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Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma
Cheng Liao1, Mu-Jun Zhao, Jing Zhao
1State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
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 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.
Results:
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.
Conclusion:
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.
Insights
Point mutations in the liver-related putative tumor suppressor gene (LPTS) are infrequent in hepatocellular carcinoma (HCC) and have minimal impact on telomerase inhibition. Epigenetic factors likely play a more significant role in LPTS gene inactivation.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The liver-related putative tumor suppressor gene (LPTS) is a novel gene identified in hepatocellular carcinoma (HCC).
- Understanding mutations in LPTS is crucial for elucidating its role in liver cancer development and its function as a telomerase inhibitor.
Purpose of the Study:
- To identify point mutations in the LPTS gene associated with its inactivation in hepatocellular carcinoma.
- To investigate the impact of identified mutations on the telomerase inhibitory activity of the LPTS protein.
Main Methods:
- Mutation screening of the entire LPTS coding sequence using single-strand conformation polymorphism (SSCP) assay and direct sequencing.
- Analysis of tumor cell lines and HCC tissue samples.
- Functional assessment of mutant LPTS protein's telomerase inhibitory activity using telomeric repeat amplification protocol (TRAP) assays.
Main Results:
- Five distinct mutations and/or polymorphisms were identified in tumor cell lines, primarily in exons 2, 5, and 7.
- The most frequent alterations were A(778)A/G and A(880)T in exon 7.
- The A(880)T mutation was observed in 10% of HCC tissue samples and did not affect the telomerase inhibitory activity.
Conclusions:
- The identified alterations in the LPTS gene are predominantly polymorphisms.
- LPTS mutations are rare in HCC and exert minimal influence on its telomerase inhibitory function.
- Epigenetic mechanisms, such as methylation and acetylation, are likely key factors in the inactivation of the LPTS gene in liver cancer.
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