Lower expression of Bax protein in various human malignancies is associated with poor response to treatment and shorter disease-free survival. We (

Cancer Lett 2002; 187:199–205
) and others (
J Clin Oncol; 23:1514–21
) have shown the association of a single nucleotide polymorphism (SNP) in the BAX promoter (G125A) with reduced protein expression and treatment resistance in chronic lymphocytic leukemia (CLL). Using luciferase reporter gene assay we demonstrated that this SNP significantly reduced BAX promoter activity (
Oncogene 2005; 24:2042–9
). Our aim was to determine the effect of this polymorphism on the binding of transcription factors. For electrophoretic mobility shift (EMSA), HeLa and K562 nuclear extracts, and for chromatin immunoprecipitation (ChIP), K562 cells were used to study their ability to bind to a radiolabeled DNA probe corresponding to the BAX promoter region with G nucleotide at the position 125 or bearing G125A SNP. Super-shift assay was performed to determine the transcription factor involved in binding. Competition assays were performed to determine differences in the binding ability of the two probes. A panel of antibodies was tested by super-shift and ChIP assays, non-specific antibodies served as negative controls. Two major band shifts were detected by EMSA. The mobility of the detected complexes was different from those observed with GC1 probe, specific for Sp1/Sp3, suggesting the involvement of transcription factors other than Sp1/ Sp3. This was confirmed in super-shift assay and ChIP assay by incubating DNA probes with Sp1 or/ and Sp3 antibodies. We also found in the competition assays that the cold probes competed differently for binding. The findings provide evidence of the ability of G125A SNP to influence transcription factor binding in vitro (as shown in EMSA experiments) and in vivo (in ChIP experiments).

Topics:
antibodies, bax protein, cancer, chromatin, chronic b-cell leukemias, chronic lymphocytic leukemia, common cold, complex, dna probes, immunoprecipitation

Disclosures: Canadian Institutes of Health Research and Regional Partnership Program, Saskatchewan Health Research Foundation.

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2006, The American Society of Hematology
2006
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