Unmethylated CpG-containing oligodeoxynucleotides inhibit apoptosis in WEHI 231 B lymphocytes induced by several agents: evidence for blockade of apoptosis at a distal signalling step

Abstract

Certain oligodeoxynucleotides (ODN) containing cytosine followed by guanosine (CpG) protect B cells from apoptosis, and induce B-cell proliferation and cytokine production. We investigated the effect of phosphorothioate CpG-containing ODNs (5'-ATAATCGACGTTCAAGCAAG-3' or 5'-TCCATGACGTTCCTGACGTT-3') and control ODNs (which did not contain CpG) on apoptosis and cell growth in WEHI 231 murine B lymphoma cells. Anti-surface (alpha-s)IgM antibody induces 40-60% DNA degradation and growth arrest of WEHI 231 cells in 24 h. Both of these effects were substantially reversed by 30 ng/ml CpG-ODN added up to 8 hr after alpha-sIgM. Control ODNs not containing the CpG motif were without effect. We explored various hypotheses to account for these effects. The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate, inhibits apoptosis induced by alpha-sIgM, but the anti-apoptotic effect of CpG-ODN was not affected by inhibitors of protein kinase C, indicating that CpG-ODN does not act via protein kinase C. CpG-ODN inhibited apoptosis and growth arrest induced by C2- and C8-ceramide, sphingomyelinase and an intracellular Ca2+ pump inhibitor thapsigargin, indicating that inhibition is not mediated via suppression of the ceramide cycle or suppression of Ca2+ mobilization. CpG-ODN partially inhibited apoptosis induced by okadaic acid, a protein phosphatase inhibitor, and by menadione, a free radical generator. CpG-ODN also inhibited apoptosis and growth arrest induced by ultraviolet-irradiation, glucocorticoid, vinca alkaloids, and doxorubicin. CpG-ODN significantly protected cells from DNA fragmentation induced by alpha-sIgM in the presence of cycloheximide, but cycloheximide itself induces apoptosis which was unaffected by CpG-ODN. These results suggest that CpG-ODNs powerfully modulate the process by which immune cells are committed to death or proliferation by a mechanism acting on distal cell signalling events. CpG-ODNs may be able to decrease immunosuppression in patients undergoing cancer chemotherapy.