Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.
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PMID:Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay. 164 64

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

Pre-treatment with low, non-toxic concentrations (0.04 microM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)-induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre-treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre-treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug-induced DNA breaks as assessed by the alkaline elution method after proteinase K digestion. The amount of DNA single-strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre-treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre-treatment were similar to those seen in cells without pretreatment. The amount of DNA double-strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VP16-induced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar to those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G1-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre-treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non-toxic MTX pre-treatment is due to a larger fraction of S-phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.
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PMID:Increase in topoisomerase-II-mediated DNA breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate. 215 35

The cyanomorpholino analog of doxorubicin (MRA-CN) is a potent cytotoxic agent which is known to cross-link DNA. A human ovarian carcinoma cell line, ES-2, was grown in increasing concentrations of MRA-CN from 0.1 to 0.5 nM. The resultant resistant subline, ES-2R, was 4-fold resistant to MRA-CN. DNA damage and repair in response to MRA-CN were compared in the parental and resistant cell lines using alkaline elution. DNA cross-links were detectable after 3-h incubation of the cells at 37 degrees C in MRA-CN at concentrations greater than or equal to 1.0 nM. Paradoxically, 2-fold more cross-links were detected in the ES-2R cells as compared with the ES-2 cells. This paradoxical difference in cross-links between the 2 cell lines was observed to increase with time of exposure to 2.5 nM of MRA-CN. Non-protein-associated DNA strand breaks were also detected in the 2 cell lines after exposure to 2.5 nM of the drug. The ES-2 cells consistently showed twice as many breaks as the ES-2R cells, which could explain the paradoxical higher apparent DNA cross-linking observed with the ES-2R cells after exposure to MRA-CN. Studies of the time course of cross-link repair after exposure to MRA-CN revealed that 75% of the DNA cross-links disappeared in the ES-2R cells by the end of 8 h in drug-free medium. In contrast, cross-links in the ES-2 cells were undetectable after 4 h, which coincided with a progressive increase in DNA strand breaks. The topoisomerase II level in the ES-2 cells was 2- to 4-fold higher than that in the ES-2R cells. However, proteinase K treatment of the lysed cells did not increase the number of apparent strand breaks produced by MRA-CN, suggesting that topoisomerase II may not be involved. These findings indicate that, in addition to DNA cross-linking, MRA-CN causes DNA strand breakage. Resistance to MRA-CN in the ES-2R cells is associated with more apparent DNA cross-linking and less DNA strand breakage, which may be a consequence of differences in DNA repair and/or nonspecific DNA degradation between the resistant and the sensitive cell lines.
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PMID:Paradoxical increase in DNA cross-linking in a human ovarian carcinoma cell line resistant to cyanomorpholino doxorubicin. 216 51

In the alkaline elution assay, expression of protein-associated DNA damage induced by topoisomerase II antagonists is facilitated by proteinase K digestion of the drug-stabilized topoisomerase-II-DNA complex. In the absence of this enzymatic deproteinization step, drug-induced DNA strand breaks are masked by the binding of the topoisomerase-II-DNA complex to the synthetic filter from which DNA is eluted subsequent to alkaline denaturation. In this manuscript, we report that as the number of cells lysed on the filter is increased, binding of the topoisomerase-II-DNA complex to the filter is compromised, permitting expression of DNA damage in the absence of enzymatic deproteinization.
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PMID:Expression of protein-associated DNA damage in the alkaline elution assay in the absence of enzymatic deproteinization. 256 38

Binding of exogenous DNA to the nuclear scaffold was investigated using a plasmid DNA (pBR322, EcoRI site deleted) of various topological forms and nuclear subfractions with different levels of nuclear DNA depletion. When supercoiled DNA was incubated with histone-depleted nuclei (nuclear halo), a dose-dependent binding of the DNA occurred, whereas no binding was observed with relaxed and linear forms of DNA. The bound DNA was released upon linearization with BamHI or digestion of the scaffolding structure with proteinase K. Extensive digestion of the halo with micrococcal nuclease generated additional sites which bind both relaxed and linear DNA. In the presence of a large excess of calf thymus DNA, these sites were effectively blocked and the specificity to supercoiled DNA was restored. The binding of all forms of DNA was abolished by heat-denatured DNA. There was no detectable change in linking number of the scaffold-associated supercoils. Competitive binding was observed between supercoiled DNAs with unrelated sequences, indicating that no specific nucleotide sequence is required for the binding. RNA was found to be a weak competitor. A DNA binding assay performed on electrophoretic blots of solubilized nuclear scaffold revealed a protein component with apparent molecular weight of 120,000 which retained selective binding to supercoils. These results suggest that the nuclear scaffold possesses DNA-binding sites for torsionally strained domains of chromatin and that an integral protein factor is involved in the binding. Implications of the findings are discussed in connection with proposed functions of the nuclear scaffold and topoisomerase II.
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PMID:The nuclear scaffold exhibits DNA-binding sites selective for supercoiled DNA. 336 76

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S1 nuclease cleavage. The S1-cleavage occurred once at these sites. In the absence of added Mg2+, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S1, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S1-cleavable, unbasepaired sites.
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PMID:Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease. 630 25

Eukaryotic topoisomerase II (Topo-II) inhibitors such as etoposide, adriamycin and mitoxantrone, which commonly stabilize the cleavable complex of the enzyme and DNA, have been found to efficiently induce chromosome-type aberrations (mainly breaks and exchanges) in cultured Chinese hamster lung fibroblastic cells (CHL cells). To clarify whether the induction of chromosome-type aberrations is mediated by stabilization of the cleavable complex, the present study investigated (1) the correlation between the induction of chromosome-type aberrations and the amount of cleavable complex formed; and (2) the ATP dependence of the Topo-II inhibitor-induced chromosome-type aberrations due to the ATP requirement of cleavable complex formation by Topo-II. First, in cells treated with the Topo-II inhibitors, (etoposide, adriamycin) and aclarubicin, an antagonist of the inhibitor of cleavable complex formation, the frequency of chromosome-type aberrations decreased dose-dependently with aclarubicin, in contrast to an increase of chromatid-type aberrations. The formation of the cleavable complex was further established by a proteinase K/SDS precipitation assay for cleaved double-strand DNA in a cell-free system and in CHL cells. Results from both experiments showed that aclarubicin caused a dose-dependent suppression of the accumulation of the cleavable complex induced by etoposide, which corresponded particularly well to the reduction of chromosome-type aberrations in etoposide-treated cells. In ATP-depleted cells simultaneously treated with etoposide and dinitrophenol (DNP), chromosome-type aberrations were reduced as compared with DNP-untreated cells, in contrast to an increase of chromatid exchanges in the cells. This means that etoposide-induced chromosome-type aberrations in ATP-depleted cells may be attributable to incompleteness of Topo-II activities to form DNA double-strand breaks. The present findings indicate that the stabilization of the cleavable complex on Topo-II is closely associated with the induction of chromosome-type aberrations.
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PMID:Efficient induction of chromosome-type aberrations by topoisomerase II inhibitors closely associated with stabilization of the cleavable complex in cultured fibroblastic cells. 773 99

Etoposide induces DNA damage to cells by interacting with the nuclear enzyme topoisomerase II. In this investigation the human lymphoblastic leukemia cell line (CEM) was used to study induction of DNA-strand breaks and cellular drug uptake after treatment with etoposide at a concentration of 0.5-2 micrograms/ml. High performance liquid chromatography was used for determination of etoposide concentrations. The alkaline elution assay and the DNA unwinding technique were compared for quantifying strand breaks in DNA induced by etoposide. The concentrations required to increase the level of DNA damage significantly was as follows: the DNA unwinding technique, 0.20 microgram/ml; the alkaline elution assay with proteinase K, 0.45 microgram/ml; the alkaline elution assay without proteinase K, 0.60 microgram/ml. When the half-life was adjusted, considering the efflux time of etoposide from cells, it was found to be only a few minutes. The present data show that the DNA unwinding technique is to be preferred for the screening of DNA damage. This technique is easier and quicker to perform than the alkaline elution technique.
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PMID:DNA damage induced by etoposide; a comparison of two different methods for determination of strand breaks in DNA. 869 38

AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta.
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PMID:A carbamate analogue of amsacrine with activity against non-cycling cells stimulates topoisomerase II cleavage at DNA sites distinct from those of amsacrine. 913 99


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