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 determine the expression differences of PDCD5 in CML patients with different genotypes. Results: Two SNPs were identified within the PDCD5 promoter. They are 27A>Gand11G >A (transcription start site as position 1), respectively. The complete linkage disequilibrium was found between these two polymorphisms. The frequencies of 27G+/11A + genotype and 27G/11A 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 27G/11A had significantly lower transcriptional activity and could not be up-regulated after apoptotic stimulations compared with the promoter with 27A/11G. PDCD5 expression analysis in mononuclear cells derived from CML patients and cell lines with different 27/11genotypes showed consistent results with the reporter assays. Conclusions:These data suggest that 27G/11Ais 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 46 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(46/28)] 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 1,013 to +91 were amplified from an individual In the present study, we screened 1.1 kb of the 5Vupstream with 27GA/11AG 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, 27A>G and 11G>A, and 11A by sequencing using T7 and SP6 primers. The pGEM-PDCD5 plasmids mutated allele disrupts an AP-2 transcription factor binding site containing 27A/11G or 27G/11A 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 (27A/11G) and pGL3-PDCD5-11A further analyzed the functional effects of the 27A>G/11G>A (27G/11A). 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 27/11 (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 5 105 cells. Cells were seeded into 12-well plates at a density of 6 104 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 were and agreed to participate in the study. normalized with equivalent quantities of green fluorescent protein (GFP). The experiment was repeated at least thrice. Isolation of genomic DNA CML bone marrow or peripheral blood samples were collected from Induction of apoptosis and luciferase assay Clinical Diagnostic Laboratory. An EDTA anticoagulated peripheral HeLa cells were used for induction of apoptosis. After 24 hours of blood sample (2 mL) was obtained from each control by venepuncture. transfection, the cultures were replaced by serum-free medium for Genomic DNA was extracted using a Blood DNA Mini kit (Watson induction of apoptosis. Assay of luciferase activity and detection of Biotechnologies, Inc., Shanghai, PRC) according to the instructions of apoptosis were carried out 0, 12, 24, and 36 hours after serum the manufacturer. DNA integrity and quantity were verified by agarose withdrawal. For tumor necrosis factor (TNF)-a-induced apoptosis (with gel electrophoresis. cycloheximide), after 24 hours of transfection, different dosages of TNF-a with constant concentration of cycloheximide (0, 5 Ag/mL Single-nucleotide polymorphism genotyping cycloheximide + 1 ng/mL TNF-a,5Ag/mL cycloheximide + 10 ng/mL Identification of PDCD5 polymorphism. DNA samples from 12 TNF-a, and 5 Ag/mL cycloheximide + 50 ng/mL TNF-a) were added to controls and 33 patients were subjected to PCR amplification of the culture plate. Cells were collected after 24 hours stimulation and 1.1 kb DNA fragment upstream of the transcriptional start site of luciferase activity was assayed as above. Apoptosis assay was carried out PDCD5. Two pairs of primers were used to amplify two overlapping by detection of phosphatidylserine externalization. In brief, HeLa fragments for accurate sequencing. They are forward 1 [5V-CTTGA- cells were cultured in serum-free medium, then were washed and GCTCAGGAGATAGAGGCC-3V (1,013/987)] and reverse 1 [5V- resuspended in binding buffer containing FITC-conjugated Annexin V TGCAATGTGCTGGTGACCTAGAG-3V (298/276)], forward 2 (25 Ag/mL) for 15 minutes before analysis. Cells were collected on a [5V-CTGCAGCCTCGAACTTCCG-3V (643/625)] and reverse 2 [5V- FACScan flow cytometer equipped with a 488 nm argon laser and CAAGCTCCTCGTCCGCCATG-3V(+72/+91)], respectively. After heating analyzed using the CellQuest software (Becton Dickinson, San Jose, at 95jC for 5 minutes, PCR amplification was done with 35 cycles: 95jC CA). The methods for the luciferase assay have been described above. for 30 seconds, 68jC for 60 seconds, and followed by a final extension step at 72jC for 7 minutes. PCR fragments were gel-purified by E.Z.N.A. Expression analysis of PDCD5 Gel Extraction kit (Omega Bio-tek, Inc., Doraville, GA) according to the Seventeen bone marrow samples from patients with wild-type instructions of the manufacturer. Sequencing analyses were done by ABI genotype and 14 from patients with 27AG/11GA genotype were

www.aacrjournals.org 8593 Clin Cancer Res 2005;11(24) December 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology subjected to mRNA expression analysis. MNCs were isolated from bone 33 CML patients, a nucleotide substitution (G>A) at 11 bp marrow aspirates by gradient centrifugation with Ficoll solution upstream from the transcriptional start site and a 27 (A > G) (Shanghai Huangjing Biotechnology Co., Shanghai, P.R. China). Total substitution were identified simultaneously (Fig. 1A). The RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) mutated allele 11A disrupts a NarI restriction enzyme site following the instructions of the manufacturer. cDNAs were synthesized (GGVCGCC, italic indicates the SNP site) and the mutated allele using the Thermoscript reverse transcription-PCR System (Invitrogen). Real-time quantitative PCR was done with the ABI 7000 Sequence 27G creates a NarI site. According to the results of direct Detection System (Applied Biosystems) using the TaqMan technology. sequencing of 45 individuals, eight heterozygous genotypes PCR variables were as follows: 50jC, 2 minutes; 95jC, 10 minutes; 11GA were always identified simultaneously with genotype followed by 50 cycles with 95jC, 15 seconds and 60jC, 1 minute. The 27AG. Sequencing analysis indicated that 11G is located in standard expression curves for ABL and PDCD5 were first constructed from the results of amplification of serial dilutions of standard cDNA. The expression levels in each patient sample were determined by reference to the corresponding expression level on the standard curve and then normalized by the ABL gene. The following primers and probes were used: PDCD5 forward, 5V-GTGATGCGGCCCAACAG- 3V; PDCD5 reverse, 5V-ATCCAGAACTTGGGCTAAGATACTG-3V; PDCD5 probe: 5V-FAM-TCTCATTTCTGCTTCCCTGTGCTTTGCT-TAMARA-3V; ABL forward Ia, 5V-TCCTCGTCCTCCAGCTGTTATC-3V; Ib, 5V-TTAT- CAAAGGAGCAGGGAAGAAG-3V; ABL reverse primer, 5V-CTCAGACCC- TGAGGCTCAAAGT-3V; ABL probe, 5V-FAM-AGCCCTTCAGCGGCCAG- TAGCATCT-TAMARA-3V.

Protein extraction and immunoblotting MNC samples from some CML patients and normal controls were collected. Genotyping was done before protein extraction. HEK293 and HeLa cells were chosen to represent wild-type cell lines, whereas human monocytic leukemia cells U937 and human Jurkat T cells were chosen as with 27AG/11GA genotype. Cells were pelleted by centrifugation, lysed in lysis buffer [0.05% SDS, 10 mmol/L HEPES, 0.15 mol/L NaCl, 1%Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, and 0.5% NP40 (pH 7.4)] and incubated for 30 minutes on ice. Lysates were centrifuged at 18,000 g for 10 minutes at 4jC and the supernatant was measured using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (10 Ag) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia, Little Chalfont, United Kingdom). Membranes were blocked in TBS containing 0.05% Tween 20 containing 5% bovine serum albumin for 1 hour and incubated overnight at 4jC with anti- PDCD5 antiserum at 1:100 dilutions. Blotting with IRDye 800- conjugated anti-IgY was done in the above buffer containing 5% (w/v) bovine serum albumin. The blots were detected by the Odyssey Imaging System (LI-COR Bioscience, Inc., Lincoln, NE).

Statistical analysis To confirm that the study groups could be regarded as Mendelian populations, the expected occurrence rates for different alleles were calculated according to the Hardy-Weinberg principle and compared with the observed occurrence rates by m2 test. Associations for genotype or allele frequencies between different groups were analyzed by m2 test. P values were two sided and defined as P V 0.05 for statistical significance. Statistical analysis for mRNA expression levels was done with SPSS (Chicago, IL) software version 11.0. Differences between groups were evaluated with adjusted t test. Significance was established if P was 0.05. Data are shown as mean, SD, and SE.

Results

Identification of single-nucleotide polymorphisms Fig. 1. A, DNA sequencing electropherogram of the region consisting of 27 and in the promoter region of PDCD5 and associations 11polymorphic sites. Top sequence, wild-type sample with the 27AA/11GG genotype. Middle sequence, 27AG/11GA double heterozygous genotype. between the 27A >G/11G>A polymorphisms and Bottom sequence, mutated homozygous genotype 27GG/11A A . B, 15 % PAGE the susceptibility of chronic myelogenous leukemia of PDCD5 DNA fragments stained with ethidium bromide. Fragment lengths are given in bp. Lane 1, DNA size standard; lane 2, undigested PCR products; lane 3, Characterization of the single-nucleotide polymorphisms. Nar I digestion of homozygous wild-type (27AA/11GG);lane4,homozygous After direct sequencing of PCR products in 12 controls and mutant (27GG/11AA); lane 5, heterozygous mutant (27AG/11G A).

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Fig. 2. Nucleotide sequence of the 5Vupstream region of the PDCD5 gene.The region includes several types of potential cis-acting regulatory elements, including multiple SP1-binding sites and ETF core sequences (double underlined), E2A binding sites, and E boxes (boxes) and several other elements (underlined), including the AP-2 binding site containing the 11polymorphism. +1, position of the transcription start site. The first exon sequence is in italics. The translation start codon is shown in boldface (from ref. 15).

CCSCRGGC (italic illustrates SNP site), an AP-2 transcription different groups. The frequency of 27G+/11A+ genotype was factor binding site, whereas the 27SNP is not in any potential significantly higher in the CML group (n = 33, 26.36%) than in transcriptional factor binding site (Fig. 2; ref. 15). the control group (n =23, 11.85%, m2 = 11.75, P < 0.01). The Genotyping and identification of linkage disequilibrium. We frequency of 27G/11A allele was significantly higher in the inferred that there may be the complete linkage disequilibrium CML group (n = 35, 13.57%) than in the control group (n = 27, between the two SNPs. After the digestion with NarI, target PCR 6.40%, m2 = 9.48, P < 0.01). The genotype and allele fragment produced two fragments (84 and 37 bp) for wild-type distributions of the two SNPs between different groups are genotype 27AA/11GG, four fragments (101, 84, 37, and presented in Table 1. 20 bp) for heterozygous genotype 27AG/11GA, and two fragments (101 and 20 bp) for homozygous mutant genotype Functional role for the 27A>G/11G>A 27GG/11AA. The 20 bp fragment was not visible due to polymorphisms in the promoter region of the its small size. We screened another 199 controls and 96 CML human PDCD5 gene by the reporter assays patients by PCR-RFLP and the fragment yields were consistent Detection of the basal promoter activity with different27/11 with our prediction of complete linkage disequilibrium variation. Having shown that the 27G/11A allele of the between 11 (G>A) and 27 (A>G; Fig. 1B). Thus, the PDCD5 gene is associated with CML, we sought to determine statistical results of the two SNPs were identical. Genotype whether the 27A>G/11G>A genetic variation has functional distributions in the CML group and the control group were consequences. Because these two SNPs lie within the promoter in Hardy-Weinberg equilibrium (control group: m2 = 1.331, region, we examined, in transiently transfected HeLa and P > 0.05; CML group: m2 = 0.797, P > 0.05). HEK293 cells, the activity of reporter constructs driven by the Association between 27/11 single-nucleotide polymorphisms human PDCD5 gene promoter containing either nucleotide and the susceptibility of chronic myelogenous leukemia. Because 27G/11A or 27A/11G. We generated two different of the low frequencies of homozygous mutated genotypes pGL3-PDCD5 constructs for 27G/11A and 27A/11G (27GG/11AA), the 27GG/11AA genotype was combined alleles (pGL3-PDCD5-11A and pGL3-PDCD5-11G, respec- with heterozygous genotype (27AG/11GA) as G/A-positive tively), and did three independent transfections with each genotype (27G+/11A+). There was a statistically significant construct. To correct for differences in transfection efficiency, difference in the distributions of the genotype and allele in the cells were cotransfected with a plasmid encoding for the GFP

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(see Materials and Methods). After normalizing luciferase Ta b l e 1. Genotype and allele distributions of activity by the internal GFP control, the average transcriptional polymorphisms in the 5Vupstream region of the activity of pGL3-PDCD5-11A was 80% less than that of pGL3- PDCD5 gene in CML patients and healthy controls PDCD5-11G in HeLa cells and HEK293 cells (Fig. 3). Up-regulation of PDCD5 gene promoter by serum withdrawal CML patients Healthy controls and tumor necrosis factor-a treatment. To assess the effects of (n = 129) (n =211) 27A>G/11G>A polymorphisms in the promoter region on n (%) the regulation of PDCD5 gene expression during cell apoptosis, we transfected pGL3-PDCD5-11A, pGL3-PDCD5-11G or a PDCD5-27/-11* c promoter control plasmid pGL3-SV40 (containing SV40 Genotype frequency promoter) into HeLa cells. Apoptosis was induced by serum AA/GG 95 (73.64) 186 (88.15) withdrawal or TNF-a treatment after 24 hours of transfection. AG/GA 33 (25.58) 23 (10.90) The detections of apoptosis were carried out at 0, 12, 24, and GG/AA 1 (0.077) 2 (0.095) 36 hours after serum withdrawal and 24 hours following G+/A+ 34 (26.36) 25 (11.85) treatment with different doses of TNF-a. Results of phospha- Allele frequency tidylserine externalization with Annexin-V-FITC are shown in A/G 223 (86.43) 395 (93.6) Fig. 4A. G/A 35 (13.57) 27 (6.4) An assay of luciferase activity was carried out 0, 12, 24, and 36 hours after serum withdrawal. The results of the luciferase *There was the complete linkage disequilibrium between 27 site and 11 s i t e assay are shown in Fig. 4B. The luciferase activity of the reporter SNPs. Genotype and allele frequencies of 27 site are listed on the left side of the slash and the corresponding data of 11site are denoted on the right. gene construct pGL3-PDCD5-11G was increased constantly cBecause of the low frequency of the homozygous polymorphic genotype, over the time course 0, 12, 24, and 36 hours after serum the GG/AA genotype was combined with the heterozygous genotype withdrawal, whereas no significant difference was observed in + + + (AG/GA) as a G/A-positive genotype (G /A ).The distributions of 27G / the report gene construct pGL3-PDCD5-11A. The observations 11A + genotype and 27G/11A allele in CML patients were significantly higher than in healthy controls (genotype: 26.36% versus 11.85%, m2 = of TNF-a-induced apoptosis support the results above (Fig. 4C). 11.75, P < 0.01;allele: 13.57% versus 6.40%, m2 =9.48,P < 0.01). The wild-type construct had greatly increased promoter activity with the elevated concentrations of TNF-a (0, 1, 10, and

Fig. 3. Transcriptional activity in HeLa and HEK293 cells transfected with luciferase reporter gene constructs driven by the PDCD5 gene promoter containing the 27G/11A o r 27A/11Gallelic variants.The luciferase activities, normalized with GFP activities, were expressed as a percentage of the negative control (i.e., pGL3-Basic plasmid). Each construct was transfected three times and assayed in duplicate.

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after apoptosis induction. Together with the above observations, it is clear that increases in luciferase activity induced by serum withdrawal and TNF-a treatment are specific for the PDCD5 promoter and that 27G/11A mutated variants are also insensitive to apoptotic induction. PDCD5 expression assays in human mononuclear cell samples and cell lines with different 27/11 genotypes To verify whether the 27/11 SNPs could influence the endogenetic expression of PDCD5, we examined the mRNA levels in bone marrow MNC cells of 31 CML patients with different genotypes by real-time quantitative PCR. The results of real-time quantitative PCR are shown in Table 2 and Fig. 5. The mean PDCD5 mRNA expression level of 14 samples with 27AG/11GA genotypes (0.300) are significantly lower than that of 17 samples with wild-type (0.735, t = 2.448, P = 0.024). It shows that 27G/11A mutated variant cannot effectively up-regulate gene expression in vivo. The results of Western blot of PDCD5 showed same trends as mRNA expression level (Fig. 6A). The bands of PDCD5 of 27AG/11GA patients (lanes 1 and 3) were much weaker than those of wild-type patients (lane 2). Interestingly, there is no obvious difference between normal controls with wild-type (lanes 5 and 6) and 27AG/11GA genotype (lane 4). We further investigated the effect of 27AG/11GA on PDCD5 expression after the induction of apoptosis. Two cell lines with wild-type (HeLa and HEK293) and two with 27AG/11GA genotype (human monocytic leukemia cells U937 and human Jurkat T cells) were induced to apoptosis by serum withdrawal. We observed prominently increased levels of PDCD5 expression in wild-type cell lines after the induction (lane 2 compared with lane 1 and lane 6 compared with lane 5), whereas slight changes happened in 27AG/11GA cell lines (lane 4 compared with lane 3 and lane 8 compared with lane 7; Fig. 6B).

Discussion

We identified new functional SNPs in the PDCD5 promoter region that are relevant to PDCD5 expression levels and that may, therefore, be useful genetic markers for detecting the susceptibility for CML. This is, to our knowledge, the first report describing associations between SNPs of apoptosis- related genes and the susceptibility to CML, which accounts for f20% of total leukemia cases in adults (16–18). Whereas the pathogenesis of CML was proposed to be involved in abnormal Fig. 4. Time course of apoptosis and the promoter activity of PDCD5 gene in HeLa cells after serum withdrawal and different doses ofTNF-a treatment with constant concentration of cycloheximide. A, the results of detection of phosphatidylserine externalization. HeLa cells were treated with serum withdrawal and phosphatidylserine externalization was determined by flow cytometry. Similar experiments were done Ta b l e 2 . Comparison of PDCD5 mRNA expression in in duplicate. B, the promoter activity of PDCD5 gene promoter containing the 27G/ CML patients with different 27/11genotypes 11A o r 27A/11Gallelic variants in HeLa cells after serum withdrawal. HeLa cells transfected with the plasmid pGL3-PDCD5-11A,pGL3-PDCD5-11G, pGL3-Basic plasmid, and pGL3-SV40 promoter were incubated in serum-free medium for 0,12, Groups No. Mean SD SE t* P 24, and 36 hours. C, the promoter activity of the PDCD5 gene promoter containing the 27G/11A o r 27A/11Gallelic variants in HeLa cells were incubated with Wild type 17 0.735 0.703 0.170 2.448 0.024 different doses ofTNF-a with 5 Ag/mL cycloheximide (CHX).Transfection efficiency 27AG/11G A 14 0.300 0.185 0.050 was normalized by GFP activity.The results of luciferase assay are shown. genotype

50 ng/mL) and the mutated one was nearly unresponsive to the *PDCD5 mRNA expression level in CML patients with 27AG/11G A induction. Furthermore, the luciferase activity of the control genotype was significantly lower than in the group with wild type by adjusted plasmid pGL3-SV40 indicates no significant effect of various t test. time intervals (Fig. 4B) or of different TNF-a dosage (Fig. 4C)

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genotype. This pilot study suggested that there was no correlation between 27/11 SNPs and +5,712 SNP in exon 5. Variations in the promoter region may potentially alter the affinities of existing protein-DNA interactions or, indeed, recruit new proteins to bind to the DNA, altering the specificity and kinetics of the transcriptional process (12, 13, 22–25). According our results, the mutated 11A allele disrupts a putative AP-2 binding site where wild-type 11G is located (CCSCRGGC, italic illustrates SNP site), whereas the 27 SNP is not in any potential transcriptional factor binding site (15). We infer that the 11 SNP may more directly affect transcriptional activity in both basal- and apoptotic-induced situations. Additional studies may be required to conclusively address whether AP-2 regulatory sites are involved in the Fig. 5. PDCD5 mRNA expression in CML samples. PDCD5 mRNA expression was different transcriptional activation of 11A versus 11G. AP-2 evaluated in 17 patients with 27AA/11GG g e n o t y p e (wild type) and 14 patients transcription factors have previously been implicated in carrying 27AG/11GA genotype (heterozygous). tumorigenesis. Moreover, they are known to play regulatory roles in apoptosis, cell cycle control, and differentiation cell differentiation, proliferation, and death that arise from (26, 27). Accordingly, regulation of AP-2 sites by different apoptosis (19–21), attempts to investigate possible defects in 11 SNP variants may affect their responses to apoptotic apoptotic mechanisms will be very helpful for formation of stimulation. effective diagnostic methods and therapeutic strategy of CML. As we now know, apoptosis is a fundamental mechanism by PDCD5 was shown to promote apoptosis in some tumor which DNA-damaging anticancer agents cause cytotoxicity cells (6) and to have lower expression level in marrow- (28). Indeed, there is now evidence that altered expression or nucleated cells of untreated CML patients than in the ones of mutation of genes encoding key apoptotic proteins can provide normal controls (9). SNPs occurring in upstream promoter cancer cells with both an intrinsic survival advantage and regions of genes can potentially affect the process of transcrip- inherent resistance to chemotherapeutic drugs. For example, it tion (10–13). The luciferase assay showed that the wild-type was verified that a polymorphism in wild-type p53 could 27A/11G variants had much higher basal promoter activity influence response in cancer chemotherapy in vitro and in vivo and could be remarkably up-regulated by apoptotic induce- (29, 30). In present study, we examined PDCD5 expression ment but the mutated 27G/11A variants were much lower under the induction of apoptosis in four cell lines with different and nearly unresponsive to the stimulation. It suggests that the 27/11 genotypes in vitro by Western blot. The results 27G/11A mutated variant of the PDCD5 gene 5Vupstream provide clues that the mutated allele carriers may be more region cannot effectively up-regulate expression of the down- resistant upon the apoptotic-inducing factors. We assume stream gene under the stress of apoptosis induction. Relevant that individuals with different PDCD5 SNPs variants may experiments in other cell lines (U937 and Raji) were done and have altered responses to chemotherapy drugs and treatment the results were similar to those presented in this report (data outcomes. not shown). The observations of endogenetic PDCD5 expression assays were consistent with reporter assays. We have shown that CML patients with 27AG/11GA genotype had statistically significantly lower mRNA expression level of PDCD5 than wild-type patients by real-time quantitative PCR. We further examined PDCD5 protein levels in cell lysates by Western blot analysis and the results also support our prediction. Thus, reduced PDCD5 expression in 27G/11A carriers might facilitate antiapoptotic effects and, therefore, accelerate CML progression and increase the susceptibility to CML. This is consistent to our association studies that found that 27G+/11A+ genotypes are more susceptible to CML. According to the database (http://www.ncbi.nlm.nih.gov/ SNP/snp_ref.cgi?rs=4723), there is a cSNP (rs4723) located in the exon 5 of PDCD5. The G-to-A substitution at position +5,712 (transcription start site as position 1) results in an amino acid change E104K in PDCD5. To investigate whether Fig. 6. PDCD5 expression in MNC samples and cell lines with different 27/11 the 27G/11A polymorphisms link to this SNP, we chose 35 genotypes. A, PDCD5 expression in MNC samples of CML patients and normal samples to screen the SNP of exon 5. Twenty individuals controls with wild-type and 27AG/11GA genotype byWestern blot. Lanes1to 3, CML patients; lanes 4 to 6, normal controls; lanes 1, 3, and 4, samples with 27AG/ (including 10 normal controls and 10 CML patients) with wild- 11GA genotype; lanes 2, 5, and 6, wild-type samples. B, PDCD5 expression after type genotype 27AA/11GG and 15 individuals (including the induction of apoptosis in cell lines with different 27/11genotypes byWestern 5 normal controls and 10 CML patients) with heterozygous blot. Lanes 1and 2, HeLa (wild type); lanes 3 and 4, U937 (27AG/11G A genotype); lanes 5 and 6, HEK293 (wild type); lanes 7 and 8, Jurkat (27AG/ genotype 27GA/11AG were sequenced and no variations 11GA genotype); lanes1,3, 5, and 7, nonapoptosis-inducing controls; lanes 2, 4, 6, were detected in exon 5. All individuals carried the wild-type and 8, cells after 24-hour serum withdrawal.

Clin Cancer Res 2005;11(24) December 15, 2005 8598 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

In summary, our findings indicate that the 27A>G/11G>A patients of psoriasis (34). A similar study in psoriasis patients SNP in the promoter region of PDCD5 gene influences PDCD5 is under way in our laboratory. We speculate that functional expression and suggest that it may be a useful genetic marker SNPs of the PDCD5 gene may participate in the pathophysi- for resolving the issue of whether a causal relationship exists ologic course of diseases involving abnormal programmed cell between PDCD5 and human disease. The identification of a death. characteristic variant will benefit individual therapy and will improve diagnosis of the disease. Moreover, recent studies Acknowledgments indicate that PDCD5 is involved in paraptosis, another form of programmed cell death (31) that may occur during carcino- We thank Professor Shan-Shan Chen and her colleagues in the Institute of Hematology at Peking University People’s Hospital for their cooperation in sample genesis (32), and neurodegeneration (33). These studies recruiting, and Dr. Mingxu Xu, Yishan Gao, Yingmei Zhang, and Ting Zhang of provide valuable insight not only for CML but also potentially Peking University Center for Human Disease Genomics for their excellent technical for other diseases, as PDCD5 expression seems to be altered in assistance.

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www.aacrjournals.org 8599 Clin Cancer Res 2005;11(24) December 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Two Single-Nucleotide Polymorphisms with Linkage Disequilibrium in the Human Programmed Cell Death 5 Gene 5 ′ Regulatory Region Affect Promoter Activity and the Susceptibility of Chronic Myelogenous Leukemia in Chinese Population

Xi Ma, Guorui Ruan, Ying Wang, et al.

Clin Cancer Res 2005;11:8592-8599.

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