The c-myb oncogene is highly expressed in many leukemias and allows leukemic cells to stay in a state of proliferation without undergoing differentiation. For this reason, downregulation of c-myb expression may be useful for leukemia therapy. The c-myb promoter contains a purine-rich region with three copies of four GGA repeats, 3(GGA)4, and transient transfection analysis showed that the deletion of all three (GGA)4 repeats abolished c-myb promoter activity in three leukemia cell lines. This result implies that the GGA repeat region in the c-myb promoter is critical for c-myb expression, probably by providing binding sites for the crucial transcription factors for c-myb transcription. Interestingly, the deletion of only one or two (GGA)4 repeat(s), including one repeat that overlaps a PU.1 binding site, led to a dramatic increase in c-myb promoter activity in CCRF-CEM T-ALL cells, but only modest effects in Jurkat T-ALL cells and K562 CML blast cells. Previous reports have shown that (GGA)4 DNA can form an unusual structure related to a guanine quadurplex and composed of a guanine tetrad stacked onto a guanine-adenine heptad. Two (GGA)4 repeats can form a higher ordered structure by dimerizing to form a tetrad:heptad:heptad:terad. To determine if the c-myb GGA repeats could form such structures, DMS footprinting and DNA polymerase arrest assays were performed with oligonucleotides containing the 3(GGA)4 from the c-myb promoter. DMS footprinting showed potassium dependent protection of all the guanines in the GGA repeats that would be involved in tetrad:hepad formation. Taq polymerase arrest assays showed potassium dependent arrests of primer elongation at the first and second (GGA)4, and polymerase arrest was enhanced by the presence of guanine quadruplex interactive compounds such as TmPyP4 and telomestatin, implying that the secondary structure formed by the c-myb GGA repeats is stabilized by small molecules that can bind to G-quadruplexes. Taq polymerase assay data from studies introducing quadruplex disrupting mutations into each (GGA)4 of the 3(GGA)4 and truncating one or two (GGA)4 demonstrated that at least two (GGA)4 are required in order to arrest primer elongation in the assay, suggesting that dimerization of two tetrad:heptad structures is necessary for stable G-quadruplex formation. Based on these data, we propose that that the GGA repeat region in the c-myb promoter plays a critical role in regulating c-myb expression and can serve both as a positive regulatory element by recruiting transcription factors and as a negative regulatory element by forming a stable secondary structure that prevents transcription factor binding. We further propose that small molecules that bind to G-quadruplexes may be able to suppress c-myb expression and prevent the proliferation of leukemia cells that require c-myb expression.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]