The cloning of a tumor suppressor gene against uterine endometrial cancer from the BAC clone that retains senescence-inducing ability to endomctrial cancer cell lines

我们已经证明，位于1号染色体长臂区域的BAC克隆E4（由STS标记D1 S459和D1 S225定义）可以诱导子宫内膜癌细胞系HHUA的复制性衰老。端粒酶活性的失活也被观察到沿着细胞死亡。E4基因的大小在STS标记D1 S437和459之间，约为1 Mb。将这些DNA片段用生物素标记后，与我们自己制备的正常人子宫内膜RNA cDNA文库进行杂交，将生物素标记的BAC DNA与磁性支撑的Fe-亲和素磁珠结合，洗脱未杂交的cDNA。第二，用接头引物通过PCR扩增杂交的cDNA。特定的连接器已经在 ...更多信息 在构建cDNA文库时，经第二轮PCR扩增后，成功分离出E4区编码的cDNA，并通过万维网基因组数据库进行鉴定和特征分析，这些cDNA为：（1）RB结合蛋白样蛋白（2）FLJ 12790（E6 BP 1同源物）（3）ENST 27746（MAP同源物（4）ORF 12（SM-20 like protein）（5）HUBCEP 80（泛素E1样）然后我们设定标准，通过该标准我们可以确定候选肿瘤抑制（衰老诱导）基因。(a)通过将该基因导入HHUA细胞，应观察到细胞死亡。(b)在临床上获得的子宫内膜癌样本中应发现任何显著的突变或表达缺失，在对每个分离的基因上的这两点进行研究后，仅ORF 12被确定为真实的候选者。目前，我们正在研究ORF 12的功能以及ORF 12参与的细胞机制。ORF 12是否真的能作为抑癌基因或细胞寿命调控基因发挥作用有待进一步研究。少

We have shown that a BAC clone designated as E4, which was located within chromosome 1 long arm region defined by STS marker D1S459 and D1S225, could induce replicative senescence of endometrial cancer cell, line HHUA. Inactivation of telomerase activity was also observed along with the cell death. The size of E4 was estimated approximately 1Mb between the STS marker D1S437 and 459.Next, we tried to identify cDNAs transcribed from the region in E4 BAG.First, chromosomal DNA was purified from the E4 by using pulse field gel electrophoresis and cut with appropriate restriction enzymes into the average size of2-3 kb. These DNA fragments were labeled with biotin and hybridized with cDNA library that was made from normal human endometrial RNAs by ourselves After combining the biotinylated BAC DNA with Fe-avidin beads which was sustained by magnet, un-hybridized CDNA was washed out. Second, hybridized cDNAs were amplified by PCR with the linker-primer. The specific linker had been already at … More tached when the CDNA library was constructed. After the second round PCR cDNAs coded within the area E4 carried were successfully isolated.These cDNAs were identified and characterized by genome data banks via world wide web.These CDNA were: (1)RB binding protein like protein (2)FLJ12790 (E6BP1 homologue) (3)ENST27746(MAP homologue (4)ORF12(SM-20 like protein) (5)HUBCEP80(Ubiquitin E1 like)Then we set the criteria by which we could determine the candidate tumor suppressor(senescence-inducing) gene.(a) By introduction the gene into HHUA cell, cell death should be observed.(b) Any significant mutation or loss of expression should be found in the clinically obtained endometrial cancer samples.After the study of these two points on each isolated gene, only ORF12 was identified as real candidate. At present we are analyzing the function of ORF12 and cell machinery in which ORF12 is involved. Whether ORF12 can really work as tumor suppressor or cell-life span controlling gene will be revealed near in future. Less