G3139 is an 18-mer phosphorothioate
oligodeoxyribonucleotide, which is targeted to the
initiation codon region of the bcl-2mRNA. Although treatment of PC3
prostate cancer cells with
G3139, which contains two CpG motifs, causes a dramatic decrease in bcl-2
protein expression after 3 days, it did not result in significant cellular apoptosis, as it does in many other cell lines. The absence of apoptosis was demonstrated by the absence of
pro-caspase 3 cleavage products and of
Annexin V cell surface expression. In addition,
ATP production and the mitochondrial membrane potential DeltaPsim were preserved. Despite this,
G3139 significantly inhibited the rate of cellular proliferation in complete media and blocked cloning in soft
agar. G4232, a variant of
G3139 that down-regulates bcl-2 expression to the same extent but has both CpG cytidines C5 methylated, was only minimally antiproliferative. A series of mismatched G3139-related oligomers were synthesized that could also substantially down-regulate bcl-2
protein expression, but only if the CpG motifs were preserved, demonstrating the presence of additional non-antisense mechanisms.
G3139 caused production of
reactive oxygen species in growth-arrested cells and oxidation of nuclear
guanosine to
8-hydroxy-2'-deoxyguanosine, as determined by 1F7
monoclonal antibody staining.
Bromodeoxyuridine incorporation studies demonstrated that
G3139 induced a G1-S entry block and an intra-S-phase block in PC3 cells that persisted as long as 3 days. This finding coincides with the observation that expression of several
proteins encoded by S-phase genes, including c-myb and
poly(ADP-ribose) polymerase, were significantly reduced. These results illustrate the complexity of the mechanism of action of
G3139 in PC3 cells.