Liao Cheng, Zhao Mu-Jun, Zhao Jing, Song Hai, Pineau Pascal, Marchio Agnes, Dejean Anne, Tiollais Pierre, Wang Hong-Yang, Li Tsai-Ping
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
World J Gastroenterol. 2003 Jan;9(1):89-93. doi: 10.3748/wjg.v9.i1.89.
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.
The entire coding sequence of LPTS gene was examined for mutations by single strand conformation polymorphism (SSCP) assay and PCR products direct sequencing in 56 liver cancer cell lines, 7 ovarian cancer and 7 head neck tumor cell lines and 70 pairs of HCC tissues samples. The cDNA fragment coding for the most frequent mutant protein was subcloned into GST fusion expression vector. The product was expressed in E.coli and purified by glutathione-agarose column. Telomeric repeat amplification protocol (TRAP) assays were performed to study the effect of point mutation to telomerase inhibitory activity.
SSCP gels showed the abnormal shifting bands and DNA sequencing found that there were 5 different mutations and/or polymorphisms in 12 tumor cell lines located at exon2, exon5 and exon7. The main alterations were 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 the mutation in position 880 was seen in 7 (10 %) cases. The mutation in the site of 880 had no effect on telomerase inhibitory activity.
Alterations identified in this study are polymorphisms of LPTS gene. LPTS mutations occur in HCC but are infrequent and of little effect on the telomerase inhibitory function of the protein. Epigenetics, such as methylation, acetylation, may play the key role in inactivation of LPTS.
寻找与肝相关的假定肿瘤抑制基因（LPTS）失活相关的点突变。LPTS是一种新发现的人类肝相关假定肿瘤抑制基因及肝细胞癌中的端粒酶抑制剂。
采用单链构象多态性分析（SSCP）及PCR产物直接测序法，检测56株肝癌细胞系、7株卵巢癌细胞系、7株头颈肿瘤细胞系及70对肝癌组织样本中LPTS基因的整个编码序列的突变情况。将编码最常见突变蛋白的cDNA片段亚克隆到GST融合表达载体中。该产物在大肠杆菌中表达，并用谷胱甘肽琼脂糖柱进行纯化。采用端粒重复序列扩增法（TRAP）研究点突变对端粒酶抑制活性的影响。
SSCP凝胶显示异常迁移条带，DNA测序发现12株肿瘤细胞系的外显子2、外显子5和外显子7存在5种不同的突变和/或多态性。主要改变为外显子7中的A(778)A/G和A(880)T。在肝癌组织样本中未发现778位点的改变，而880位点的突变在7例（10%）病例中可见。880位点的突变对端粒酶抑制活性无影响。
本研究中鉴定出的改变是LPTS基因的多态性。LPTS突变在肝癌中发生，但频率较低，对该蛋白的端粒酶抑制功能影响较小。表观遗传学，如甲基化、乙酰化，可能在LPTS失活中起关键作用。
