| Human chromosome 8p23 is known as a region that is associated with loss of heterozygosity (LOH), which is frequently deleted in hepatocellular carcinoma (HCC)tissues. We reported here the identification and characterization of a liver-relatedputative tumor suppressor gene, LPTS, localized at 8p23, which was isolated by allelic-loss mapping and positional candidate cloning. The expression of LPTS was ubiquitous in normal human tissues, albeit at relatively low levels, and appeared to be significantly reduced, or sometimes undetectable in HCC cells and neoplastic tissues. Thus, it appeared that LPTS might be involved in the control of cell proliferation. Indeed, the significant suppression of growth of SMMC-7721 HCC cells had been observed after the introduction of LPTS gene. Furthermore, the antisense oligodeoxynucleotides (AS-ODNs) had been used to suppress the expression of LPTS in normal liver cells L02, and several AS-ODNs specific for LPTS mRNA significantly enhanced cell growth, while control oligodeoxynucleotides (ODNs) did not. Our results suggest that LPTS might be a growth-inhibitory protein in human hepatocytes. (Work above was published in HEPATOLOGY, 2000, 32: 721-727) Genomic structure analysis showed that there were 7 exons totally in LPTS gene, and two mRNA transcripts, referred to LPTS-S and LPTS-L, had been identified in the normal and cancerous liver cells, as well as other tumors. Noobvious difference in ratio of distribution between LPTS-S and LPTS-L could be observed among HCC, normal liver, or other tumors. The exon 6 was deleted in ivtranscript LPTS-S, which encoded a 174-a.a. protein, while LPTS-L contained all 7 exons and encoded a 328-a.a. protein. These two proteins shared the same 132-a.a N terminal fragment, and showed the same suppressing activity to the colony formation in HCC cells. The phylogenetic analysis of LPTS proteins in yeast, C. elegans, D. melanogaster, mouse and human showed a pattern with very high homology, suggesting that LPTS might be a gene of critical important function in the evolution. Western Blot and histoimmunochemical analysis showed that the proteins encoded by LPTS gene were expressed in noncancerous livers, while no positiveexpression can be detected in cancerous liver tissues. The subcellular localizationstudy through EGFP-LPTS fusion expression in cells and immunofluroescence assay revealed that LPTS-L was limited in the nucleolus, and the sequence determining the nucleolar localization was in the C terminal of the LPTS-L, while LPTS-S could be found both in the nuclei and cytoplasm. Functional study of LPTS gene in HCC cells suggested that LPTS could inhibit the proliferation of cells and induce cells into death. Cell enlargement and flatten had been shown in HCC cell line SMMC-7721 upon the over-expression of LPTS gene, the morphologic characteristic always associated with crisis, and arrest in proliferation. The further functional analysis showed that LPTS-S and LPTS-L could inhibit the activity of telomerase. GST-LPTS-S and GST-LPTS-L fusion protein were expressed in E.coli and purified by glutathione-agarose column to perform the assay of the effect of LPTS to telomerase activity in vitro. The results showed clearly that LPTS-S and LPTS-L proteins could inhibit the telomerase activity in SMMC-7721HCC cells and 293 fetal kidney cells. The same inhibitory results were acquired through analysis the telomerase activity in SMMC-7721 cells stably transfected with LPTS. The experiments in vivo andin vitro suggested that LPTS was a telomerase inhibitor, and LPTS-L protein had very potent telomerase inhibitory activity with the vactivity domain localized in the C terminal fragment. Point mutations assay through sing...
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