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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (2) : 148-152     DOI: 10.1007/s11684-009-0032-7
Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines
Qiong CHEN, Qing YU, Yuhu SONG, Peiyuan Li, Ying CHANG, Zhijun WANG, Lifeng LIU, Wei WU, Jusheng LIN()
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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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 down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.

Keywords gene      X-linked inhibitor of apoptosis protein associated factor-1 (XAF1)      promoter      transcription regulation     
Corresponding Authors: LIN Jusheng,   
Issue Date: 05 June 2009
URL:     OR
Fig.1  1395-bp fragment of XAF1 gene promoter amplified by PCR. M: marker DL2000; 1, 2, 3: 1395-bp fragment of XAF1 gene promoter.
Fig.2  Agarose gel electrophoresis identification of (a) pGL3-XAF1p and (b) pEGFP-XAF1p. M: DNA marker 3; 1: pGL3-XAF1p; 2: pGL3-XAF1p double digested by I and I; 3: pEGFP-XAF1p; and 4: pEGFP-XAF1p double digested by I and H I.
Fig.3  The activity of pGL3-XAF1p in a variety of cell lines. 293: human embryonic kidney 293 cells; L02: human embryonic liver cell line L02 cells; HepG2: human hepatoma cell line HepG2; AGS: human gastric cancer cell line AGS; and LoVo: human colon cancer cell line LoVo.
Fig.4  The activity of pGL3-XAF1p in the cancer cell lines. HepG2: human hepatoma cell line HepG2; AGS: human gastric cancer cell line AGS; LoVo: human colon cancer cell line LoVo.
1 Jacobson M D, Weil M, Raff M C. Programmed cell death in animal development. Cell , 1997, 88(3): 347-354
doi: 10.1016/S0092-8674(00)81873-5
2 Horvitz H R. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res , 1999, 59(7): 1701-1706
3 Thompson C B. Apoptosis in the pathogenesis and treatment of disease. Science , 1995, 267(5203): 1456-1462
doi: 10.1126/science.7878464
4 Hanahan D, Weinberg R A. The hallmarks of cancer. Cell , 2000, 100(1): 57-70
doi: 10.1016/S0092-8674(00)81683-9
5 Deveraux Q L, Reed J C. IAP family proteins-suppressors of apoptosis. Genes Dev , 1999, 13(3): 239-252
doi: 10.1101/gad.13.3.239
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
doi: 10.1006/geno.2000.6364
7 Liston P, Fong W G, 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
doi: 10.1038/35055027
8 Plenchette S, Cheung H H, Fong W G, LaCasse E C, Korneluk R G. The role of XAF1 in cancer. Curr Opin Investig Drugs , 2007, 8(6): 469-476
9 Byun D S, Cho K, Ryu B K, Lee M G, Kang M J, 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
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
doi: 10.1111/j.0022-202X.2004.23467.x
11 Green M, Schuetz T J, Sullivan E K, Kingston R E. A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function. Mol Cell Biol , 1995, 15(6): 3354-3362
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
doi: 10.1074/jbc.M505890200
13 Smale S T. Core promoters: active contributors to combinatorial gene expression. Genes Dev , 2001, 15(19): 2503-2508
doi: 10.1101/gad.937701
14 Naylor L H. Reporter gene technology: The future looks bright. Biochem Pharmacol , 1999, 58(5): 749-757
doi: 10.1016/S0006-2952(99)00096-9
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
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
17 Hightower L E. Heat shock, stress proteins, chaperones and proteotoxieity. Cell , 1991, 66(2): 191-197
doi: 10.1016/0092-8674(91)90611-2
18 Trautinger F, Kind?s-Mügge I, Knobler R M, H?nigsmann H. Stress proteins in the cellular response to ultraviolet radiation. J Photochem Photobiol B , 1996, 35(3): 141-148
doi: 10.1016/S1011-1344(96)07344-7
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
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
doi: 10.1007/s00280-003-0726-5
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
22 Wei H. Activation of oncogenes and/or inactivation of anti-oncogenes by reactive oxygen species. Med Hypotheses , 1992, 39(3): 267-270
doi: 10.1016/0306-9877(92)90120-2
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
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
doi: 10.1093/nar/22.15.3113
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
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