Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Phos-tag analyses reveal that DNA Top2 Ser 1363 and Ser 1364 in the C-terminal charged region are phosphorylated. A , schematic of the fission yeast Top2 polypeptide. Top2 consists of a catalytic core and acidic/basic clusters at both ends of the protein. The catalytic core contains two evolutionarily conserved domains, an ATP-binding hydrolytic domain ( green ), and a DNA-binding cleavage domain ( red ). B , Phos-tag analysis of FLAG-tagged Top2 protein. Extracts of a cs nda3-KM311 β-tubulin mutant strain expressing Top2–3FLAG were prepared from asynchronously cultured ( AS ) and mitotically arrested cells ( M ) and run on SDS-PAGE gels in the presence or absence of 25 μ m Phos-tag. The untagged strain was used as a negative control. Each sample was preincubated with phage λ PPase (+) or buffer (−). Ponceau staining served as a loading control. Anti-FLAG antibodies detected phosphorylated Top2 proteins ( Top2-P ) in asynchronous and mitotically arrested cells. The position of the marker band is not indicated in the phos-tag blot because the marker proteins were also highly phosphorylated, and precise positions were unclear. C , Phos-tag analysis of truncated Top2 proteins. Top panel , N- or C-terminally truncated Top2-FLAG proteins were mildly overproduced under the inducible nmt promoter (plasmid Rep81) in the absence of thiamine in WT S. pombe cells. A strain containing only the vector was used as a control. Positions of unphosphorylated bands are indicated by red arrowheads. Bottom panel , truncated fragments and their phosphorylation ( Phospho ) status are indicated (+, phosphorylated; −, unphosphorylated). A red dashed box shows the predicted region of phosphorylation. aa , amino acids. D , C-terminally truncated Top2 proteins were expressed under the native promoter with the chromosomally integrated FLAG-tagged gene. Top2 C terminus phosphorylation is predicted between residues 1352 and 1391 ( double-headed arrow in the bottom panel ). E , the amino acid sequence around Ser 1363 and Ser 1364 of S. pombe Top2, with seven-amino-acid sequences. The consensus target sequence for CKII is shown. Xl , Xenopus laevis ; Hs , Homo sapiens ; Dm , Drosophila melanogaster ; Ce , Caenorhabditis elegans ; At , Arabidopsis thaliana ; Sp , Schizosaccharomyces pombe ; Sc , Saccharomyces cerevisiae. F , the Phos-tag–mediated mobility shift of the Top2 protein disappeared as a result of alanine substitutions for Ser 1363 and Ser 1364 . The intensity of the smeary phosphorylated Top2 bands was quantified and is indicated relative to the background.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Binding Assay, Mutagenesis, Expressing, Cell Culture, SDS Page, Negative Control, Staining, Marker, Plasmid Preparation, Sequencing, Mobility Shift

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Top2 Ser 1363 and Ser 1364 are phosphorylated by CKII throughout the cell cycle. A and B , the specificity of polyclonal antibodies against phospho-Ser 1363 –containing ( A ) and phospho-Ser 1364 –containing ( B ) phosphopeptides was examined. Asynchronous extracts of the strain expressing FLAG-tagged WT Top2 protein or nonphosphorylatable alanine mutants of Top2 (S1363A or S1364A) proteins were prepared and run on SDS-PAGE. Ponceau staining was used for the loading control of extracts. C , Ser 1363 and Ser 1364 were phosphorylated by CKII in vitro . Immunoprecipitated Top2-FLAG proteins were dephosphorylated by λ protein phosphatase and then incubated with CKII. Anti-Top2 phospho-Ser 1363 and -phospho-Ser 1364 antibodies were used to detect rephosphorylation by CKII. D , phosphorylation of Ser 1363 and Ser 1364 was diminished in two distinct alleles of CKII ts mutants, cka1-372 and orb5/cka1-19 , at the restrictive temperature (36 °C for 6 h). Cell extracts were prepared in WT, cka1-372 , orb5/cka1-19 and tor2-S mutants expressing Top2-FLAG protein at 26 °C and 36 °C (6 h) along with the untagged strain. The tor2-S mutant was used as a control strain , which shows small cells as observed in cka1-372 and cka1/orb5-19 mutant cells at the restrictive temperature ( Fig. S3 A ). Top2 proteins were detected with anti-FLAG antibodies in the presence ( Phos-tag ) or absence ( normal ) of 25 μ m Phos-tag. Top2 Ser 1363 and Ser 1364 phosphorylation was detected using anti-phospho-Ser 1363 and anti-phospho-Ser 1364 antibodies, respectively. Anti-PSTAIR (Cdc2) antibody was used for a loading control of extracts. The asterisk indicates nonspecific bands that probably appear under delay or arrest of cell-cycle progression, such as under nitrogen starvation ( F ), UV irradiation ( Fig. S5 ), and low-glucose conditions ( Figs. S4 A and S5 ). Because FLAG tagging partly reduces the Top2 protein level ( Fig. S4 B ), both phospho-specific antibodies give a weaker signal in FLAG-tagged strains relative to untagged strains. E , Ser 1363 and Ser 1364 were phosphorylated throughout the cell cycle. Block and release of cdc25-22 mutant cells expressing Top2–3FLAG was done for synchronous culture commencing from late G 2 phase to mitosis. Immunoblotting was performed with antibodies against FLAG, Top2 phospho-Ser 1363 and phospho-Ser 1364 . Cut2 (securin) and Cdc13 (mitotic cyclin) are shown as mitotic progression markers. Cell cycle progression was monitored by counting the number of binucleate cells lacking ( blue , anaphase–telophase) and possessing septa ( red , G 1 /S phase). F , top panel , Top2 phosphorylation was examined in nitrogen-starved, WT, G 0 -arrested cells, which were then permitted to proliferate by addition of a nitrogen source . Immunoblotting was performed with antibodies against FLAG, Top2 phospho-Ser 1363 , and tubulin (a loading control) as shown in D. Bottom panel , FACScan analysis indicating the timing of S phase (5–6 h). Top2 phosphorylation did not change during nitrogen starvation or after release.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Expressing, SDS Page, Staining, In Vitro, Immunoprecipitation, Incubation, Mutagenesis, Irradiation, Blocking Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Top2–2A (S1363A,S1364A) mutant protein maintains decatenation activity but has reduced ATPase activity. A , SDS-PAGE patterns of WT Top2 and alanine substitution mutant Top2-2A (S1363A and S1364A). FLAG-tagged Top2-WT and -2A proteins were overproduced under the inducible nmt promoter (plasmid Rep41) in WT S. pombe cells and immunoprecipitated using anti-FLAG antibody. A strain containing only the vector was used as a control. Immunoprecipitated FLAG-tagged Top2 proteins are indicated by arrows . The position of the protein marker bands ( M ) is indicated. B , Top2 decatenation assay. kDNA (153 ng) was incubated with immunoprecipitated Top2 fractions in ATP-containing reaction buffer (see “Experimental procedures”) for 1–30 min at 37 °C. An immunoprecipitated fraction from cell extracts containing only the empty vector was used for a mock reaction ( vector IP ). The reaction was terminated using stop buffer and loaded onto a 1% agarose gel followed by ethidium bromide staining. Only the catenated kDNA ( kDNA ) and decatenated kDNA ( decat. kDNA ) were loaded as controls, along with λDNA digested by EcoT14I (λ -EcoT14I ). Positions of catenated and decatenated kDNA are indicated by an arrow and a vertical line , respectively. The ratio of decatenated DNA to total DNA (catenated + decatenated) was quantified. Phosphorylation of Ser 1363 and Ser 1364 does not affect Top2 decatenation activity. C , Top2 ATPase assay. Immunoprecipitated Top2-WT or -2A mutant proteins were incubated with ATP and kDNA in the presence or absence of the 5 μ m anti-cancer topo II inhibitor ICRF-193 for 30 min at 30 °C. Free phosphate produced by ATP hydrolysis was measured by malachite green colorimetric reagent (see “Experimental procedures”). Error bars represent the standard deviation for each experiment performed in triplicate. p values for comparison among four conditions were calculated using one-way analysis of variance with Holm multi-comparison correction. *, p < 0.05.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Mutagenesis, Activity Assay, SDS Page, Plasmid Preparation, Immunoprecipitation, Marker, Incubation, Agarose Gel Electrophoresis, Staining, ATPase Assay, Produced, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Defective chromosome segregation induced by an anti-cancer catalytic topo II inhibitor, ICRF-193, is exacerbated in cka1-372 and top2-2A (S1363A,S1364A) mutant cells. A , CKII ts mutant cka1-372 cells showed more severe defects in mitotic chromosome segregation than WT cells in the presence of ICRF-193. Left panel , DAPI-stained micrographs of WT and cka1-372 mutant cells were obtained at the restrictive temperature (36 °C) for 3 h in the presence of DMSO or ICRF-193 (5 and 10 μ m ). A displaced nuclear phenotype was frequently observed in ICRF-193–treated cka1 mutant cells ( arrows ). Right panel , frequencies of defective phenotypes categorized as lagging-like ( blue ), streaked chromosomes ( red ), and displaced nucleus ( green ). More than 200 anaphase cells were counted for each sample. Error bars represent the standard deviation for each defective phenotypes. p values for comparison between the drug-treated WT and cka1 mutant were calculated using a Student's t test. *, p < 0.05; **, p < 0.01 ( black , total frequency of abnormal phenotypes; color , each phenotype). B , defects in chromosome segregation increased significantly in unphosphorylatable top2-2A mutants compared with WT and phosphomimetic top2–2E mutant cells in the presence of ICRF-193. Cells were asynchronously cultured at 26 °C for 2 h in the presence of DMSO or 5 μ m ICRF-193. Left panel , representative micrographs of DMSO- and 5 μ m ICRF-193–treated top2-2A cells. Chromatin DNA was stained with DAPI. Abnormally streaked chromosomes in anaphase are indicated ( arrows ). Scale bar = 10 μm. Right panel , frequencies of anaphase cells with abnormally streaked chromosomes. More than 200 anaphase cells were counted for each sample. Error bars represent the standard deviation for each experiment performed in biological triplicates. Significant differences among the three strains were examined using one-way analysis of variance with Bonferroni multi-comparison correction. *, p < 0.05; **, p < 0.01; n.s. , not significant.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Mutagenesis, Staining, Standard Deviation, Cell Culture

Journal: Frontiers in Oncology

Article Title: Discovery of New Catalytic Topoisomerase II Inhibitors for Anticancer Therapeutics

doi: 10.3389/fonc.2020.633142

Figure Lengend Snippet: The spatial relationship of the docking pocket with TOP2 and DNA. Both top and side views show the relative position of TOP2A dimer (cyan), DNA substrate (yellow), and the molecular surface of the docking pocket (red) used for in silico drug screening.

Article Snippet: Human TOP2 (431–1193), TOP2A (TopoGEN) or TOP2B (Inspiralis) proteins were incubated with 300 nM of 5ZRF oligo in a standard 20 μl EMSA reaction in a reaction buffer containing 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.05% NP-40, protease inhibitors, and 0.1 μg poly[dI:dC].

Techniques: In Silico, Drug discovery

Journal: Frontiers in Oncology

Article Title: Discovery of New Catalytic Topoisomerase II Inhibitors for Anticancer Therapeutics

doi: 10.3389/fonc.2020.633142

Figure Lengend Snippet: In silico screening for TOP2 inhibitors. (A) Molecular dynamics simulation of the docking pocket (blue) and T60 inside the TOP2A docking pocket (red) in 30ns. (B) A computer-aided drug design (CADD)_pipeline was applied to screen for TOP2 binders. Two rounds of virtual screening protocol combined with K-DNA decatenation and relaxation bioassays led to the discovery of the lead compound, T60. (C) A bar chart of protein–ligand contacts during the 30 ns simulation period. The interchanging bond formation (circled) between Gln773 and Gly777 is corresponding to T60 RMSD (circled) from (A) in the first 8 ns. (D) A schematic diagram shows T60 atoms interacting with surrounding protein residues of TOP2A. Bond energies were shown in kcal/mol.

Article Snippet: Human TOP2 (431–1193), TOP2A (TopoGEN) or TOP2B (Inspiralis) proteins were incubated with 300 nM of 5ZRF oligo in a standard 20 μl EMSA reaction in a reaction buffer containing 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.05% NP-40, protease inhibitors, and 0.1 μg poly[dI:dC].

Techniques: In Silico

Journal: Frontiers in Oncology

Article Title: Discovery of New Catalytic Topoisomerase II Inhibitors for Anticancer Therapeutics

doi: 10.3389/fonc.2020.633142

Figure Lengend Snippet: T60 is an inhibitor of TOP2A and TOP2B. K-DNA decatenation assays measured TOP2A (A) and TOP2B (C) activities that were inhibited by T60. The decatenation assays were repeated three times per concentration, and the densitometry of the D-DNA bands was used to establish an inhibition curve for IC 50 calculation. Etoposide and ICRF193 were used as positive controls. Plasmid DNA relaxation assay measured TOP2A (B) and TOP2B (D) activities that were inhibited by T60. The relaxation assays were repeated three times per concentration and the densitometry of the SC bands was used to establish an inhibition curve for IC 50 calculation. K, catenated kinetoplast DNA; D, decatenated K-DNA; SC, supercoiled forms of the plasmid; and R, relaxed forms of the plasmid.

Article Snippet: Human TOP2 (431–1193), TOP2A (TopoGEN) or TOP2B (Inspiralis) proteins were incubated with 300 nM of 5ZRF oligo in a standard 20 μl EMSA reaction in a reaction buffer containing 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.05% NP-40, protease inhibitors, and 0.1 μg poly[dI:dC].

Techniques: Concentration Assay, Inhibition, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Discovery of New Catalytic Topoisomerase II Inhibitors for Anticancer Therapeutics

doi: 10.3389/fonc.2020.633142

Figure Lengend Snippet: T60 is a catalytic TOP2 inhibitor (A) In ethidium bromide displacement assays, 100 µg/ml salmon sperm DNA was mixed with or without 2 µg/ml ethidium bromide together with the indicated chemicals. Reactions were subjected to fluorescence emission of 590 nm and excitation of 535 nm. Obtained signals were detected by using the Multimode Microplate Reader (Infinite F500). (B) DNA cleavage assays were performed using human TOP2A or TOP2B incubated with the supercoiled pHOT plasmid in the presence of the vehicle, 200 µM of etoposide, ICRF193, or T60 in a reaction buffer without ATP. (C) DNA cleavage assays were performed using full-length human TOP2A and TOP2B incubated with the supercoiled pHOT plasmid in the presence of 200 µM of etoposide plus 1, 100, and 200 µM of ICRF193 or T60. Reactions from (B, C) were separated on a DNA gel with ethidium bromide. (D, E) EMSA was performed by incubating 30 µM of the purified human TOP2A(431-1193) with 300nM of IRDay700 labeled 5ZRF DNA oligo. Vehicle or 200 µM of etoposide, ICRF193, or T60 was added as shown in (D) , or 0 – 200 µM of T60 was added in the reactions as shown in (E) . (F) EMSA was performed by incubating 2 µM of full-length TOP2A or TOP2B with 300 nM of IRDay700 labeled 5ZRF DNA oligo in the presence of vehicle or 200 µM of ICRF193 or T60. All experiments were repeated three times, and one representative image was shown. N, nicked DNA; L, linear DNA; and SC, supercoiled DNA.

Article Snippet: Human TOP2 (431–1193), TOP2A (TopoGEN) or TOP2B (Inspiralis) proteins were incubated with 300 nM of 5ZRF oligo in a standard 20 μl EMSA reaction in a reaction buffer containing 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.05% NP-40, protease inhibitors, and 0.1 μg poly[dI:dC].

Techniques: Fluorescence, Incubation, Plasmid Preparation, Purification, Labeling

Journal: Frontiers in Oncology

Article Title: Discovery of New Catalytic Topoisomerase II Inhibitors for Anticancer Therapeutics

doi: 10.3389/fonc.2020.633142

Figure Lengend Snippet: Structure–activity relationship (SAR) between T60 and TOP2. (A) T60 derivatives were custom synthesized (>95% purity) and tested for their effects on TOP2 by both relaxation and K-DNA decatenation assays. (B, C) Representative results of relaxation (B) and K-DNA decatenation (C) assays were presented. The densitometry of SC and D-DNA bands were used to establish the efficacy of the suppressive effects of T60 and its derivatives to TOP2A. Experiments were repeated three times, and results were presented as mean ± SD.

Article Snippet: Human TOP2 (431–1193), TOP2A (TopoGEN) or TOP2B (Inspiralis) proteins were incubated with 300 nM of 5ZRF oligo in a standard 20 μl EMSA reaction in a reaction buffer containing 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.05% NP-40, protease inhibitors, and 0.1 μg poly[dI:dC].

Techniques: Activity Assay, Synthesized

Journal: Frontiers in Oncology

Article Title: Discovery of New Catalytic Topoisomerase II Inhibitors for Anticancer Therapeutics

doi: 10.3389/fonc.2020.633142

Figure Lengend Snippet: T60 targets TOP2 in cells and suppress cell proliferation. (A) K562 cells were treated with 20 µM of etoposide, ICRF193, or T60 for 0, 24, 48, and 72 h. (B) K562 cells were treated with 0, 5, 10, 20 µM of etoposide, ICRF193, or T60 for 48 h. Cell proliferation rates were measured by MTS assays. (C) HeLa cells were treated with the vehicle or 20 µM of T60, etoposide, or ICRF193 for 48 h. BrdU incorporation rates were measured. (D) HeLa cells were treated with 0 – 20 µM of etoposide, ICRF193, or T60 for 48 h. Cytotoxicity was evaluated by measuring the LDH levels from the culture media. (E) K562 cells were treated with the vehicle or 10 – 20 µM of etoposide, ICRF193, or T60 for 4 h. Immunoblotting measured p-H2AX γ levels in cells with vinculin as a loading control. (F) K562 cells were treated with the vehicle or 10 – 20 µM of etoposide and co-treated with vehicle, 20 µM ICRF193, or 20 µM T60 for 4 h. Immunoblotting measured p-H2AX γ levels in cells with beta-actin as the loading control. (G) HeLa cells were treated with either vehicle or 20 µM T60 for 48 h, before co-stained with DAPI (blue) and phalloidin (red) and examined by confocal microscopy. Fifty cells from five random high power fields were used to analyze nuclei sizes by the Image J software. All results were repeated in three independent experiments. (H) K562 cells were treated with the vehicle or 20 µM T60 for 2 h and used to perform cellular thermal shift assays between 37 and 46°. Protein levels of TOP2A and TOP2B were measured by immunoblotting with beta-actin as the loading control. All experiments were repeated three times, and results were presented as mean ± SD. One-way ANOVA followed by t -test was used to determine differences between groups with the level of significance set at *p < 0.05 and **p < 0.01. One-way ANOVA followed by t-test was used to determine differences between groups with the level of significance set at p < 0.001 as ***.

Article Snippet: Human TOP2 (431–1193), TOP2A (TopoGEN) or TOP2B (Inspiralis) proteins were incubated with 300 nM of 5ZRF oligo in a standard 20 μl EMSA reaction in a reaction buffer containing 20 mM HEPES-NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.05% NP-40, protease inhibitors, and 0.1 μg poly[dI:dC].

Techniques: BrdU Incorporation Assay, Western Blot, Control, Staining, Confocal Microscopy, Software