Earlier we had reported a guanine to adenosine substitution at position 125 (G125A) in the BAX promoter, and its association with higher stage of the disease and failure to achieve complete response to treatment in chronic lymphocytic leukemia (CLL) patients. The aim of this study was to test the hypothesis that G125A leads to a reduction in the transcription of the BAX gene and that this is a direct cause of altered BAX mRNA and protein expression. In lymphocytes of CLL patients, BAX mRNA expression was determined by RNase protection assay and Bax protein was detected by immunoblotting. The presence of G125A in the BAX promoter was associated with lower BAX mRNA (P=0.004) and protein (P=0.024) levels. In transient expression assays using wild-type and mutant BAX promoter sequences linked to Luciferase as a reporter, the G125A polymorphism reduced expression of the BAX promoter by 2.6-fold. These studies suggest a mechanism for the biological effect of the G125A.
BAX is a tumor suppressor gene, which codes for the proapoptotic Bax protein, which belongs to the Bcl-2 family (Green and Reed, 1998). By creating channels in mitochondria, Bax plays a role in the release of cytochrome c and other apoptosis-related factors (Green and Reed, 1998), leading to the activation of caspases (Green and Reed, 1998) (Gross et al., 1999). Oligomerization of Bax molecules and interactions between Bax and other members of the Bcl-2 family (homo-/heterodimerization) can influence the activity of the proteins (Adams and Cory, 1998). The effect of these interactions are more likely to affect the regulation of apoptosis, and the relative ratios of these proteins may be more important than absolute levels (Korsmeyer et al., 1993).
Resistance to apoptotic death to a variable degree is an attribute of most human tumors and aberrant expression of Bax has been demonstrated in many human tumors (Bargou et al., 1995; Krajewski et al., 1995; Tai et al., 1998, 1999; Kokawa et al., 1999a, 1999b; Sakuragi et al., 2002). Bax expression is reduced or functionally altered in ovarian (Tai et al., 1998, 1999), breast (Krajewski et al., 1995), and colon cancers (LeBlanc et al., 2002), and in hematolymphoid malignancies (Meijerink et al., 1998; Peng et al., 1998; Saxena et al., 2004). Lower expression of Bax protein in ovarian (Tai et al., 1998, 1999), cervical (Kokawa et al., 1999b) and uterine (Kokawa et al., 1999a) carcinomas, and reduced BAX RNA and protein in metastatic breast carcinomas (Bargou et al., 1995; Krajewski et al., 1995) are associated with poor response to chemotherapy and shorter disease-free survival. Low Bax protein in p53-positive colon cancer cell lines (LS174T, LoVo, LS513, HCT116) correlates with resistance of tumor cells to 5-fluorouracil-induced apoptosis (Violette et al., 2002). Additional evidence for the role of Bax in malignancies is provided by animal models; the loss of BAX gene was found to be associated with lymphoid hyperplasia in mice (Knudson et al., 1995), and in the murine model of choroid plexus tumor development (Yin et al., 1997).
In chronic lymphocytic leukemia (CLL), the Bcl-2/Bax ratio determines cell survival and is a better predictor of resistance to treatment and progressive disease than the levels of the Bcl-2 and Bax proteins alone (Pepper et al., 1996; Thomas et al., 1996; Pepper et al., 1997; Saxena et al., 2004). The antiapoptotic Bcl-2 family member, Mcl-1, has been shown to be of both pathogenic and clinical significance in CLL (Kitada et al., 1998; Saxena et al., 2004). Bax has been shown to heterodimerize with both Bcl-2 (Zha et al., 1996) and Mcl-1 (Bodrug et al., 1995), and its apoptosis-promoting function may be compromised by overexpression of Bcl-2 and Mcl-1 or a reduction in Bax expression. Thus, Bax appears to be of importance in regulating cell survival in CLL, an essential attribute of this malignancy (Caligaris-Cappio and Hamblin, 1999).
The mechanisms underlying aberrant expression of Bax include structural changes in the gene and p53-mediated downregulation of Bax protein. Mutations in the third and second exons of the BAX affect protein expression and have been identified in different tumors, for example, colon (Rampino et al., 1997; Ouyang et al., 1998), endometrium (Ouyang et al., 1998; Catasus et al., 2000; Sakuragi et al., 2002), stomach (Yamamoto et al., 1997; Ouyang et al., 1998; Lee et al., 2002), and lymphomas (Peng et al., 1998), and in human hematopoietic cell lines (Brimmell et al., 1998; Meijerink et al., 1998; Ouyang et al., 1998). Association between BAX mutations and resistance to apoptosis has been observed in colon carcinoma (LeBlanc et al., 2002). However, mutations in BAX exons have not been detected in CLL (Saxena et al., 2002). P53-mediated downregulation of Bax has been shown in vitro in different tissues (Miyashita et al., 1994) and in vivo in a transgenic tumor murine model (Yin et al., 1997). Approximately half of the p53-dependent cell deaths require Bax protein (Yin et al., 1997) and p53 mutations are a late event in CLL (mostly in advanced stages of the disease) (Cordone et al., 1998).
There could be other mechanisms for altered Bax expression as well. We were the first to describe an alteration in the BAX promoter associated with reduced Bax protein expression. We found a single-nucleotide polymorphism (SNP), a G to A transition, 125 nucleotides upstream from the start of transcription and 248 nucleotides from the start of translation of Bax. This SNP was associated with reduced protein expression, higher stage of the disease, and failure to achieve complete response to treatment in patients with CLL (Saxena et al., 2002). Although we were able to demonstrate the association of the polymorphism with reduced protein expression and a poor clinical outcome, we did not know how this polymorphism affects Bax expression (Saxena et al., 2002). The G125A polymorphism is located within the 5′-untranslated region (5’-UTR) of the BAX promoter. Since the site where the polymorphism is located lies outside of the open codon reading frame in the gene, this SNP would not affect the amino-acid sequence of Bax, but it may affect gene expression at the transcriptional level. The aim of this study was to test the hypothesis that the G125A transition affects transcription of the BAX gene and is a direct cause of altered Bax protein expression in tumor cells.
G125A SNP in the BAX promoter in CLL patients
In all, 71 patients with CLL were screened for the presence of G125A SNP in the BAX promoter, using restriction enzyme AciI. Out of 77 patients, 24 (31.2%) had this polymorphism in their lymphocytes, and it was absent in the remaining 53 patients.
Theoretical prediction of promoter activity
The genetic algorithm analysis using RegScan program (http://www.mgs.bionet.nsc.ru/mgs/) for the analysis of the promoter sequence was used. The RegScan program recognizes eukaryotic gene promoters based on the assessment of dinucleotide frequencies distribution by discriminant analysis. The analysis predicted the highest promoter activity at positions 724 and 725 of the sequence (GenBank Accession numbers U17193 and AY217036) with score 0.94 at both positions (Figure 1). The position 725 at these sequences corresponds to the position 125 from the start of the BAX gene transcription and where the polymorphism G125A occurs. The nucleotide A that replaces G at the location of the G125A transition causes the decrease of predicted promoter activity score from 0.94 to 0.87 with confidence interval (CI) levels of 95%.
Association of BAX promoter G125A SNP with BAX mRNA and protein levels in human CLL cells
All samples were obtained from untreated patients at diagnosis. BAX and Bcl-2 mRNA levels were determined by RNase protection assay in cells from 21 CLL patients (Figure 2a), and Bcl-2/Bax ratios were calculated. Samples bearing the SNP had statistically significantly (P=0.004, t-test) lower levels of BAX mRNA (mean – 0.078 relative units, 95% CI=0–0.17) than samples without it (mean − 0.29, 95% CI=0.21–0.38) (Figure 2b). A statistically significant correlation existed between mRNA levels and the BAX SNP (P=0.016, logistic regression analysis). Bcl-2/Bax ratio of mRNA expression was statistically higher (P=0.002, t-test) in samples with the SNP (mean − 17.87 relative units, 95% CI=1.94–33.80) than those without it (mean − 2.44, 95% CI=1.31–3.57). A statistical correlation was found between the presence of BAX SNP and higher Bcl-2/Bax ratio (P=0.018, logistic regression analysis). Bax protein levels were determined in 23 CLL samples using Western blotting (Figure 3a). Samples bearing the SNP had significantly (P=0.024, t-test) lower Bax protein expression (mean – 0.22, 95% CI=0.095–0.35) than samples without it (mean – 0.42, 95% CI=0.30–0.54) (Figure 3b). The significance of the correlation was established by logistic regression analysis (P=0.047). There was a correlation between mRNA and protein levels (P=0.001, Fisher's test); eight out of eight samples with low mRNA had low protein levels and 10 out of 13 samples with high mRNA had also high levels of Bax protein.
We constructed reporter plasmids with the mutant and wild-type BAX promoter linked to Luciferase. The pGL3-BAXpr(w), with the part of the BAX gene promoter that includes wild-type BAX promoter fragment with TATA box and 5′-UTR with the G nucleotide at the position 125, and the pGL3-BAXpr(m) plasmid containing the same promoter region with the G125A SNP (human BAX gene, 5′-region, GenBank Accession number U17193) (Table 1, Figure 3a). Restriction enzyme analysis (REA) with KpnI and HindIII (Figure 4b) and sequencing (Figure 4a) provided evidence for the successful insertion of the BAX promoter into the pGL3-Basic vector. Firefly Luciferase activity in each individual transfection was normalized to corresponding Renilla Luciferase activity in order to account for variability in transfection efficiency. The results were compared to the expression of pGL3-Basic plasmid alone (accepted as equal to 1), and presented as fold activity (Figure 4c). The transfection was considered successful only if Luciferase activity of the positive control (pGL3-Control vector) was at least 10-fold higher than of the pGL3-Basic vector (Figure 4c). In six independent transfections, the Luciferase activity for HeLa cells transfected with pGL3-BAXpr(w) was significantly (2.6-fold) higher than for cells transfected with pGL3-BAXpr(m) (P=0.0202, t-test) (Figure 4c). This provides experimental evidence of the negative influence of the G125A SNP on BAX gene expression.
Programmed cell death requires Bax protein (Green and Reed, 1998) and the protein is frequently reduced in different tumors including CLL (Saxena et al., 2002), colon (LeBlanc et al., 2002), stomach, breast (Krajewski et al., 1995), and endometrium (Tai et al., 1998, 1999) (Kokawa et al., 1999a, 1999b) cancers. Mutations found in the third or second exon of BAX in different tumors are associated with resistance to apoptosis (Rampino et al., 1997; Brimmell et al., 1998; Meijerink et al., 1998; Ouyang et al., 1998; Peng et al., 1998; LeBlanc et al., 2002).
We reported earlier a novel SNP, G125A, in the BAX promoter in lymphocytes of CLL patients. This SNP was associated with more rapid disease progression and resistance to treatment (Saxena et al., 2002). The SNP is located within 5′-UTR region of the promoter and it may potentially affect the BAX gene expression. Indeed, we had identified its association with reduced protein expression in a small sample (n=6) (Saxena et al., 2002). In this report, we confirm this association showing reduced gene expression at both mRNA and protein levels in 21 and 23 CLL patients. Our results showing that the SNP reducing the efficacy of the BAX promoter suggested a relationship for the association between the SNP and reduced Bax protein level. The next logical step is to provide an evidence for a cause–effect relationship between this SNP and its effect on the gene expression. We do so in this paper by functional studies assaying promoter activity using recombinant plasmids.
Analysis of the BAX promoter sequence using the RegScan program (Levitsky et al., 2001) predicted the highest promoter activity at the location of the SNP. Interestingly, the RegScan program predicted that G to A transition would lead to a decrease in the promoter activity of Bax. This supports our results showing decreased mRNA and protein levels in CLL samples with transition.
CLL is the most common leukemia in the Western world and is incurable by current treatment modalities (Kay et al., 2002). Since apoptosis resistance is a fundamental attribute of CLL (Caligaris-Cappio and Hamblin, 1999), identifying key molecules regulating this process is crucial for developing more effective treatments. In addition to Bcl-2 and Mcl-1 (Kitada et al., 1998; Saxena et al., 2004), the role of Bax in prolonged cell survival in CLL has been proven by many investigators (Pepper et al., 1996, 1997; Thomas et al., 1996; Saxena et al., 2004). Although Bak, another proapoptotic member of the Bcl-2 family is expressed in CLL cells (Kitada et al., 1998, 1999; Sanz et al., 2004), it may not be able to substitute for the loss or reduction in Bax. This has been shown in the BAX-knockout human colon carcinoma cell line that depends completely on Bax for apoptosis initiation (LeBlanc et al., 2002).
A reduction in the amount of Bax protein consequent to the presence of BAX SNP will not only decrease its ability to act directly on the mitochondrial membrane but also offer advantage to the antiapoptotic Bcl-2 and Mcl-1 as Bax can heterodimerize with both of these Bcl-2 family members. Further, increased Bcl-2 relative to Bax may block Bax homodimerization (Gross et al., 1998). The clinical impact of G125A SNP on CLL patients includes progression beyond Rai stage 0 (P=0.00018) and failure to achieve complete response to standard chlormabucil/prednisone treatment (P=0.038) as shown by us earlier (Saxena et al., 2002).
In this paper, we have provided conclusive evidence that G125A SNP underlies aberrant (low) Bax expression in CLL providing justification for its exploitation as a potential target for treatment.
Materials and methods
The research was approved with the University of Saskatchewan Advisory Committee on Ethics in Human Experimentation. Blood samples were obtained from 77 CLL patients (age range=34–86 years; median=70; 50 men and 27 women) registered at the Saskatoon Cancer Center, and processed as described earlier (Moshynska et al., 2004). All samples were obtained from untreated patients at diagnosis; this is relevant since chemotherapy selects clones with overexpressed Bax protein (Pepper et al., 1999).
The human epithelial cancer cell line HeLa (CCL-2, American Type Culture Collection, Manassas, VA, YSA) was maintained in Dulbecco's modified Eagle's medium (Sigma, St Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen), at 37°C under 5% CO2.
RNA was analysed using ribonuclease (RNase) protection assay as we described earlier (Moshynska et al., 2004). RNA was extracted from isolated lymphocytes using TRIzol (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions, quantified by measuring its absorbance at 260 nm and stored at −70°C. Each RNA sample (10 μg) was processed using the In Vitro Transcription kit (Pharmingen, Mississauga, ON, USA), and was hybridized with the human apoptosis multiprobe template set (hApo-2c, Pharmingen) as described earlier (Moshynska et al., 2004). The level of each mRNA sample (intensity × mm2) was normalized against the corresponding levels of the GAPDH housekeeping gene product, and relative expression levels were calculated (Figure 2).
REA for BAX promoter SNP, G125A
DNA was extracted from isolated blood lymphocytes using the QIAamp Tissue Kit (Qiagen, Mississauga, Ontario, Canada), the BAX promoter region was amplified (Saxena et al., 2002), and digested with AciI restriction enzyme (New England BioLabs Inc., Mississauga, Ontario, Canada) as described earlier (Moshynska et al., 2003).
Cell lysates were prepared and protein concentration was determined as described earlier (Saxena et al., 2004). Proteins (20 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to a nitrocellulose membrane using a wet transfer apparatus (Bio-Rad), blocked, and exposed to anti-Bax primary antibody (rabbit anti-human polyclonal, Santa Cruz, Santa Cruz, CA, USA). The blots were washed and incubated with a goat anti-rabbit (Santa Cruz, Santa Cruz, CA, USA) horseradish peroxidase-conjugated secondary antibody, washed, and processed for chemiluminescence detection. Blots were reprobed with mouse monoclonal anti-β-actin primary and goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Saxena et al., 2004). Bax expression levels were normalized to actin, and relative expression levels were calculated (Figure 3).
Luciferase reporter gene construct
The plasmid constructs used in this study are listed in the Table 1. The following Luciferase reporter vectors were obtained from Promega (Madison, WI, USA): pGL3-Basic, pGL-3-Control, and pRL-SV40. Constructs were prepared using standard recombination techniques (Sambrook and Maniatis, 1989). Briefly, the genomic DNA was extracted from peripheral blood lymphocytes, and BAX promoter was PCR amplified and sequenced as described earlier (Saxena et al., 2002), and two samples were selected, one with (subsequently referred to as ‘mutant’) and the other without G125A SNP (subsequently referred to as ‘wild type’). The samples did not have any other genetic changes in the amplified region (human BAX gene, 5′-region, GenBank Accession number U17193). Then, the genomic DNA was amplified using BLRGA-F (IndexTermAAGGTACCAGGGGCGGTGTAGT) and BLRGA-R (IndexTermCCAAGCTTGACTGTCCAATGAGCATC) primers: a site for KpnI is italicized and HindIII site is underlined (Table 1). The size of the amplified products was verified by agarose gel electrophoresis (Figure 4b), the PCR fragments were digested with KpnI (Promega, Madison, WI, USA) and HindIII (Promega), and then cloned between the KpnI and HindIII sites of the pGL3-Basic vector. The ligation mixture (5 μl) was electroporated into Escherichia coli strain DH5α using standard procedure and the Gene Pulser apparatus (Bio-Rad, 12 000 V/cm, 200 Ω). Recombinant plasmids were selected in the presence of Ampicillin (100 μg/ml), purified using Plasmid Purification kit (Qiagen Inc., Mississauga, Ontario, Canada), and screened for the presence of the KpnI/HindIII fragment by the agarose gel electrophoresis (Figure 4b). Two plasmids were created, the plasmid pGL3-BAXpr(w), with G nucleotide at the position 125, and the plasmid pGL3-BAXpr(m), with A at position 125 (Figure 4a, Table 1). The cloned fragments were verified by sequencing (Figure 4a) using 377 ABI Prism DNA Sequencer (Perkin-Elmer Inc., Foster City, CA, USA). The RVprimer 3 (clockwise primer) and GLprimer2 (counter clockwise primer) (Promega) were used (Figure 4a, Table 1).
Transfection by Luciferase reporter gene constructs
HeLa cells were transfected with 15 μg of plasmid DNA (pGL3-BAXpr(w), pGL3-BAXpr(m), pGL3-Basic (internal negative control), or pGL3-Control (positive control)) using calcium phosphate transfection method (Chen and Okayama, 1988). Briefly, HeLa cells were washed with 0.1 M phosphate-buffered saline (PBS), trypsinized (Trypsin–EDTA; Invitrogen, Grand Island, NY, USA). Cells were transferred into a six-well plate (1 × 106 cells in 2.5 ml of media) 24 h prior to transfection, and were fed 2 h prior to transfection. The cells were cotransfected with 3 μg of the reporter plasmid pRL-SV40 (this plasmid's Renilla Luciferase is enhanced by SV40 promoter, it was added to each transfection as transfection efficiency control, Table 1) into the HeLa cell line. The transfected cells were maintained for 48 h with the media replaced once after 16 h after transfection. Transfection efficiencies were normalized to Renilla Luciferase activity in the Luciferase reporter gene assay (Promega) according to the manufacturer's instructions. Six independent transfections were performed. Cell viability was determined by trypan blue exclusion (1 : 1) (Sanchez et al., 1986). The total amount of cells was calculated in three different grids of Hematocytometer (0.1 mm deep, Bright Line, Reichert, Buffalo, NY, USA) as recommended by the manufacturer.
Luciferase expression assay
Cells were washed with 0.1 M PBS, harvested with 1 ml of cell harvesting buffer (0.04 M Tris (pH 7.5), 0.001 M EDTA, 0.15 M NaCl), centrifuged, resuspended in 0.25 M Tris (pH 7.5), diluted to 3 × 106/ml in 0.25 M Tris, and stored at −70°C. Cells were lysed in 100 μl of lysis buffer provided with the kit and 70 μl of cell lysates were used for each assay. The Luciferase activity (relative light units) was measured using Dual-Glo Luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions in luminescence counter (Packard Bioscience, Meriden, CT, USA).
Statistical analysis was performed using SPSS software version 11.5 (Chicago, IL, USA). The independent t-test analysis was used to determine the statistical significance of differences in mean values; all tests were two-sided. Logistic regression analysis was used to determine the association between BAX mRNA and protein levels and presence of BAX SNP. Differences were considered statistically significant at P-values less than 0.05. The theoretical prediction of promoter activity was carried out using publicly available online RegScan algorithm at the molecular biology server (http://www.mgs.bionet.nsc.ru/mgs/) (Levitsky et al., 2001).
Adams JM and Cory S . (1998). Science, 281, 1322–1326.
Bargou RC, Daniel PT, Mapara MY, Bommert K, Wagener C, Kallinich B, Royer HD and Dorken B . (1995). Int. J. Cancer, 60, 854–859.
Bodrug SE, Aime-Sempe C, Sato T, Krajewski S, Hanada M and Reed JC . (1995). Cell Death Differ., 2, 173–182.
Brimmell M, Mendiola R, Mangion J and Packham G . (1998). Oncogene, 16, 1803–1812.
Caligaris-Cappio F and Hamblin TJ . (1999). J. Clin. Oncol., 17, 399–408.
Catasus L, Matias-Guiu X, Machin P, Zannoni GF, Scambia G, Benedetti-Panici P and Prat J . (2000). Cancer, 88, 2290–2297.
Chen CA and Okayama H . (1988). Biotechniques, 6, 632–638.
Cordone I, Masi S, Mauro FR, Soddu S, Morsilli O, Valentini T, Vegna ML, Guglielmi C, Mancini F, Giuliacci S, Sacchi A, Mandelli F and Foa R . (1998). Blood, 91, 4342–4349.
Green DR and Reed JC . (1998). Science, 281, 1309–1312.
Gross A, Jockel J, Wei MC and Korsmeyer SJ . (1998). EMBO J., 17, 3878–3885.
Gross A, McDonnell JM and Korsmeyer SJ . (1999). Genes Dev., 13, 1899–1911.
Kay NE, Hamblin TJ, Jelinek DF, Dewald GW, Byrd JC, Farag S, Lucas M and Lin T . (2002). Hematology (Am Soc Hematol Educ Program), 193–213.
Kitada S, Andersen J, Akar S, Zapata JM, Takayama S, Krajewski S, Wang HG, Zhang X, Bullrich F, Croce CM, Rai K, Hines J and Reed JC . (1998). Blood, 91, 3379–3389.
Kitada S, Zapata JM, Andreeff M and Reed JC . (1999). Br. J. Haematol., 106, 995–1004.
Knudson CM, Tung KS, Tourtellotte WG, Brown GA and Korsmeyer SJ . (1995). Science, 270, 96–99.
Kokawa K, Shikone T, Otani T and Nakano R . (1999a). Cancer, 85, 1799–1809.
Kokawa K, Shikone T, Otani T and Nakano R . (1999b). Cancer, 86, 79–87.
Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE and Oltvai ZN . (1993). Semin. Cancer Biol., 4, 327–332.
Krajewski S, Blomqvist C, Franssila K, Krajewska M, Wasenius VM, Niskanen E, Nordling S and Reed JC . (1995). Cancer Res., 55, 4471–4478.
LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J, Schow P, Fong S, Schwall R, Sinicropi D and Ashkenazi A . (2002). Nat. Med., 8, 274–281.
Lee HS, Choi SI, Lee HK, Kim HS, Yang HK, Kang GH, Kim YI, Lee BL and Kim WH . (2002). Mod. Pathol., 15, 632–640.
Levitsky VG, Podkolodnaya OA, Kolchanov NA and Podkolodny NL . (2001). Bioinformatics, 17, 998–1010.
Meijerink JP, Mensink EJ, Wang K, Sedlak TW, Sloetjes AW, de Witte T, Waksman G and Korsmeyer SJ . (1998). Blood, 91, 2991–2997.
Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B and Reed JC . (1994). Oncogene, 9, 1799–1805.
Moshynska O, Sankaran K and Saxena A . (2003). Mol. Pathol., 56, 205–209.
Moshynska O, Sankaran K, Pahwa P and Saxena A . (2004). J. Natl. Cancer Inst., 96, 673–682.
Ouyang H, Furukawa T, Abe T, Kato Y and Horii A . (1998). Clin. Cancer Res., 4, 1071–1074.
Peng H, Aiello A, Packham G, Isaacson PG and Pan L . (1998). J. Pathol., 186, 378–382.
Pepper C, Bentley P and Hoy T . (1996). Br. J. Haematol., 95, 513–517.
Pepper C, Hoy T and Bentley DP . (1997). Br. J. Cancer, 76, 935–938.
Pepper C, Thomas A, Hoy T and Bentley P . (1999). Br. J. Haematol., 104, 581–588.
Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC and Perucho M . (1997). Science, 275, 967–969.
Sakuragi N, Salah-eldin AE, Watari H, Itoh T, Inoue S, Moriuchi T and Fujimoto S . (2002). Gynecol. Oncol., 86, 288–296.
Sambrook JFE and Maniatis T . (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press: Cold Spring Harbor, New York.
Sanchez MS, Ford CW and Yancey Jr RJ . (1986). Antimicrob Agents Chemother, 29, 634–638.
Sanz L, Garcia-Marco JA, Casanova B, de La Fuente MT, Garcia-Gila M, Garcia-Pardo A and Silva A . (2004). Biochem. Biophys. Res. Commun., 315, 562–567.
Saxena A, Moshynska O, Sankaran K, Viswanathan S and Sheridan DP . (2002). Cancer Lett., 187, 199–205.
Saxena A, Viswanathan S, Moshynska O, Tandon P, Sankaran K and Sheridan DP . (2004). Am. J. Hematol., 75, 22–33.
Tai YT, Lee S, Niloff E, Weisman C, Strobel T and Cannistra SA . (1998). J. Clin. Oncol., 16, 2583–2590.
Tai YT, Strobel T, Kufe D and Cannistra SA . (1999). Cancer Res., 59, 2121–2126.
Thomas A, El Rouby S, Reed JC, Krajewski S, Silber R, Potmesil M and Newcomb EW . (1996). Oncogene, 12, 1055–1062.
Violette S, Poulain L, Dussaulx E, Pepin D, Faussat AM, Chambaz J, Lacorte JM, Staedel C and Lesuffleur T . (2002). Int. J. Cancer, 98, 498–504.
Yamamoto H, Sawai H and Perucho M . (1997). Cancer Res., 57, 4420–4426.
Yin C, Knudson CM, Korsmeyer SJ and Van Dyke T . (1997). Nature, 385, 637–640.
Zha H, Aime-Sempe C, Sato T and Reed JC . (1996). J. Biol. Chem., 271, 7440–7444.
We thank Dr WF Dong for his advice during construction of the pGL3-based plasmids, Ms H Neufeld for assisting with Western blotting experiments, and Mr T Reichert and Ms M Hesson for assisting with preparing illustrations.
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Cite this article
Moshynska, O., Moshynskyy, I., Misra, V. et al. G125A single-nucleotide polymorphism in the human BAX promoter affects gene expression. Oncogene 24, 2042–2049 (2005). https://doi.org/10.1038/sj.onc.1208377
- single-nucleotide polymorphism
- Luciferase activity
- gene expression
- chronic lymphocytic leukemia
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