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Article Figures & Data

Figures

  • Fig. 1.

    Effect of flavopiridol on cell growth. (A) SUDHL4 cell line. Cultures were exposed to various concentrations of flavopiridol and at the indicated points counted by electronic counter at 24 hours (▪), 48 hours (□), and 72 hours (□). The data are shown as percentage of untreated control. Total untreated control cell number/culture at 24 hours was 104,040 ± 3,750; 48 hours, 183,880 ± 6,687; and 72 hours, 371,670 ± 11,956. (B) PC3 cell line. Exponentially growing cells were incubated with flavopiridol at the indicated concentrations for 48 hours, and cell growth was assessed by the colorimetric SRB assay, as described in Materials and Methods. The data are the mean of four determinations ± standard deviation (SD) and are representative of three experiments for each cell line.

  • Fig. 2.

    Morphology of SUDHL4 and PC-3 cells after exposure to flavopiridol. (A) Untreated control SUDHL4s. (B) SUDHL4s after 12 hours exposure to flavopiridol at 1,000 nmol/L. (C) Untreated control PC-3s. (D) PC-3s after 12 hours exposure to flavopiridol at 1,000 nmol/L. Photography was at 1,000 ×, oil immersion microscope, after cytospin preparation as described in Materials and Methods.

  • Fig. 3.

    Effect of flavopiridol on growth of SUDHL4, HL60, MOLT4, K562, and PC3 cells. Cells (2 × 104 cells/mL) were plated into each well of a six-well plate. After 24 hours, cells were treated in triplicate either with vehicle or with 50, 100, and 500 nmol/L of flavopiridol for 48 hours. Cell growth was determined by counting live (Trypan Blue excluded) and dead (Trypan Blue stained) cells on a hemacytometer. (A) Represents growth inhibition as percent of control for SUDHL4 (○), HL60 (□), MOLT4 (▵), K562 (▿), and PC3 (◊) cells after 48 hours drug exposure. (B) Presents the results as percent of total live cells (open bar) and dead cells (hatched bar) after 500 nmol/L flavopiridol exposure for 48 hours. The experiments represent the mean of three determinations ± SD.

  • Fig. 4.

    DNA fragmentation after exposure to flavopiridol. (A) Exponentially growing SUDHL4, SUDHL6, MOLT4, and Jurkat cells were exposed to the indicated concentrations of flavopiridol for 14 hours and genomic DNA extracted as described in Materials and Methods before electrophoresis in a 1.6% agarose gel. (B) SUDHL4 cells were exposed to the following concentrations of flavopiridol for the indicated periods after prelabelling DNA with [14C]-thymidine. The fraction of DNA eluting from filters is indicated. Untreated control (▵); 100 nmol/L (▪); 300 nmol/L (▴); 500 nmol/L (□); 1,000 nmol/L (•). (C) PC-3 cells were exposed to either untreated control (○); 1,000 nmol/L (•), or 3,000 nmol/L (▿) flavopiridol-containing medium for 24 or 48 hours. Each symbol represents the mean of three independent experiments.

  • Fig. 5.

    Effect of flavopiridol on p53, bcl2, and bax proteins. Exponentially growing SUDHL4 cells (A) and PC3 cells (B) were treated with 500 nmol/L of flavopiridol for 1, 3, 6, 18, and 24 hours. Cells were washed with PBS, lysed, and Western blot analysis performed as described in Materials and Methods. Proteins were visualized by autoradiography using ECL. The arrows indicate the position of the bax protein.

  • Fig. 6.

    Cell cycle distribution and effect on CDK1 activity after exposure to flavopiridol. In (A), the fraction of PC3 cells in G1, S, and G2/M is indicated after 12 hours exposure to 300 nmol/L flavopiridol. The experiment shown is the mean of duplicate samples with a range of < 5% and is representative of two experiments. In (B), SUDHL4 cells were exposed to 500 nmol/L flavopiridol (•) or vehicle (▪) for the indicated time periods and CDK1 activity assayed. The experiment shown is representative of two experiments, with each kinase determination the average ± SD of three determinations. In (C), SUDHL4 cells (A) were exposed to vehicle (solid line) or 300 nmol/L flavopiridol (dashed line) for 8 hours and cell cycle distribution assayed by flow cytometry.