Fig. 1.
Fig. 1. Comparison of topo II–mediated events in HL-60 and K562 cells. (A) Examination of topo II polypeptide content in HL-60 and K562 cells. Whole cell extracts containing polypeptides from 3 × 105 (lanes 1 and 5), 1.5 × 105 (lanes 2 and 6), 0.75 × 105 (lanes 3 and 7) and 0.3 × 105 (lanes 4 and 8) K562 cells (lanes 1 through 4) or HL-60 cells (lanes 5 through 8) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies that recognize both topo II isoforms (upper panel) or, as a loading control, histone H1 (lower panel). Scanning of the blots (not shown) showed that topo IIβ levels in the two cell lines were equal after correction for differences in loading, whereas levels of topo IIα were twofold higher in K562 cells. (B) Formation of topo II–mediated strand breaks in the presence of etoposide. HL-60 cells (•) and K562 cells (○) were incubated with the indicated concentration of etoposide at 37°C for 30 minutes before application to Nucleopore filters. After elution for 7 hours, the fraction of DNA retained on the filters was compared to the fraction retained after irradiation of HL-60 or K562 cells. (C) Kinetics of resealing of etoposide-induced DNA single-strand breaks. HL-60 cells treated with 10.5 μmol/L etoposide (•) or K562 cells treated with 6.8 μmol/L etoposide (○) for 30 minutes at 37°C were diluted 30-fold and incubated at 37°C for the indicated length of time before deposition on Nucleopore filters. The fraction of DNA retained on the filters after elution under alkaline conditions were compared with the fraction retained after irradiation of HL-60 or K562 cells. (D) Effect of a 1-hour etoposide exposure on clonogenic survival of HL-60 (•) and K562 cells (○). Cells were simultaneously treated for 1 hour with the indicated concentration of etoposide, washed, and plated in 0.3% agar as described in Materials and Methods. Colony formation was assessed 10 to 14 days later. Bars, ± standard deviation of quadruplicate samples. Results in (A), (B), and (D) are representative of four separate experiments. Results in (C) are means of two separate experiments.

Comparison of topo II–mediated events in HL-60 and K562 cells. (A) Examination of topo II polypeptide content in HL-60 and K562 cells. Whole cell extracts containing polypeptides from 3 × 105 (lanes 1 and 5), 1.5 × 105 (lanes 2 and 6), 0.75 × 105 (lanes 3 and 7) and 0.3 × 105 (lanes 4 and 8) K562 cells (lanes 1 through 4) or HL-60 cells (lanes 5 through 8) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies that recognize both topo II isoforms (upper panel) or, as a loading control, histone H1 (lower panel). Scanning of the blots (not shown) showed that topo IIβ levels in the two cell lines were equal after correction for differences in loading, whereas levels of topo IIα were twofold higher in K562 cells. (B) Formation of topo II–mediated strand breaks in the presence of etoposide. HL-60 cells (•) and K562 cells (○) were incubated with the indicated concentration of etoposide at 37°C for 30 minutes before application to Nucleopore filters. After elution for 7 hours, the fraction of DNA retained on the filters was compared to the fraction retained after irradiation of HL-60 or K562 cells. (C) Kinetics of resealing of etoposide-induced DNA single-strand breaks. HL-60 cells treated with 10.5 μmol/L etoposide (•) or K562 cells treated with 6.8 μmol/L etoposide (○) for 30 minutes at 37°C were diluted 30-fold and incubated at 37°C for the indicated length of time before deposition on Nucleopore filters. The fraction of DNA retained on the filters after elution under alkaline conditions were compared with the fraction retained after irradiation of HL-60 or K562 cells. (D) Effect of a 1-hour etoposide exposure on clonogenic survival of HL-60 (•) and K562 cells (○). Cells were simultaneously treated for 1 hour with the indicated concentration of etoposide, washed, and plated in 0.3% agar as described in Materials and Methods. Colony formation was assessed 10 to 14 days later. Bars, ± standard deviation of quadruplicate samples. Results in (A), (B), and (D) are representative of four separate experiments. Results in (C) are means of two separate experiments.

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