DOI QR코드

DOI QR Code

Expression of hPOT1 in HeLa Cells and the Probability of Gene Variation of hpot1 Exon14 in Endometrial Cancer are Much Higher than in Other Cancers

  • Liu, Fei (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Pu, Xiao-Yun (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Huang, Shao-Guang (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Xiang, Gui-Ming (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Jiang, Dong-Neng (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Hou, Gou (Institute of Biochemistry and Molecular Biology, Guangdong Medical College) ;
  • Huang, Di-Nan (Institute of Biochemistry and Molecular Biology, Guangdong Medical College)
  • 발행 : 2012.11.30

초록

To investigate the expression of hPOT1 in the HeLa cell line and screen point mutations of hpot1 in different tumor tissues a two step osmotic method was used to extract nuclear proteins. EMSA was performed to determine the expression of hPOT1 in the HeLa cell line. PCR was also employed to amplify the exon14 sequence of the hpot1 gene in various of cancer tissues. A SV gel and PCR clean-up system was performed to enrich PCR products. DNAStar was used to analyse the exon14 sequence of the hpot1 gene. hPOT1 was expressed in the HeLa cell line and the signal was gradually enhanced as the amount of extracted nuclear proteins increased. The DNA fragment of exon14 of hpot1 was successfully amplified in the HeLa cell line and all cancer tissues, point mutations being observed in 2 out of 3 cases of endometrial cancer (66.7%) despite the hpot1 sequence being highly conserved. However, the sequence of hpot1 exon14 do not demonstrate point mutations in most cancer tissues. Since hPOT1 was expressed in HeLa cell and the probability of gene point variants was obviously higher in endometrial cancer than other cancers, it may be involved in the pathogenesis of gynecological cancers, especially in cervix and endometrium.

키워드

참고문헌

  1. Baumann P, Cech TR (2001). Pot1, the putative telomere endbinding protein in fission yeast and humans. Science, 5519, 1171-5.
  2. Baumann P, Podell E, Cech TR (2002). Human Pot1 (protection of telomeres) protein: cytolocalization, gene structure, and alternative splicing. Mol Cell Biol, 22, 8079-87. https://doi.org/10.1128/MCB.22.22.8079-8087.2002
  3. Cantara S, Capuano S, Capezzone M, et al (2012). Lack of mutations of the telomerase RNA component in familial papillary thyroid cancer with short telomeres. Thyroid, 22, 363-8. https://doi.org/10.1089/thy.2011.0109
  4. Choi JE, Kang HG, Jang JS, et al (2009). Polymorphisms in telomere maintenance genes and risk of lung cancer. Cancer Epidemiol Biomarkers Prev, 10, 2773-81.
  5. Colgin LM, Baran K, Baumann P, et al (2003). Human POT1 facilitates telomere elongation by telomerase. Curr Biol, 11, 942-6.
  6. de Lange T (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev, 18, 2100-10.
  7. Diotti R,Loayza D (2011). Shelterin complex and associated factors at human telomeres. Nucleus, 2, 119-35. https://doi.org/10.4161/nucl.2.2.15135
  8. Fujii K, Sasahira T, Moriwaka Y, et al (2008). Protection of telomeres 1 protein levels are associated with telomere length in gastric cancer. Int J Mol Med, 5, 599-604.
  9. Hanahan D,Weinberg RA (2011). Hallmarks of cancer: the next generation. Cell, 5, 646-74.
  10. He H, Multani AS, Cosme-Blanco W, et al (2006). POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologous recombination. EMBO J, 21, 5180-90.
  11. Hellman LM, Fried MG (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc, 8, 1849-61.
  12. Hosgood HD, 3rd, Cawthon R, He X, et al (2009). Genetic variation in telomere maintenance genes, telomere length, and lung cancer susceptibility. Lung Cancer, 2, 157-61.
  13. Kelleher C, Kurth I,Lingner J (2005). Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro. Mol Cell Biol, 2, 808-18.
  14. Latrick CM,Cech TR (2010). POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J, 5, 924-33.
  15. Lei M, Podell ER,Cech TR (2004). Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection. Nat Struct Mol Biol, 12, 1223-9.
  16. Lin X, Gu J, Lu C, et al (2006). Expression of telomereassociated genes as prognostic markers for overall survival in patients with non-small cell lung cancer. Clin Cancer Res, 19, 5720-5.
  17. Loayza D, Parsons H, Donigian J, et al (2004). DNA binding features of human POT1: a nonamer 5'-TAGGGTTAG-3' minimal binding site, sequence specificity, and internal binding to multimeric sites. J Biol Chem, 13, 13241-8.
  18. Mason M, Schuller A,Skordalakes E (2011). Telomerase structure function. Curr Opin Struct Biol, 1, 92-100.
  19. Nan H, Qureshi AA, Prescott J, et al (2011). Genetic variants in telomere-maintaining genes and skin cancer risk. Hum Genet, 3, 247-53.
  20. Poncet D, Belleville A, t'kint de Roodenbeke C, et al (2008). Changes in the expression of telomere maintenance genes suggest global telomere dysfunction in B-chronic lymphocytic leukemia. Blood, 4, 2388-91.
  21. Porquet N, Poirier A, Houle F, et al (2011). Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFkappaB survival axis downstream of Death receptor-3. BMC Cancer, 285.
  22. Savage SA, Chanock SJ, Lissowska J, et al (2007). Genetic variation in five genes important in telomere biology and risk for breast cancer. Br J Cancer, 6, 832-6.
  23. Shen J, Gammon MD, Wu HC, et al (2010). Multiple genetic variants in telomere pathway genes and breast cancer risk. Cancer Epidemiol Biomarkers Prev, 1, 219-28.
  24. Smith AJ,Humphries SE (2009). Characterization of DNAbinding proteins using multiplexed competitor EMSA. J Mol Biol, 3, 714-7.
  25. Tang T, Zhou FX, Lei H, et al (2009). Increased expression of telomere-related proteins correlates with resistance to radiation in human laryngeal cancer cell lines. Oncol Rep, 6, 1505-9.
  26. Wan SM, Tie J, Zhang YF, et al (2011). Silencing of the hPOT1 gene by RNA inference promotes apoptosis and inhibits proliferation and aggressive phenotype of gastric cancer cells, likely through up-regulating PinX1 expression. J Clin Pathol, 12, 1051-7.
  27. Wu L, Multani AS, He H, et al (2006). Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell, 1, 49-62.
  28. Yang Q, Zheng YL,Harris CC (2005). POT1 and TRF2 cooperate to maintain telomeric integrity. Mol Cell Biol, 3, 1070-80.

피인용 문헌

  1. Shelterin Proteins and Cancer vol.16, pp.8, 2015, https://doi.org/10.7314/APJCP.2015.16.8.3085