The bcl-2 (B-cell CLL/lymphoma 2) gene product is a mitochondrial membrane protein that functions as an inhibitor of apoptosis and has been classified as a proto-oncogene. Overexpression of bcl-2 has been reported in follicular lymphoma and most solid tumors, including lung, breast, and prostate cancer. The bcl-2 gene contains a highly GC-rich region upstream of the P1 promoter, and deletion or mutation of this region has been shown to increase promoter activity by 2.1-fold and 2.6-fold, respectively. This GC-rich element is a 39-base-pair sequence containing seven polypurine (guanine) tracts separated by one or more bases, which corresponds to the general motif capable of forming an intramolecular G-quadruplex structure. Initial studies revealed that incubation of the G-rich strand of this element in 100 mM K+ resulted in a band that runs anomalously on a nondenaturing gel. We further demonstrate, using DMS footprinting and polymerase stop assays, that the bcl-2 G-rich sequence can form three distinct G-quadruplex structures, which are further stabilized by G-quadruplex-interactive agents, such as TMPyP4, Se2SAP, and telomestatin. Specific mutations within the G-tracts lead to the disappearance of one or more of these G-quadruplexes formed within this region. Comparative CD spectroscopy and DMS footprinting predict a core bcl-2 G-quadruplex structure consisting of three stacked G-tetrads formed by at least two parallel G-stretches connected through at least two double-chain-reversal loops. Our collective results suggest that the multiple G-quadruplex structures identified in the promoter region of the bcl-2 gene are likely to play a similar role to the c-myc G-quadruplex in that their formation could serve to modulate gene transcription.

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