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AIM:To find the point mutations meaningful for inactivationof liver-related putative tumor suppressor gene(LPTS)gene,a human novel liver-related putative tumor suppressor geneand telomerase inhibitor in hepatocellular carcinoma.METHODS:The entire coding sequence of LPTS genewas examined for mutations by single strand conformationpolymorphism(SSCP)assay and PCR products directsequencing in 56 liver cancer cell lines,7 ovarian cancerand 7 head & neck tumor cell lines and 70 pairs of HCCtissues samples.The cDNA fragment coding for the mostfrequent mutant protein was subcloned into GST fusionexpression vector.The product was expressed in E.coliand purified by glutathione-agarose column.Telomericrepeat amplification protocol(TRAP)assays wereperformed to study the effect of point mutation totelomerase inhibitory activity.RESULTS:SSCP gels showed the abnormal shifting bandsand DNA sequencing found that there were 5 differentmutations and/or polymorphisms in 12 tumor cell lineslocated at exon2,exon5 and exon7.The main alterationswere A(778)A/G and A(880)T in exon7.The change in siteof 778 could not be found in HCC tissue samples,while themutation in position 880 was seen in 7(10%)cases.Themutation in the site of 880 had no effect on telomeraseinhibitory activity.CONCLUSION:Alterations identified in this study arepolymorphisms of LPTS gene.LPTS mutations occur in HCCbut are infrequent and of little effect on the telomeraseinhibitory function of the protein.Epigenetics,such asmethylation,acetylation,may play the key role in inactivationof LPTS.
AIM: To find the point mutations meaningful for inactivation of liver-related putative tumor suppressor gene (LPTS) gene, a human novel liver-related putative tumor suppressor gene and telomerase inhibitor in hepatocellular carcinoma. METHODS: The entire coding sequence of LPTS gene as examined for mutations by single strand conformation polymorphism (SSCP) assay and PCR products direct sequencing in 56 liver cancer cell lines, 7 ovarian cancerand 7 head & neck tumor cell lines and 70 pairs of HCCtissues samples. The cDNA fragment coding for the most frequent variant protein was subcloned into GST fusion expression vector. The product was expressed in E. coli and purified by glutathione-agarose chromatography. TRAP assays were developed to study the effect of point mutation totelomerase inhibitory activity. RESULTS: SSCP gels showed the abnormal shifting bands and DNA sequencing found that there were 5 different motifs and / or polymorphisms in 12 tumor cell lineslocated at exon2 , exon5 and exon7. The main alterationswere A (778) A / G and A (880) T in exon7.The change in site of 778 could not be found in HCC tissue samples, while themutation in position 880 was seen in 7 (10% ) cases.Themutation in the site of 880 had no effect on telomerase inhibition activity. CONCLUSION: Alterations identified in this study are polymorphisms of LPTS gene. LPTS mutations occur in HCCbut are infrequent and of little effect on the telomerase inhibition function of the protein. Epigenetics, such asmethylation, acetylation, may play the key role in inactivationof LPTS.