. In SNU16 gastric cancer cells, ATBF1 and RUNX3 were cytoplasmic before TGF-beta1 stimulation, but after 24h of TGF-beta1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus.

CONCLUSION

ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-beta signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator.

摘要

背景与目的

AT motif 结合因子 1（ATBF1）是一种同源转录因子，被鉴定为肿瘤抑制因子，在胃癌中经常发生 ATBF1 基因座的杂合性缺失。我们之前的研究表明，ATBF1 的表达与胃癌的恶性特征呈负相关，并且 ATBF1 增强了 p21Waf1/Cip1 的启动子活性。我们还发现 ATBF1 在细胞质和细胞核之间移动，但易位的精确机制尚不清楚。在这项研究中，我们与 TGF-β信号转导合作，研究了与 runt 域转录因子 3（RUNX3）一起向核内转位的机制。

材料和方法

为了分析胃癌细胞中 ATBF1 和 RUNX3 的表达，我们对 98 例切除的胃癌组织样本进行了免疫组织化学染色，并将核染色强度评为 0 至 5 级。进行了 ATBF1 和 RUNX3 的共免疫沉淀（co-IP）。通过用 p21Waf1/Cip1 报告载体转染 ATBF1 和 RUNX3 进行双荧光素酶测定。为了研究 TGF-β 信号刺激下内源性 ATBF1 和 RUNX3 的核转位，我们使用共聚焦激光扫描显微镜检查了重组 TGF-β1 处理的胃癌细胞中亚细胞定位的 ATBF1 和 RUNX3。

结果

在 37 例（37.8%）胃癌组织样本中观察到 ATBF1 的强免疫组织化学核染色，在 15 例（15.3%）中观察到 RUNX3 的核染色。ATBF1 和 RUNX3 的核定位存在统计学显著相关性（rs=0.433，p<0.001）。co-IP 揭示了 ATBF1 和 RUNX3 之间的物理关联。ATBF1 和 RUNX3 协同上调 p21Waf1/Cip1 启动子活性。在 SNU16 胃癌细胞中，ATBF1 和 RUNX3 在 TGF-β1 刺激前位于细胞质中，但在 TGF-β1 刺激 24 小时后，内源性 ATBF1 和 RUNX3 转位到细胞核。