BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

• MeSH
• DNA vazebné proteiny * genetika   metabolismus   MeSH
• DNA-topoisomerasy I genetika   metabolismus   MeSH
• endonukleasy * genetika   metabolismus   MeSH
• fosfodiesterasy * genetika   metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• Saccharomyces cerevisiae - proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a-2c) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (hTOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (1c, 1d = tacrine-(CH2)8-9-coumarin). The most active of the tested compounds, derivatives 1c and 1d, both display longer chains.

• MeSH
• antitumorózní látky chemie   farmakologie   MeSH
• buňky A549 MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• DNA-topoisomerasy typu II metabolismus   MeSH
• inhibitory topoisomerasy I chemie   farmakologie   MeSH
• inhibitory topoisomerasy II chemie   farmakologie   MeSH
• kumariny chemie   farmakologie   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádorové buňky kultivované MeSH
• proliferace buněk účinky léků   MeSH
• proteiny vázající poly-ADP-ribosu antagonisté a inhibitory   metabolismus   MeSH
• takrin chemie   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A series of new 3,6,9-trisubstituted acridine derivatives with fluorine substituents on phenyl ring were synthesized and their interaction with calf thymus DNA was investigated. Analysis using UV-Vis absorbance spectra provided valuable information about the formation of the acridine-DNA complex. In addition, compounds 8b and 8d were found to display an increased binding affinity (K = 2.32 and 2.28 × 106 M-1, respectively). Topo I/II inhibition mode assays were also performed, and the results verify that the novel compounds display topoisomerase I and II inhibitory activity; compounds 8a, 8b and 8c completely inhibited topoisomerase I activity at a concentration of 60 × 10-6 M, but only compound 8d showed partial ability to inhibit topoisomerase II at concentrations of 30 and 50 × 10-6 M. The ability of the derivatives to impair cell proliferation was tested through an analysis of cell cycle distribution, quantification of cell number, viability studies, metabolic activity measurement and clonogenic assay. The content and localization of the derivatives in cells were analyzed using flow cytometry and fluorescence microscopy. The compounds 8b and 8d altered the physiochemical properties and improved antiproliferative activity in A549 human lung carcinoma cells (compound 8d displayed the highest level of activity, 4.25 × 10-6 M, after 48 h).

• MeSH
• akridiny chemická syntéza   chemie   farmakologie   MeSH
• antitumorózní látky chemická syntéza   chemie   farmakologie   MeSH
• buňky A549 MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• DNA-topoisomerasy typu II metabolismus   MeSH
• DNA účinky léků   MeSH
• halogenace MeSH
• inhibitory topoisomerasy I chemická syntéza   chemie   farmakologie   MeSH
• inhibitory topoisomerasy II chemická syntéza   chemie   farmakologie   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• molekulární struktura MeSH
• proliferace buněk účinky léků   MeSH
• skot MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cruciform structures are preferential targets for many architectural and regulatory proteins, as well as a number of DNA binding proteins with weak sequence specificity. Some of these proteins are also capable of inducing the formation of cruciform structures upon DNA binding. In this paper we analyzed the amino acid composition of eighteen cruciform binding proteins of Homo sapiens. Comparison with general amino acid frequencies in all human proteins revealed unique differences, with notable enrichment for lysine and serine and/or depletion for alanine, glycine, glutamine, arginine, tyrosine and tryptophan residues. Based on bootstrap resampling and fuzzy cluster analysis, multiple molecular mechanisms of interaction with cruciform DNA structures could be suggested, including those involved in DNA repair, transcription and chromatin regulation. The proteins DEK, HMGB1 and TOP1 in particular formed a very distinctive group. Nonetheless, a strong interaction network connecting nearly all the cruciform binding proteins studied was demonstrated. Data reported here will be very useful for future prediction of new cruciform binding proteins or even construction of predictive tool/web-based application.

• MeSH
• aminokyseliny chemie   MeSH
• chromatin MeSH
• chromozomální proteiny, nehistonové chemie   MeSH
• DNA vazebné proteiny chemie   MeSH
• DNA-topoisomerasy I chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• onkogenní proteiny chemie   MeSH
• protein HMGB1 chemie   MeSH
• proteiny vázající poly-ADP-ribosu chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A series of new Zn(II) complexes with flufenamic acid (flu) has been synthesized, namely [Zn3(dmso)2(flu)6] (1), [Zn3(flu)6(py)2] (2), [Zn(flu)2(tmen)] (3), [ZnCl(flu)(neo)] (4), and [Zn(cyclam)(flu)2] (5), where py=pyridine, tmen=N,N,N',N'-Tetramethylethylene diamine, neo=2,9-Dimethyl-1,10-phenanthroline and cyclam=1,4,8,11-Tetraazacyclotetradecane. These complexes have been characterized by infrared spectroscopy, single-crystal X-ray structure analysis, elemental and thermal analysis. All complexes contain deprotonated flufenamic acid coordinated via carboxylato group to zinc atoms, but their structures differ in the carboxylato binding mode, the coordination number of the central atom, the shape of the coordination polyhedra and the resultant supramolecular structures. Furthermore, an interaction of complexes with calf-thymus DNA (CT DNA) and human serum albumin (HSA) has been investigated by spectroscopic techniques. Moreover, the complexes 1 and 2 inhibit the catalytic activity of topoisomerase I at 60μM.

• MeSH
• antiflogistika nesteroidní chemická syntéza   chemie   MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• DNA chemie   MeSH
• inhibitory topoisomerasy I chemická syntéza   chemie   farmakologie   MeSH
• komplexní sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• kyselina flufenamová chemická syntéza   chemie   MeSH
• lidé MeSH
• lidský sérový albumin chemie   MeSH
• ligandy MeSH
• molekulární struktura MeSH
• skot MeSH
• zinek chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: Prospective-retrospective assessment of the TOP1 gene copy number and TOP1 mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan. TOP1 copy number status was analyzed by fluorescence in situ hybridization (FISH) using a TOP1/CEN20 dual-probe combination. TOP1 mRNA data were available from previous analyses. RESULTS: TOP1 FISH and follow-up data were obtained from 534 patients. TOP1 gain was identified in 27% using a single-probe enumeration strategy (≥4 TOP1 signals per cell) and in 31% when defined by a TOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent on TOP1 FISH status.TOP1 mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized by TOP1 mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment by TOP1 mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasing TOP1 mRNA values appeared to be associated with increasing benefit of irinotecan. CONCLUSIONS: In contrast to the TOP1 copy number, a trend was demonstrated for a predictive property of TOP1 mRNA expression. On the basis of TOP1 mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.

• MeSH
• adjuvantní chemoterapie MeSH
• antitumorózní látky fytogenní terapeutické užití   MeSH
• biologické markery MeSH
• DNA-topoisomerasy I genetika   MeSH
• exprese genu * MeSH
• genová dávka * MeSH
• hybridizace in situ fluorescenční MeSH
• inhibitory topoisomerasy I terapeutické užití   MeSH
• kamptothecin analogy a deriváty   terapeutické užití   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nádory tračníku diagnóza   farmakoterapie   genetika   mortalita   MeSH
• prognóza MeSH
• staging nádorů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

In this paper, we describe the biochemical properties and biological activity of a series of cholinesterase reactivators (symmetrical bisquaternary xylene-linked compounds, K106-K114) with ctDNA. The interaction of the studied derivatives with ctDNA was investigated using UV-Vis, fluorescence, CD and LD spectrometry, and electrophoretic and viscometric methods. The binding constants K were estimated to be in the range 1.05 × 10(5)-5.14 × 10(6) M(-1) and the percentage of hypochromism was found to be 10.64-19.28% (from UV-Vis titration). The used methods indicate that the studied samples are groove binders. Electrophoretic methods proved that the studied compounds clearly influence calf thymus Topo I (at 5 μM concentration, except for compounds K107, K111 and K114 which were effective at higher concentrations) and human Topo II (K110 partially inhibited Topo II effects even at 5 μM concentration) activity.

• MeSH
• cirkulární dichroismus MeSH
• denaturace nukleových kyselin MeSH
• DNA-topoisomerasy I chemie   metabolismus   MeSH
• DNA-topoisomerasy typu II chemie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• molekulární struktura MeSH
• reaktivátory cholinesterázy chemie   farmakologie   MeSH
• spektrální analýza MeSH
• vazba proteinů MeSH
• viskozita MeSH
• Publikační typ
• časopisecké články MeSH

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.

• MeSH
• DNA nádorová genetika   MeSH
• DNA-topoisomerasy I genetika   MeSH
• dusík MeSH
• fixace tkání metody   MeSH
• karcinom chemie   chirurgie   MeSH
• kolon chemie   MeSH
• kryoprezervace přístrojové vybavení   metody   MeSH
• kvantitativní polymerázová řetězová reakce přístrojové vybavení   metody   MeSH
• lidé MeSH
• nádorové proteiny biosyntéza   genetika   MeSH
• nádory tračníku chemie   chirurgie   MeSH
• ochrana biologická přístrojové vybavení   metody   MeSH
• odběr biologického vzorku přístrojové vybavení   metody   MeSH
• regulace genové exprese u nádorů MeSH
• reprodukovatelnost výsledků MeSH
• ribozomální DNA genetika   MeSH
• řízení kvality MeSH
• RNA nádorová analýza   genetika   izolace a purifikace   MeSH
• RNA ribozomální 18S genetika   MeSH
• roztoky pro uchovávání orgánů MeSH
• rychlé screeningové testy přístrojové vybavení   metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Three new diphenylsubstituted spirotriazolidine- and thiazolidinone-acridines were prepared and their interaction with calf thymus DNA investigated with UV-vis, fluorescence, circular dichroism spectroscopy and viscometry. The binding constants K were estimated to range from 0.34 to 0.93 × 10(4) M(-1). UV-vis, fluorescence and circular dichroism measurements indicated that the compounds act as effective DNA-interacting agents. Electrophoretic separation proved that ligands inhibited topoisomerase I and II. The biological activity of compounds 3, 5 &6 at several different concentrations (10, 20 and 50 μM) was evaluated both 48 h and 72 h following their addition to HL-60 cancer cells. The results were analysed using various different techniques (MMP detection, changes in metabolic activity/viability and analysis of cell cycle distribution). Acridine was also used as the positive control in these assays. The results from MMP analysis demonstrate the strong effect of 3-diphenylamino-2-(acridin-9-yl)imino-1,3-thiazolidin-4-one (5) on mitochondrial physiology. Cell viability analysis showed that acridine derivatives 3 and 6 were less effective than derivative 5 and the acridine control.

• MeSH
• akridiny chemie   MeSH
• antitumorózní látky chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• DNA-topoisomerasy typu II metabolismus   MeSH
• DNA metabolismus   MeSH
• HL-60 buňky MeSH
• inhibitory topoisomeras chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• lidé MeSH
• skot MeSH
• spirosloučeniny chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Metastatic pheochromocytoma continues to be an incurable disease, and treatment with conventional cytotoxic chemotherapy offers limited efficacy. In the present study, we evaluated a novel topoisomerase I inhibitor, LMP-400, as a potential treatment for this devastating disease. We found a high expression of topoisomerase I in human metastatic pheochromocytoma, providing a basis for the evaluation of a topoisomerase 1 inhibitor as a therapeutic strategy. LMP-400 inhibited the cell growth of established mouse pheochromocytoma cell lines and primary human tumor tissue cultures. In a study performed in athymic female mice, LMP-400 demonstrated a significant inhibitory effect on tumor growth with two drug administration regimens. Furthermore, low doses of LMP-400 decreased the protein levels of hypoxia-inducible factor 1 (HIF-1α), one of a family of factors studied as potential metastatic drivers in these tumors. The HIF-1α decrease resulted in changes in the mRNA levels of HIF-1 transcriptional targets. In vitro, LMP-400 showed an increase in the growth-inhibitory effects in combination with other chemotherapeutic drugs that are currently used for the treatment of pheochromocytoma. We conclude that LMP-400 has promising antitumor activity in preclinical models of metastatic pheochromocytoma and its use should be considered in future clinical trials.

• MeSH
• antitumorózní látky farmakologie   MeSH
• benzodioxoly aplikace a dávkování   farmakologie   MeSH
• buňky PC12 MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa genetika   metabolismus   MeSH
• feochromocytom farmakoterapie   enzymologie   patologie   MeSH
• hypoxie buňky MeSH
• inhibitory topoisomerasy I aplikace a dávkování   farmakologie   MeSH
• isochinoliny aplikace a dávkování   farmakologie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nádory jater farmakoterapie   enzymologie   sekundární   MeSH
• nádory nadledvin farmakoterapie   enzymologie   patologie   MeSH
• nádory plic farmakoterapie   enzymologie   sekundární   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proliferace buněk účinky léků   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• synergismus léků MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Intramural MeSH