Cell cycle-coupled relocation of types I and II topoisomerases and modulation of catalytic enzyme activities

Abstract

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10-15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (alpha) or reticular (beta) nuclear patterns throughout interphase. In contrast to topoisomerase IIalpha, topoisomerase IIbeta was completely excluded from nucleoli. In mitosis, topoisomerase IIbeta diffused completely into the cytosol, whereas topoisomerases I and IIalpha remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIalpha accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2-3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIalpha escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIalpha, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIbeta remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIbeta is released from the heterochromatin, whereas topoisomerase I and IIalpha remain chromosome bound. Scaffold-associated topoisomerase IIalpha appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.

Figure 1

Reactivity of topoisomerase antibodies in Western blotting. 50 ng of purified recombinant human topoisomerase I (lanes 2–4), IIα (lanes 9 and 10), or IIβ (lanes 14 and 15) or 5 × 10⁵ A431 cells lysed in hot SDS (lanes 5–7, 11, 12, 16, and 17) were separated by SDS-PAGE and blotted onto Immobilon-P membranes. Lanes 1, 8, and 13 show Coomassie blue staining of 300 ng of purified topoisomerases I, IIα, and IIβ, respectively. Blot membranes were probed with antibodies, as follows. Lanes 2 and 5: Scl-70, 1:2,000; lanes 3 and 6: rabbit anti–peptide directed against human topoisomerase I-COOH terminus, 1:5,000; lanes 4 and 7: rabbit anti–peptide directed against human topoisomerase I-NH₂ terminus, 1:1,000; lanes 9 and 11: Mouse monoclonal antibody Ki-S1, 1:500; lanes 10 and 12: rabbit anti–peptide antibody directed against human topoisomerase IIα-COOH terminus, 1: 5,000; lanes 14 and 16: Mouse monoclonal antibody 3H10, 1:800; lanes 15 and 17: Rabbit anti–peptide antibody directed against human topoisomerase IIβ-COOH terminus, 1:5,000. Strips represent the whole running distance of the gel, excluding the stacking portion.

Figure 2

Specificity controls of indirect immunofluorescence microscopy of topoisomerases. A431 cells were grown on microscope slides, fixed, permeabilized, and double stained with topoisomerase antibodies and bisbenzimide (Hoechst Frankfurt, Germany). Images of immunofluorescence (top) and DNA (bottom) are paired. (a) Immunostaining of topoisomerase I with Scl-70 autoantibodies; (b) as a, but Scl-70 autoantibodies were preincubated with 1 μg of heat-inactivated (60°C, 5 min) human topoisomerase I for 1 h at 20°C; (c) Immunostaining of topoisomerase IIα with Ki-S1 mouse monoclonal antibody; (d) as c, but Ki-S1 antibody was preincubated with 1 mg of heat-inactivated (60°C, 5 min) human topoisomerase IIα for 1 h at 20°C; (e) as c, but Ki-S1 antibody was preincubated with 1 μg of heat-inactivated (60°C, 5 min) human topoisomerase IIβ for 1 h at 20°C; (f) immunostaining of topoisomerase IIβ with 3H10 mouse monoclonal antibody; (g) as f, but 3H10 antibody was preincubated with 1 μg of heat-inactivated (60°C, 5 min) human topoisomerase IIβ for 1 h at 20°C; (h) as f, but 3H10 antibody was preincubated with 1 mg of heat-inactivated (60°C, 5 min) human topoisomerase IIα for 1 h at 20°C.

Figure 3

Fluorescent images of topoisomerases in human A431 cells. Monolayers of A431 cells grown on micoscopic slides were fixed, permeabilized, and doublestained with bisbenzimide (Hoechst; right) and topoisomerase antibodies (left). (a) Immunostaining of topoisomerase I with Scl-70 autoantibodies. (b) Immunostaining of topoisomerase IIα with Ki-S1 mouse monoclonal antibody. (c) Immunostaining of topoisomerase IIβ with 3H10 mouse monoclonal antibody.

Figure 4

Localization of topoisomerases in interphase and mitosis. Close-up pictures of representative cells in interphase (a–c) or mitosis (d–f) immunostained for topoisomerase I (a and d), topoisomerase IIα (b and e), or topoisomerase IIβ (c and f). The left of each pair of images represents immunostaining. The right shows the corresponding DNA pattern (Hoechst).

Figure 5

Colocalization of topoisomerases with DNA in interphase cells. Pseudo-color coded fluorescent images of topoisomerases (red) were stacked with corresponding patterns of bisbenzimide-stained DNA (blue). (Middle) immunostaining of topoisomerase I with Scl70 (a), topoisomerase IIα with Ki-S1(b), and topoisomerase IIβ with 3H10 (c). (Left) Corresponding image of bisbenzimide-stained DNA (Hoechst). (Right) Stacked image of immunostaining (red) and DNA (blue). Arrows indicate the positions of nucleoli.

Figure 6

Localization of topoisomerases in isolated chromosomes. Isolated chromosomes were doublestained for DNA and topoisomerases I (a), IIα (b), or IIβ (c), as in Fig. 3. Paired images of immunofluorescence (left) and corresponding DNA pattern (right) are shown.

Figure 7

Drug-induced topoisomerase band-depletion in mitosis and interphase. Cells in logarithmic growth (Log) or blocked in metaphase by demecolcine (M) were treated with 30 μM camptothecin (line 1, +Drug) or 200 μM etoposide (lines 2 and 3, +Drug) for 1 h at 37°C. Controls were incubated without drug. Subsequently, the cells were harvested and lysed with hot SDS, and lysate equivalent of 5 × 10⁵ cells was loaded onto each lane. Western blots were probed with rabbit peptide antibodies directed against the COOH termini of human topoisomerase I (top), IIα (middle), or IIβ (bottom). This is a representative example of at least three identical experiments with similar outcome. A quantitative analysis of band intensities is given in Table I. The observed differences in band intensity between treated and untreated cells were significant on the 0.01 level (Wilcoxon's signed rank test).

Figure 8

Cell cycle–coupled expression of topoisomerases. Cells were synchronized by demecolcine treatment and harvested at the indicated cell cycle stages (G₁, S, G₂/M). Mitotic cells (M) were harvested directly after treatment with demecolcine for 16 h. Log-phase cells not treated with demecolcine (Log) served as controls. 5 × 10⁵ nuclei isolated from the cells were lysed with hot SDS and applied to each lane. Western blots were probed with rabbit anti–peptide antibodies directed against the COOH termini of human topoisomerases I (top), IIα (middle), or IIβ (bottom), respectively. This is a representative example of at least three identical experiments with similar outcomes. A quantitative analysis of band intensities is given in Table I.

Figure 9

Cell cycle–coupled changes in DNA extractability of topoisomerases. (a) Nuclei were isolated from cells in logarithmic growth (Log) or blocked in metaphase by demecolcine (M). Nuclei were either lysed in hot SDS (N) or were first extracted with 350 mM NaCl, and the nuclear remnant (R) was lysed in hot SDS after digestion with DNase I (50 U/10⁶ nuclei) for 20 min at 37°C. 7 × 10⁵ nuclei (N) or an equivalent amount of DNase-digested nuclear remnant (R) was applied to Western blotting. Blots were probed with rabbit peptide antibodies directed against the COOH terminus of human topoisomerase I (lanes 1–4), IIα (lanes 5–8), or IIβ (lanes 9–12). This is a representative example of three identical experiments with similar outcomes. (b) The procedure described in a was performed with synchronized cells harvested in G₁, S, or G₂ phase and with cells blocked in metaphase by demecolcine (M). The relative amounts of topoisomerases I, IIα, and IIβ that were nonextractable by 350 mM NaCl as compared to total nuclear content, were determined by comparative videodensitometry of the immunoblots. Mean values of three identical experiments are plotted. Bars represent standard errors of the mean. (c) Nuclei were isolated from cells blocked in metaphase by demecolcine and extracted with 350 mM NaCl. Nonextractable nuclear remnants were dissolved in an equal volume of 3.5-fold diluted extraction buffer and incubated with (lane 2) and without (lane 1) 200 μM etoposide in the presence of 1 mM ATP. Extracts were diluted 3.5-fold and incubated with (lane 4) and without (lane 3) 200 μm etoposide in the presence of 1 mM ATP and 4 μg calf thymus DNA. After 30 min the reaction was stopped by addition of hot SDS. Samples were sheared with a syringe and applied to Western blotting, and blots were probed with peptide antibodies directed against the COOH terminus of human topoisomerase IIα. A representative example of three identical experiments with similar outcomes is shown.

Figure 9

Cell cycle–coupled changes in DNA extractability of topoisomerases. (a) Nuclei were isolated from cells in logarithmic growth (Log) or blocked in metaphase by demecolcine (M). Nuclei were either lysed in hot SDS (N) or were first extracted with 350 mM NaCl, and the nuclear remnant (R) was lysed in hot SDS after digestion with DNase I (50 U/10⁶ nuclei) for 20 min at 37°C. 7 × 10⁵ nuclei (N) or an equivalent amount of DNase-digested nuclear remnant (R) was applied to Western blotting. Blots were probed with rabbit peptide antibodies directed against the COOH terminus of human topoisomerase I (lanes 1–4), IIα (lanes 5–8), or IIβ (lanes 9–12). This is a representative example of three identical experiments with similar outcomes. (b) The procedure described in a was performed with synchronized cells harvested in G₁, S, or G₂ phase and with cells blocked in metaphase by demecolcine (M). The relative amounts of topoisomerases I, IIα, and IIβ that were nonextractable by 350 mM NaCl as compared to total nuclear content, were determined by comparative videodensitometry of the immunoblots. Mean values of three identical experiments are plotted. Bars represent standard errors of the mean. (c) Nuclei were isolated from cells blocked in metaphase by demecolcine and extracted with 350 mM NaCl. Nonextractable nuclear remnants were dissolved in an equal volume of 3.5-fold diluted extraction buffer and incubated with (lane 2) and without (lane 1) 200 μM etoposide in the presence of 1 mM ATP. Extracts were diluted 3.5-fold and incubated with (lane 4) and without (lane 3) 200 μm etoposide in the presence of 1 mM ATP and 4 μg calf thymus DNA. After 30 min the reaction was stopped by addition of hot SDS. Samples were sheared with a syringe and applied to Western blotting, and blots were probed with peptide antibodies directed against the COOH terminus of human topoisomerase IIα. A representative example of three identical experiments with similar outcomes is shown.

Figure 9

Cell cycle–coupled changes in DNA extractability of topoisomerases. (a) Nuclei were isolated from cells in logarithmic growth (Log) or blocked in metaphase by demecolcine (M). Nuclei were either lysed in hot SDS (N) or were first extracted with 350 mM NaCl, and the nuclear remnant (R) was lysed in hot SDS after digestion with DNase I (50 U/10⁶ nuclei) for 20 min at 37°C. 7 × 10⁵ nuclei (N) or an equivalent amount of DNase-digested nuclear remnant (R) was applied to Western blotting. Blots were probed with rabbit peptide antibodies directed against the COOH terminus of human topoisomerase I (lanes 1–4), IIα (lanes 5–8), or IIβ (lanes 9–12). This is a representative example of three identical experiments with similar outcomes. (b) The procedure described in a was performed with synchronized cells harvested in G₁, S, or G₂ phase and with cells blocked in metaphase by demecolcine (M). The relative amounts of topoisomerases I, IIα, and IIβ that were nonextractable by 350 mM NaCl as compared to total nuclear content, were determined by comparative videodensitometry of the immunoblots. Mean values of three identical experiments are plotted. Bars represent standard errors of the mean. (c) Nuclei were isolated from cells blocked in metaphase by demecolcine and extracted with 350 mM NaCl. Nonextractable nuclear remnants were dissolved in an equal volume of 3.5-fold diluted extraction buffer and incubated with (lane 2) and without (lane 1) 200 μM etoposide in the presence of 1 mM ATP. Extracts were diluted 3.5-fold and incubated with (lane 4) and without (lane 3) 200 μm etoposide in the presence of 1 mM ATP and 4 μg calf thymus DNA. After 30 min the reaction was stopped by addition of hot SDS. Samples were sheared with a syringe and applied to Western blotting, and blots were probed with peptide antibodies directed against the COOH terminus of human topoisomerase IIα. A representative example of three identical experiments with similar outcomes is shown.

Figure 10

Treatment of nuclear extracts with alkaline phosphatase. Nuclei of mitotic (lanes 3 and 4) and interphase cells (lanes 1 and 2) were extracted with 800 mM NaCl. Extracts were precipitated with 3 M ammonium sulfate, renatured with diethanolamine buffer, pH 9.8, and incubated with 30 U of alkaline phosphatase at 37°C for 90 min (lanes 2 and 4). Controls (lanes 1 and 3) were incubated with an equivalent amount of phosphatase storage buffer without enzyme. Extracts were subsequently precipitated with 15% trichloroacetic acid. Precipitates were washed with acetone, dissolved in SDS sample buffer containing 5 M urea, separated on 5.5% polyacrylamide gels with a 3% stacking portion, transferred to Immobilon P membranes, and probed with peptide antibodies specific for the COOH-terminal portions of topoisomerase I (row 1), IIα (row 2), or IIβ (row 3). Molecular weight values of enzyme specific protein bands indicated on the right margin were derived from Rf-plots of migration distances of marker proteins (see Materials and Methods) run in the same gel. The coefficient of variance of these molecular weight values was <20% in a run of three similar experiments, of which this one is a representative example. The observed differences in apparent molecular weight were significant on the 0.05 level (Wilcoxon's signed rank test).