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

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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