Seki Yoshiyuki, Suico Mary Ann, Uto Ayako, Hisatsune Akinobu, Shuto Tsuyoshi, Isohama Yoichiro, Kai Hirofumi
Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
Cancer Res. 2002 Nov 15;62(22):6579-86.
Although X chromosome transfer experiments indicated that tumor suppressor genes are present on the X chromosome, they have not been previously identified. In this report, we show that the ETS transcription factor MEF (ELF4), which is located on chromosome Xq26.1, possesses tumor suppressive capability. MEF expression was up-regulated by 5-azacytidine in some cancer cell lines. MEF overexpression induced morphological changes, such as the conversion of normally loose cell-cell contacts to strong interactions similar to those seen in the presence of matrix metalloproteinase (MMP) inhibitor BB94. In the colony formation assay, A549 cells, but not MEF-overexpressing cells, formed colonies in soft agar culture. Furthermore, MEF-overexpressing cells s.c. injected in the nude mice did not grow, whereas the control cells did. The A549 tumors were poorly differentiated, whereas the MEF-overexpressing tumors were well differentiated. By immunostaining with CD31, a marker on vascular endothelial cells, we found that tumor angiogenesis was significantly suppressed in the tumors formed from MEF-overexpressing cells. In addition, the conditioned media from A549 cell cultures stimulated the migration of human umbilical vein endothelial cells, whereas conditioned media from MEF-overexpressing cell cultures had less of an effect. By gelatin zymography, Western blotting analysis, and immunohistochemistry, we found that the expression levels of MMP-9 and MMP-2 were significantly reduced in MEF-overexpressing tumors. Immunohistochemical analyses showed that interleukin (IL)-8 expression was reduced in the MEF-overexpressing tumors in nude mice. Furthermore, IL-8 mRNA expression in vitro was significantly down-regulated in MEF-overexpressing cells, compared with A549 cells. MEF suppressed the transcription and promoter activities of the genes encoding MMP-9 and IL-8, whereas ETS-2 up-regulated these activities. Therefore, we propose that MEF is a candidate tumor suppressor gene on the X chromosome with activities that are opposite to those of ETS-2.
尽管X染色体转移实验表明X染色体上存在肿瘤抑制基因，但此前尚未鉴定出这些基因。在本报告中，我们表明位于Xq26.1染色体上的ETS转录因子MEF（ELF4）具有肿瘤抑制能力。在某些癌细胞系中，5-氮杂胞苷可上调MEF的表达。MEF的过表达诱导了形态学变化，例如正常松散的细胞间接触转变为类似于基质金属蛋白酶（MMP）抑制剂BB94存在时所观察到的强相互作用。在集落形成试验中，A549细胞在软琼脂培养中形成集落，而过表达MEF的细胞则不能。此外，皮下注射到裸鼠体内的过表达MEF的细胞不生长，而对照细胞则生长。A549肿瘤分化不良，而过表达MEF的肿瘤分化良好。通过用血管内皮细胞标志物CD31进行免疫染色，我们发现过表达MEF的细胞形成的肿瘤中肿瘤血管生成明显受到抑制。此外，A549细胞培养的条件培养基刺激人脐静脉内皮细胞迁移，而过表达MEF的细胞培养的条件培养基的作用较小。通过明胶酶谱法、蛋白质印迹分析和免疫组织化学，我们发现过表达MEF的肿瘤中MMP-9和MMP-2的表达水平显著降低。免疫组织化学分析表明，裸鼠过表达MEF的肿瘤中白细胞介素（IL）-8表达降低。此外，与A549细胞相比，过表达MEF的细胞在体外IL-8 mRNA表达显著下调。MEF抑制编码MMP-9和IL-8的基因的转录和启动子活性，而ETS-2上调这些活性。因此，我们认为MEF是X染色体上的一个候选肿瘤抑制基因，其活性与ETS-2相反。
