CCDC6 inhibits the phosphatase activity of PP4c.

<p>(<b>a</b>) PP4 complex immunopurified from HeLa cells transfected with CCDC6-specific shRNAs (shCCDC6) or with non-targeting control shRNAs (shCTRL), was incubated for 30 minutes at 30°C with pH2AX S139-enriched chromatin purified from irradiated cells. The phosphatase reactions were followed by western blot and probed with the indicated antibodies. (<b>b</b>) PP4c phosphatase was immunoprecipitated from shCCDC6 or shCTRL. 1, 0,7, 0,3 voulmes of total PP4c immunoprecipitated from 3 mg of total cell extract were mixed with 3 µg histones purified from cells exposed to 10 Gy IR and incubated in phosphatase buffer at 30°C for 30 minutes. Phosphatase reaction was terminated by the addition of 100 µl of Malachite Green solution and absorbance was measured at 630 nm. After the phosphatase assay, the actual amount of PP4c in each immunoprecipitate was determined by Western Blotting with the indicated antibody. PP4c activity is represented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 630 nm) and PP4c densitometric signal at western blot. (<b>c</b>) Enzimatic activity of PP4c immunopurified from HeLa cells transfected with CCDC6-specific shRNA (shCCDC6) or with non-targeting control sh-RNAs (shCTRL) was assessed by Malachite Green phosphatase assay. 1, 0,7 and 0,3 volumes of total PP4c immunoprecipitated from 3 mg of total cell extract were incubated with 175 µM of RKpTIRR synthetic peptide for 30 minutes at 30°C. Phosphatase reaction was terminated by the addition of 100 µl of Malachite Green solution and absorbance was measured at 630 nm. After the phosphatase assay, the actual amount of PP4c in each immunoprecipitate was determined by Western Blotting with the indicated antibody. PP4c activity is represented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 630 nm) and PP4c densitometric signal at western blot. (<b>d</b>) PP4 complex immunopurified from TPC-1 cells transfected with CCDC6 wt, CCDC6 (1–223) and (1–101) truncated mutants, was incubated for 30 minutes at 30°C with pH2AX S139-enriched chromatin, purified from irradiated cells. The phosphatase reactions were followed by immunoblotting and probed with the indicated antibodies. (<b>e</b>) (<b>f</b>) Enzimatic activity of PP4c immunopurified from TPC-1 cells transfected with epitope-tagged CCDC6 wt or empty vector, was determined as described in (<b>b</b>) and (<b>c</b>).</p

Loss of CCDC6 increases cell growth and confers resistance to genotoxic stress.

<p>(<b>a</b>) HeLa CCDC6 depleted clones were obtained after transfection of a plasmid pool of mission ShRNA (pLKo.1 puro ShCCDC6 NM_005436, Sigma-Aldrich) after two weeks puromycin selection. Immunoblot with anti CCDC6 and a-tubulin were shown. (<b>b</b>) CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL) were plated at 10×10⁵/dish in triplicate and counted at the indicated times (<b>c</b>) Cell cycle distribution of a stable HeLa CCDC6 silenced clone (shCCDC6 #1) and control HeLa cells (shCTRL) after release from double thymidine block (TT-block) (<b>d</b>) CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL) were plated at 10×10⁵, treated with the indicated doses of Etoposide and collected at 48 hours. The histograms are representative of three independent experiments and error bars indicate the standard error mean.</p

CCDC6 in the genome stability control.

<p>(<b>a</b>) shCCDC6 and shCTRL HeLa cells were depleted of PP4c by shRNA (48 hours) and were exposed to 1 Gy of IR, as indicated (−/+). Phosphorylation of H2AX, PP4c, CCDC6, total H2AX and tubulin amount were revealed at IB of WCL. (<b>b</b>) In the cell extract of CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL), after double thymidine block (TT block) and release in presence of 1 µM Etoposide at several time point, as indicated, phosphorylation levels of H2AX and of RPA2 were revealed with anti-pH2AX S139 and with anti-p-RPA2 by western blot. Anti-total H2AX and anti-total RPA were shown as loading control. (<b>c</b>) Schematic diagram of CCDC6 function in modulating PP4c activity on the phosphorylation status of H2AX in the DNA-damage response.</p

Loss of CCDC6 affects the DNA damage induced G2 arrest.

<p>(<b>a</b>) Mitotic entry of the stable HeLa CCDC6 silenced clone #1 (shCCDC6) and control HeLa cells (shCTRL) after TT-block and release in 1 µM Etoposide for one hour where indicated, in presence of 50 ng/ml Nocodazole was monitored by western blot using the anti-p-S/T-MPM2 antibody. Sketch of the cells treatment is shown in the bottom panel. <b>(b)</b> Percentage of mitotic cells was monitored, by FACS analysis, with anti-p-Ser10-histone H3 staining, in stable CCDC6 silenced and control HeLa cells treated as in (a) at 8 hours, as indicated. <b>(c)</b> in HeLa CCDC6 silenced clone #1 (shCCDC6) and control HeLa cells (shCTRL) growth on coverslips and collected at several time points following G1/S syncronization by double thymidine block (TT-block) in the presence of 1 µM Etoposide, as indicated, mitotic figures were counted after nuclear counterstaining with Dapi. Magnification_ was at 40x. The histograms are representative of three independent experiments and error bars indicate the standard error mean. (<b>d</b>) After TT-block and release in 1 µM Etoposide stable HeLa CCDC6 silenced clone #1 (shCCDC6) and control HeLa cells (shCTRL) were collected at several time points as indicated. Checkpoint activity was monitored by western blot using the anti-pSer317-chk1 antibody. Total chk1 is shown at bottom of the figure.</p

Loss of CCDC6 affects H2AX phosphorylation after DSBs.

<p>(<b>a</b>) In the WCL of two representative CCDC6-depleted HeLa clones (shCCDC6 #1 and #2) and control HeLa cells (shCTRL), thirty minutes after 1–5 Gy IR exposure, the phosphorylation of H2AX was detected with the mouse anti-pH2AX S139 by western blot. Anti-total H2AX was used as a loading control. The immunoblots with anti-CCDC6 and α-tubulin antibodies were shown in the bottom. (<b>b</b>) H2AX phosphorylation detection with mouse anti-pH2AX S139 by WCL analysis of CCDC6-depleted Hela clone #1 (shCCDC6) and control cells (shCTRL) at several time points as indicated after exposure to 1Gy of IR. Anti total H2AX is shown as loading control. (<b>c</b>) Immunofluorescence analysis of pH2AX S139 foci in CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL), thirty minutes after 1, and 5 Gy IR exposure. Nuclei were counterstained with DAPI. Magnification_ was at 63x. (<b>d</b>) Quantification of pH2AX S139 foci number. At least 300 cells were analysed per experiment. Error bars indicate the standard mean error. (<b>e)</b> CCDC6-depleted clone #1 (shCCDC6) and control HeLa cells (shCTRL) transfected with expression vectors encoding CCDC6wt, CCDC6T434A or the empty vector were treated with etoposide at 1, 2,5 and 5 µM for 8 h and western blot analysis of pH2AX S139 and myc-tagged proteins were performed. (<b>f</b>) H2AX phosphorylation detection with mouse anti-pH2AX S139 by WCL analysis of CCDC6-depleted Hela clone #1 (shCCDC6) and control cells (shCTRL) at several time points as indicated after exposure to 1Gy of IR. Anti total H2AX is shown as loading control. The anti-pSer-1981-ATM and the ATM hybridization are shown at bottom of the figure.</p

Loss of CCDC6 affects DSBs repair.

<p>(<b>a</b>) Detection of DSBs by PFGE. After 10 Gy IR exposure CCDC6-depleted (shCCDC6) and CCDC6-proficient (shCTRL) HeLa cells have been collected at different time points (1, 2, 4, 24 hours). Densitometric analysis of DSBs bands were plotted as percentage of total DNA. <b>(b</b>) The percentages of GFP positive cells, compared to controls, have been plotted on the histograms that are representative of three independent experiments. Error bars indicate the standard error mean. The anti-HA-I-Sce1 and anti-tubulin immunoblots are shown at bottom of the figure. <b>(c</b>) HeLa cells, bearing the doxycycline-inducible I-Sce1 DNA repair construct, have been transfected with control shRNAs (shCTRL) or sh-CCDC6 by Microporator MP-100 transfection system (Digital Bio, Korea). The CCDC6 protein depletion was assessed by western blot analysis for every rate of transfection (#1, #2, #3, #4). The percentages of protein expression, compared to controls, have been plotted on the histogram below.</p

Gold(III) Macrocycles: Nucleotide-Specific Unconventional Catalytic Inhibitors of Human Topoisomerase I

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au³⁺ macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, <b>3</b>, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by <b>3</b> was 2 orders of magnitude weaker than its inhibition of Top1, confirming that <b>3</b> is a type I-specific catalytic inhibitor. Importantly, Au³⁺ is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that <b>3</b> intercalates directly at the 5′-TA-3′ dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π–π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations

Gold(III) Macrocycles: Nucleotide-Specific Unconventional Catalytic Inhibitors of Human Topoisomerase I

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au³⁺ macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, <b>3</b>, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by <b>3</b> was 2 orders of magnitude weaker than its inhibition of Top1, confirming that <b>3</b> is a type I-specific catalytic inhibitor. Importantly, Au³⁺ is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that <b>3</b> intercalates directly at the 5′-TA-3′ dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π–π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations

Gold(III) Macrocycles: Nucleotide-Specific Unconventional Catalytic Inhibitors of Human Topoisomerase I

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au³⁺ macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, <b>3</b>, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by <b>3</b> was 2 orders of magnitude weaker than its inhibition of Top1, confirming that <b>3</b> is a type I-specific catalytic inhibitor. Importantly, Au³⁺ is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that <b>3</b> intercalates directly at the 5′-TA-3′ dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π–π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations

Gold(III) Macrocycles: Nucleotide-Specific Unconventional Catalytic Inhibitors of Human Topoisomerase I

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au³⁺ macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, <b>3</b>, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by <b>3</b> was 2 orders of magnitude weaker than its inhibition of Top1, confirming that <b>3</b> is a type I-specific catalytic inhibitor. Importantly, Au³⁺ is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that <b>3</b> intercalates directly at the 5′-TA-3′ dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π–π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations