Skip to main content
SpringerLink
Log in

Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

  • Research Article
  • Published:
Frontiers of Medicine in China Aims and scope Submit manuscript

Abstract

To investigate the regulation of tumor suppressor XAF1 gene expression in digestive system cancers, we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines (human hepatoma cell line HepG2, human colon cancer cell line LoVo, and human gastric cancer cell line AGS) and nontumor cell lines (human embryonic liver cell line L02 (L02 cells) and human embryonic kidney 293 cells [HEK293 cells]) as controls. 1395-bp-promoter fragment of XAF1 gene was amplified by polymerase chain reaction (PCR) and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription activity. The plasmids were transfected into a variety of cell lines by lipofectamine 2000. The promoter transcription activity was determined by dual-luciferase report assay, and enhanced green fluorescent protein (EGFP)-positive cells were detected by fluorescence microscope. The expression of XAF1 mRNA in HEK293 and L02 were significantly higher than that in any of the three digestive system cancer cell lines. The dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 cells. Expression of green fluorescent protein (GFP) under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 cells. The activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were significantly lower than those in the unstressed cells. The results suggested that remarkably downregulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Jacobson M D, Weil M, Raff M C. Programmed cell death in animal development. Cell, 1997, 88(3): 347–354

    Article  PubMed  CAS  Google Scholar 

  2. Horvitz H R. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res, 1999, 59(7): 1701–1706

    Google Scholar 

  3. Thompson C B. Apoptosis in the pathogenesis and treatment of disease. Science, 1995, 267(5203): 1456–1462

    Article  PubMed  CAS  Google Scholar 

  4. Hanahan D, Weinberg R A. The hallmarks of cancer. Cell, 2000, 100 (1): 57–70

    Article  PubMed  CAS  Google Scholar 

  5. Deveraux Q L, Reed J C. IAP family proteins-suppressors of apoptosis. Genes Dev, 1999, 13(3): 239–252

    Article  PubMed  CAS  Google Scholar 

  6. Fong W G, Liston P, Rajcan-Separovic E, St Jean M, Craig C, Korneluk R G. Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer cell lines. Genomics, 2000, 70(1): 113–122

    Article  PubMed  CAS  Google Scholar 

  7. Liston P, Fong WG, Kelly N L, Toji S, Miyazaki T, Conte D, Tamai K, Craig C G, McBurney M W, Korneluk R G. Identification of XAF1 as an antagonist of XIAP anti-caspase activity. Nat Cell Biol, 2001, 3(2): 128–133

    Article  PubMed  CAS  Google Scholar 

  8. Plenchette S, Cheung H H, Fong WG, LaCasse E C, Korneluk R G. The role of XAF1 in cancer. Curr Opin Investig Drugs, 2007, 8(6): 469–476

    PubMed  CAS  Google Scholar 

  9. Byun D S, Cho K, Ryu B K, Lee MG, Kang MJ, Kim H R, Chi S G. Hypermethylation of XIAP-associated factor 1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res, 2003, 63(21): 7068–7075

    PubMed  CAS  Google Scholar 

  10. Ng K C, Campos E I, Martinka M, Li G. XAF1 expression is significantly reduced in human melanoma. J Invest Dermatol, 2004, 123(6): 1127–1134

    Article  PubMed  CAS  Google Scholar 

  11. Green M, Schuetz T J, Sullivan E K, Kingston R E. A heat shockresponsive domain of human HSF1 that regulates transcription activation domain function. Mol Cell Biol, 1995, 15(6): 3354–3362

    PubMed  CAS  Google Scholar 

  12. Wang J, He H, Yu L, Xia H H, Lin M C, Gu Q, Li M, Zou B, An X, Jiang B, Kung H F, Wong B C. HSF1 down-regulates XAF1 through transcriptional regulation. J Biol Chem, 2006, 281(5): 2451–2459

    Article  PubMed  CAS  Google Scholar 

  13. Smale S T. Core promoters: active contributors to combinatorial gene expression. Genes Dev, 2001, 15(19): 2503–2508

    Article  PubMed  CAS  Google Scholar 

  14. Naylor L H. Reporter gene technology: The future looks bright. Biochem Pharmacol, 1999, 58(5): 749–757

    Article  PubMed  CAS  Google Scholar 

  15. Jang J Y, Kim H J, Chi S G, Lee K Y, Nam K D, Kim N H, Lee S K, Joo K R, Dong S H, Kim B H, Chang Y W, Lee J I, Chang R. Frequent epigenetic inactivation of XAF1 by promoter hypermethylation in human colon cancers. Korean J Gastroenterol, 2005, 45(4): 285–293

    PubMed  Google Scholar 

  16. Carper S W, Duffy J J, Gerner E W. Heat shock proteins in thermotolerance and other cellular processes. Cancer Res, 1987, 47 (20): 5249–5255

    PubMed  CAS  Google Scholar 

  17. Hightower L E. Heat shock, stress proteins, chaperones and proteotoxieity. Cell, 1991, 66(2): 191–197

    Article  PubMed  CAS  Google Scholar 

  18. Trautinger F, Kindås-Mügge I, Knobler RM, Hönigsmann H. Stress proteins in the cellular response to ultraviolet radiation. J Photochem Photobiol B, 1996, 35(3): 141–148

    Article  PubMed  CAS  Google Scholar 

  19. Szatrowski T P, Nathan C F. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res, 1991, 51(3): 794–798

    PubMed  CAS  Google Scholar 

  20. Hileman E O, Liu J, Albitar M, Keating M J, Huang P. Intrinsic oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity. Cancer Chemother Pharmacol, 2004, 53(3): 209–219

    Article  PubMed  CAS  Google Scholar 

  21. Wang D, Kreutzer D A, Essigmann J M. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat Res, 1998, 400(1,2): 99–115

    PubMed  CAS  Google Scholar 

  22. Wei H. Activation of oncogenes and/or inactivation of antioncogenes by reactive oxygen species. Med Hypotheses, 1992, 39 (3): 267–270

    Article  PubMed  CAS  Google Scholar 

  23. Kroeger P E, Morimoto R I. Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity. Mol Cell Biol, 1994, 14(11): 7592–7603

    PubMed  CAS  Google Scholar 

  24. Wang Y, Morgan W D. Cooperative interaction of human HSF1 heat shock transcription factor with promoter DNA. Nucleic Acids Res, 1994, 22(15): 3113–3118

    Article  PubMed  CAS  Google Scholar 

  25. Birch-Machin I, Gao S, Huen D, McGirr R, White R A, Russell S. Genomic analysis of heat-shock factor targets in Drosophila. Genome Biol, 2005, 6(7): R63

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Qiong Chen, Qing Yu, Yuhu Song, Peiyuan Li, Ying Chang, Zhijun Wang, Lifeng Liu, Wei Wu & Jusheng Lin

Authors
  1. Qiong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qing Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuhu Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peiyuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ying Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhijun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lifeng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wei Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jusheng Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jusheng Lin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, Q., Yu, Q., Song, Y. et al. Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines. Front. Med. China 3, 148–152 (2009). https://doi.org/10.1007/s11684-009-0032-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11684-009-0032-7

Keywords

Search

Navigation