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

我们发现，位于1号染色体长臂区（由STS标记D1S459和D1S225定义）的BAC克隆E4可以诱导子宫内膜癌细胞HHUA细胞系的复制性衰老。随着细胞死亡，端粒酶活性失活。估计E4的大小在STS标记D1S437和459之间约为1Mb。接下来，我们试图鉴定从E4 BAG区域转录的cdna。首先，用脉冲场凝胶电泳法纯化E4染色体DNA，并用合适的限制性内切酶切成平均大小为2-3 kb的片段。这些DNA片段用生物素标记，并与我们自制的正常人子宫内膜rna cDNA文库杂交。将生物素化的BAC DNA与磁铁维持的铁亲和蛋白珠结合，洗去未杂交的cDNA。其次，用连接引物进行PCR扩增。当构建CDNA文库时，特异的连接子已经在…More的位置。第二轮PCR后，成功分离出E4携带区编码的cdna。通过万维网的基因组数据库对这些dna进行了鉴定和表征。这些CDNA分别为：(1)RB结合蛋白样蛋白(2)FLJ12790 （E6BP1同源物）(3)ENST27746（MAP同源物）(4)ORF12（SM-20样蛋白）(5)HUBCEP80（泛素E1样基因），并以此为标准确定候选肿瘤抑制（衰老诱导）基因。(a)将基因导入HHUA细胞，观察到细胞死亡。(b)应在临床获得的子宫内膜癌样本中发现任何显著的突变或表达缺失。在对每个分离基因的这两点进行研究后，只有ORF12被确定为真正的候选基因。目前我们正在分析ORF12的功能及其参与的细胞机制。ORF12是否真的可以作为肿瘤抑制基因或细胞寿命控制基因，将在不久的将来揭晓。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