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Two Single-Nucleotide Polymorphisms with Linkage Human Cancer Biology Two Single-Nucleotide Polymorphisms with Linkage Disequilibrium in the Human Programmed Cell Death 5 Gene 5VRegulatory Region Affect Promoter Activity and the Susceptibility of Chronic Myelogenous Leukemia in Chinese Population Xi Ma,1Guorui Ruan,4 Ying Wang,2 Qiyan Li,3 Ping Zhu,5 Ya-Z h e n Q in, 4 Jin-Lan Li,4 Yan-Rong Liu,4 Dalong Ma,1and Hongshan Zhao1 Abstract Purpose: Chronic myelogenous leukemia (CML) is a disease characterized cytogenetically by the presence of the Philadelphia chromosome. Recent studies suggested that altered PDCD5 expression may have significant implications in CML progression. The aim of this study was to identify single-nucleotide polymorphisms (SNP) within the programmedcelldeath5(PDCD5) promoter region and show their functional relevance to PDCD5 expression as well as their genetic susceptibility to CML. Experimental Design: One hundred twenty-nine CML subjects and 211healthy controls were recruited for identification of SNPs and subsequent genetic analysis. Luciferase reporter assays were carried out to show the functional significance of the SNPs located in the promoter region to PDCD5 expression. Real-time quantitative PCR andWestern blot analysis were done to deter- mine the expression differences of PDCD5 in CML patients with different genotypes. Results: Two SNPs were identified within the PDCD5 promoter. They are À27A>GandÀ11G >A (transcription start site as position 1), respectively. The complete linkage disequilibrium was found between these two polymorphisms. The frequencies of À27G+/À11A + genotype and À27G/À11A allele were significantly higher in CML patients than in healthy controls (genotype: 26.36% versus 11.85%, m2=11.75, P < 0.01; allele: 13.57% versus 6.40%, m2 =9.48,P < 0.01). Luciferase reporter assays revealed that the promoter with À27G/À11A had significantly lower transcriptional activity and could not be up-regulated after apoptotic stimulations compared with the promoter with À27A/À11G. PDCD5 expression analysis in mononuclear cells derived from CML patients and cell lines with different À27/À11genotypes showed consistent results with the reporter assays. Conclusions:These data suggest that À27G/À11Ais associated with reduced PDCD5 promoter activity and increased susceptibility to CML. Chronic myelogenous leukemias (CML) are caused by exhibit enhanced proliferative capacity, altered adhesion constitutively activated tyrosine kinase BCR/ABL that leads properties, and reduced apoptosis (2, 3). It is believed that to a proliferative and survival advantage to hematopoietic BCR/ABL–dependent inhibition of apoptosis plays an impor- progenitors (1). In particular, BCR/ABL–transformed cells tant role in leukemic cell growth and accumulation in CML patients. For the past few years, a number of attempts have been Authors’ Affiliations: 1Department of Immunology, School of Basic Medicine, made to identify critical genes and factors that regulate survival Peking University Center for Human Disease Genomics; Departments of and apoptosis in CML cells (4). Apart from external factors 2 3 Prosthodontics and Periodontology, School of Stomatology, Peking University; (such as cytokines; ref. 5), a number of cellular molecules have 4Institute of Hematology, Peking University People’s Hospital; and 5Department of Hemoltology, Peking University First Hospital, Beijing, P.R. China been implicated in the regulation of apoptosis in BCR/ABL– Received 1/6/05; revised 8/24/05; accepted 8/26/05. transformed cells. However, little is known about the relative Grant support: National Natural Science Foundation of China grant 30470844 contribution of each of these molecules to the survival of and the Chinese HighTech Program (863) grant 2002BA711A01. leukemic cells. The costs of publication of this article were defrayed in part by the payment of page Programmed cell death 5 (PDCD5), also designated TF-1 cell charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. apoptosis–related gene-19 (TFAR19), is a gene cloned from TF-1 Note: X. Ma and G. Ruan contributed equally to this work. cells undergoing apoptosis in our laboratory (6). PDCD5 is Requests for reprints: Hongshan Zhao, Department of Immunology, School of evolutionarily conserved among diverse species ranging from Basic Medicine, Peking University Center for Human Disease Genomics, Peking yeast to mammals (6, 7). Initial functional studies indicated University, 38 Xueyuan Road, 100083 Beijing, P.R. China. Phone: 86-10- 82802846, ext. 420; Fax: 86-10-82801149; E-mail: [email protected]. that recombinant human PDCD5 could accelerate apoptosis F 2005 American Association for Cancer Research. of some tumor cells (e.g., HeLa, TF-1, HL60, MCG-803, and doi:10.1158/1078-0432.CCR-05-0039 MCF-7; refs. 6, 8). A recent study revealed that PDCD5 Clin Cancer Res 2005;11(24) December 15, 2005 8592 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. SNPs in PDCD5 Gene Associated with CML Susceptibility expression was lower in marrow nucleated cells in CML patients Big Dye terminator reagents 2.0 (Applied Biosystems, Foster City, CA) than that in normal controls (9), suggesting that reduced on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). PDCD5 expression may play an important role in the pro- PCR-RFLP. PCR-RFLP was used for genotyping of SNPs within the gression of CML. PDCD5 promoter. The shorter fragment (121 bp from À46 to +75, transcription site as position 1) of the experimental subjects was Single-nucleotide polymorphisms (SNP) are the most com- amplified by forward 3 [5V-GCTCCGGGCTGGATTGGTG-3V(À46/À28)] mon type of human genetic variations. They not only serve and reverse 3 [5V-CATGGCTCGGCGTCAGCG-3V (+58/+75)] primers. as markers for constructing dense genetic maps but also PCR products were digested by NarI (New England Biolabs, Beverly, potentially have direct roles in complex diseases as well as in MA) according to the instructions of the manufacturer. Fragments were differential drug responses between individuals. Increasing separated on a 15% polyacrylamide gel stained by ethidium bromide. evidence shows that SNPs present in noncoding regions can To verify the genotyping results, direct sequencing of PCR products was influence gene expression by affecting regulatory elements. done in 20% random samples using the reverse 3 primer. Functional SNPs in promoter regions have been shown to alter the activity of the promoter and subsequent levels of mRNA Construction of PDCD5 luciferase reporter vector V expression (10–13). DNA fragments from the 5 upstream region of the PDCD5 gene between nucleotides À1,013 to +91 were amplified from an individual In the present study, we screened 1.1 kb of the 5Vupstream with À27GA/À11AG genotype. The PCR products were subcloned into region of the PDCD5 gene to search common genetic variants pGEM-T Easy vector (Promega, Madison, WI) to obtain plasmid pGEM- with distinct effects on the transcriptional activity of the gene. PDCD5. The DNA sequences and orientations of inserts were verified We identified two SNPs, À27A>G and À11G>A, and À11A by sequencing using T7 and SP6 primers. The pGEM-PDCD5 plasmids mutated allele disrupts an AP-2 transcription factor binding site containing À27A/À11G or À27G/À11A were digested with EcoRI within the promoter. Association studies were done to show the and subcloned into the SmaI site of the pGL3-Basic plasmid (Promega) susceptibility of this SNP to CML in a Chinese population. We to generate pGL3-PDCD5-11G (À27A/À11G) and pGL3-PDCD5-11A further analyzed the functional effects of the À27A>G/À11G>A (À27G/À11A). polymorphisms by luciferase reporter assay in HeLa and Cell culture, transfection, and luciferase assay HEK293 cells after nonapoptotic and apoptotic stimulations. HeLa and HEK293 cells were cultured in DMEM supplemented with PDCD5 expression analysis was also done in human mono- 10% FCS at 37jCin5%CO2. Cells were transfected by electroporation nuclear cells (MNC) and cell lines with different À27/À11 (120 V, 20 ms, BTX ECM830, Genetronics, Inc., San Diego, CA). Ten genotypes. micrograms total of plasmid DNA consisting of 9 Ag of reporter plasmid and 1 Ag of pGFP-N1 (Clontech, Mountain View, CA; for normalization Materials and Methods of transfection efficiency) were used for transfection for 5 Â 105 cells. Cells were seeded into 12-well plates at a density of 6 Â 104 cells per Subjects well in 1 mL medium after electroporation. Luciferase activity was A total of 129 CML subjects were recruited from the Institute of assayed 24 hours after transfection. Cells were washed with PBS and Hematology at Peking University People’s Hospital for the study. All lysed with 50 AL of freshly diluted reporter lysis buffer (Promega). CML cases were classified according to morphology, immunology, and After centrifugation, 10 AL of supernatant was added to 50 ALof cytogenetics classification (14). Ethnically and geographically matched the luciferase assay substrate (Promega) and the luminescence of the 211 healthy controls were obtained from blood DNA bank of Peking samples were read immediately with a POLARstar galaxy spectrometer University First Hospital and staff of Peking University Center for (BMG Labtechnologies, Offenburg, Germany), in which light produc- Human Disease Genomics. Patients and controls were well informed tion (relative light units) was measured for 10 seconds. The results
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