CREB antisense oligonucleotides induce non-apoptotic cell death in proliferating leukemia cells, but not normal hematopoietic cells, by a bizarre non-antisense mechanism.

We report that antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) against cyclic AMP response element-binding protein (CREB) induce the death of human leukemia cell lines including HL-60, Kasumi-1 and K562, OCI-AML1a and also primary leukemia cells isolated from patients with acute myelocytic leukemia and chronic myelocytic leukemia in blastic crisis. In contrast, normal human bone marrow CD34+ cells and normal peripheral blood lymphocytes were resistant to the antisense-mediated cell death. We found that antisense-treated HL-60 cells had prominent nuclear fragmentations but lacked apoptotic features including internucleosomal DNA cleavage and TUNEL positivity. Cell cycle analysis demonstrated a remarkable reduction in G1 phase population along with a mild accumulation of S phase and good preservation of G2/M phase, indicating cells died at G2/M without cycling into G1 phase. None of the sense-sequenced PS-ODNs induced cell death. Further, neither the expression nor the message of CREB protein was reduced by antisense treatment, indicating that cell death was mediated by a non-antisense mechanism. On the other hand, no consensus oligonucleotide sequence for cell death induction was detected. Rather, we found a good correlation between the melting temperatures and the anti-proliferative activities of the oligonucleotides. Thus, CREB antisense PS-ODNs selectively induce a non-apoptotic cell death in leukemic cells by an unknown hybridization-dependent mechanism.