Article Figures & Data
Figures
- Figure 1.
Glucocorticoids inhibit Vinca alkaloid-induced DNA fragmentation. BCap37 cells and KB cells were treated with different concentrations of vinblastine (Vinb.) or vincristine (Vinc.) for 48 h with or without pretreatment of 10−7 m TA (Gluco.). Cells were then harvested and fragmented DNA was extracted and analyzed by electrophoresis in 1.2% agarose gel containing 0.1% ethidium bromide.
- Figure 2.
Glucocorticoids do not affect mitotic arrest induced by Vinca alkaloids. Cells were exposed to 100 nm vinblastine (Vinb.) and vincristine (Vinc.) with or without pretreatment of glucocorticoids (Gluco.) for 24 h before they were harvested for preparation of cytospin slides as described in Materials and Methods. Three hundred cells/slide were counted and only those cells with typical morphological features of condensed chromosomes were identified as mitotically arrested cells. Columns, mean percentage of mitotic cells (independent experiments performed in triplicate); bars, SD.
- Figure 3.
Vinca alkaloids and glucocorticoids possess opposite effects on IκBα. BCap37 cells were incubated with different concentrations of vinblastine (Vinb.) or vincristine (Vinc.) for 24 h with or without pretreatment of glucocorticoids (10−7 m TA, 24 h; Gluco.). Equal amounts of cellular proteins (50 μg/lane) were fractionated on a 12.5% SDS-PAGE gel and transferred to PVDF membranes followed by immunoblotting with anti-IκBα polyclonal antibody. β-actin proteins were blotted as a control.
- Figure 4.
Effects of Vinca alkaloids and glucocorticoids on IKK activity. BCap37 cells were treated with the indicated concentrations of vinblastine (Vinb.) or vincristine (Vinc.) for 24 h with or without pretreatment of glucocorticoids (10−7 m TA, 24 h; Gluco.). IKK complex was immunoprecipitated with an anti-IKKα antibody, and the immune complex was subject to in vitro kinase assay (KA) using GST-IκBα fusion protein as the substrate. Following SDS-PAGE, the portion of the gel containing the substrate was dried and processed for autoradiography. The portion containing IKK was analyzed by Western blotting for IKKα protein.
- Figure 5.
Transfection with antisense IκBα sensitizes Vinca alkaloid-induced apoptosis. BCap37 cells transfected with vector (pcDNA3) only and antisense IκBα (IκBα-ANT5) were incubated with the indicated concentrations of vinblastine (Vinb.) or vincristine (Vinc.) for 48 h. A, DNA fragmentation assays. Cells were harvested and fragmented DNA was analyzed by electrophoresis in 1.2% agarose gel containing 0.1% ethidium bromide. B, MTT assays. Points, mean of three separate experiments; bars, SD. There is a statistically significant difference between antisense IκBα-transfected cells and vector-transfected cells treated by Vinca alkaloids (P < 0.01, Student's t test).
- Figure 6.
Vinca alkaloids do not degrade mutant IκBα. Equal amounts of cellular proteins (50 μg/lane) from wild-type BCap37 cells (WT), cells transfected with empty pcDNA3 vector (Vector), and cells transfected with mutant IκBα (Mutant IκBα) treated with different concentrations of vinblastine (A) and vincristine (B) for 24 h were fractionated on 12.5% SDS-PAGE gel and transferred to PVDF membranes followed by immunoblotting with anti-IκBα polyclonal antibody. β-actin protein was used as a control.
- Figure 7.
Overexpression of mutant IκBα blocks Vinca alkaloid-induced NF-κB activation. BCap37 cells transfected with pcDNA3 vector only (Vector) or mutant IκBα (Mutant IκBα) were treated with different concentrations of vinblastine (Vinb.; A) and vincristine (Vinc.; B) for 24 h. Equal amounts of nuclear cell extracts were subjected to EMSAs with [γ-32P]-labeled oligonucleotide encompassing the NF-κB binding site. NS, nonspecific site.
- Figure 8.
Overexpression of mutant IκBα suppresses vinblastine- and vincristine-induced apoptosis. Wild-type BCap37 cells (WT) and cells transfected with pcDNA3 vector only (Vector) or mutant IκBα (Mutant IκBα) were treated with 50 or 100 nm vinblastine (A) and vincristine (B) for 48 h. Cells were harvested and stained with propidium iodide for flow cytometric analysis. The peaks corresponding to G1 and G2-M phases of the cell cycle are indicated. The sub-G1 peaks (AP) represent apoptotic cells.
Volume 3, Issue 3
