Leptomycin B (LMB) induces apoptosis in a cyclin B1–dependent manner.

(A) LMB (3 ng/mL) induces apoptosis in NIH3T3 cells as measured by annexin V staining. (B) LMB (3 ng/mL) causes nuclear accumulation of the cyclin B1 protein. Original magnification × 400. Cyclin B1 antibody, green; Hoechst 33342 DNA, blue. (C) Treatment of NIH3T3 cells with cyclin B1 antisense oligonucleotides inhibits LMB-induced apoptosis. Sense oligonucleotides had no effect on apoptosis. Apoptosis is measured in the presence and absence of 3 ng LMB. Error bars represent the SEM of no less than 20 000 cells and is a representative of triplicate experiments. (D) Cyclin B1 protein levels indicating that the antisense treatment from Figure 2C lowers cyclin B1 protein levels in the presence or absence of LMB. Protein (10 μg/well) is loaded and cdc2 staining is used as a loading control. (E) Cyclin A antisense has no affect on LMB-induced apoptosis, as measured by annexin staining. (F) Cyclin A antisense reduces cyclin A but not cyclin B1 protein levels. (G) LMB sensitizes NIH3T3 and Cos-1 cells to radiation-induced apoptosis. Cells were treated with 0.05 ng/mL LMB and annexin V binding measured after 200 cGy γ radiation. Errors are the mean of triplicate experiments. (H) LMB (0.05 ng/mL) induces apoptosis in Ramos cells (0.05 ng/mL), as measured by annexin V staining.