Hakuro Okano

Requirement of AP-1 for Ceramide-induced Apoptosis in Human Leukemia HL-60 Cells

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 μM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 μMN-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 μMN-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius.

We cloned and sequenced the dextranase (Dex) (1,6-alpha-glucanhydrolase; EC 3.2.1.11)-encoding gene from Streptococcus salivarius (Ss) strain M-33. Recombinant clones from an Ss genomic library specifying Dex activity were identified as colonies surrounded by transparent halos on blue dextran plates. One of the clones had a 4.3-kb KpnI fragment containing the gene coding for an 826-amino-acid polypeptide with a molecular mass of 87.9 kDa, which corresponds well to that of native Dex from the Ss culture supernatant. There was no sequence homology between the gene encoding Ss Dex and the gene encoding dextran glucosidase of S. mutans, or between their protein products.