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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform. 165 25

The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM). ADM and MX2 showed an equivalent antitumor effect against K562. K562/ADM was highly resistant to ADM. In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM. The efflux of ADM was rapid in K562/ADM but not in K562. On the other hand, the efflux of MX2 was rapid in both cell lines. The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562. On the other hand, formation of those breaks by MX2 was not decreased. Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells. On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed. The topoisomerase II activity in K562 and K562/ADM was not significantly different. It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells.
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PMID:3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin conquers multidrug resistance by rapid influx following higher frequency of formation of DNA single- and double-strand breaks. 216 45

Topoisomerase II alpha is an essential nuclear enzyme involved in DNA replication and a target for many of the clinically useful antineoplastic agents. In a mitoxantrone-selected human leukemia cell line, HL-60/MX2, cellular topoisomerase II (topo II) catalytic activity is decreased, in association with the finding of reduced nuclear topo II alpha and beta protein levels. In addition, HL-60/MX2 cells contain a novel M(r) 160,000 topo II alpha-related protein that localizes predominantly to the cell cytoplasm (W. G. Harker et al., Biochemistry, 30: 9953-9961, 1991). In these studies, we have investigated the molecular mechanisms underlying the altered expression of the topo II alpha protein(s) in these cells. Three topo II alpha mRNAs, 7.2, 6.3, and 4.8 kb, were identified in the HL-60/MX2 cells, with the 6.3 and 4.8 kb transcripts being present in roughly equivalent amounts, while the 7.2-kb mRNA represents < 7% of the total topo II alpha-specific mRNA. Portions of the 3'-coding and 3'-untranslated regions were found to be missing from the 7.2- and 4.8-kb topo II alpha mRNAs by Northern blot analysis. Sequences encoding the 3' regions of the normal and truncated forms of the topo II alpha enzyme were obtained from the HL-60/MX2 cells through the use of a 3'-rapid amplification of cDNA ends strategy. Approximately 1321 nucleotides are missing from the 3'-coding and 3'-untranslated regions of the 4.8-kb mRNA and are replaced by 122 nucleotides that contain an in-frame stop codon and consensus polyadenylation signal. The translation product of the truncated 4388-bp topo II alpha transcript would have a predicted M(r) of 157,850, with 108 COOH-terminal amino acids being replaced by 13 novel residues. Immunoblot analysis confirmed that amino acids in the COOH-terminal region of topo II alpha were missing from the M(r) 160,000 HL-60/MX2 protein, and antisera generated to a synthetic peptide representing the 13 unique amino acids identified a M(r) 160,000 protein in nuclear extracts from these cells. PCR evaluation of the organization of the 3' region of the topo II alpha gene revealed that the 4.8-kb mRNA found in HL-60/MX2 cells diverges from that of the 6.3-kb mRNA at a consensus exon-intron splice donor site. The 122-bp novel nucleotides identified in the truncated transcript appear to originate from an adjacent intron as a result of altered RNA processing. These studies suggest that as a result of the disruption of the carboxy terminus of the topo II alpha protein and the putative nuclear targeting sequences identified therein, cellular localization of the protein is altered, which may confer a growth advantage for the HL-60/MX2 cells in the presence of mitoxantrone.
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PMID:Selective use of an alternative stop codon and polyadenylation signal within intron sequences leads to a truncated topoisomerase II alpha messenger RNA and protein in human HL-60 leukemia cells selected for resistance to mitoxantrone. 758 37

Resistance to the classical anthracyclines may be due to one or several mechanisms, most notably p-glycoprotein (pGP) associated multidrug resistance (mdr1, "typical mdr") and altered activity of topoisomerase II (topo II) ("atypical mdr"). Modulators of mdr1 will be of limited value in case of combined forms of resistance. A Friend murine erythroleukemia cell line (F4-6R) carrying both mdrl and topo II mediated anthracycline resistance was used to determine the efficacy of structurally altered anthracyclines against such extended multidrug resistance. Proliferation assays showed that 3'N-morpholinyl substituted anthracyclines were able to retain much of their activity even in this setting. MX2 (KRN8602; 3'-deamino-3'-[4-morpholinyl]-13-deoxo-10-hydroxycarminomycin+ ++), which is 9-alkylated in addition to carrying a 3'N-morpholinyl group, was the most promising agent tested.
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PMID:Structurally modified anthracyclines retain activity in a cell line with simultaneous typical and atypical multidrug resistance. 765 9

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.
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PMID:Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells. 838 9

The morpholinyl analogues of doxorubicin (DOX) have previously been reported to be non-cross-resistant in multidrug resistant (MDR) cells due to a lower affinity for P-glycoprotein relative to the parent compound. In order to further investigate the mechanisms of action of these morpholinyl anthracyclines, we examined their ability to cause DNA single- and double-strand breaks (SSB, DSB) and their interactions with topoisomerases. Alkaline elution curves were determined after 2-h drug treatment at 0.5, 2 and 5 microM, while neutral elution was conducted at 5, 10 and 25 microM in a human ovarian cell line, ES-2. A pulse-field gel electrophoresis assay was used to confirm the neutral elution data under the same conditions. Further, K-SDS precipitation and topoisomerase drug inhibition assays were used to determine the effects of DOX and the morpholinyl analogues on topoisomerase (Topo) I and II. Under deproteinated elution conditions (pH 12.1), DOX, morpholinyl DOX (MRA), methoxy-morpholinyl DOX (MMDX) and morpholinyl oxaunomycin (MX2) were equipotent at causing SSB in the human ovarian carcinoma cell line, ES-2. However, neutral elution (pH 9.6) under deproteinated conditions revealed marked differences in the degree of DNA DSB. After 2-h drug exposures at 10 microM, DSBs were 3300 rad equivalents for MX2, 1500 for DOX and 400 for both MRA and MMDX in the ES-2 cell line. Pulse-field data substantiated these differences in DSBs, with breaks easily detected after MX2 and DOX treatment, but not with MRA and MMDX. DOX and MX2 thus cause DNA strand breaks selectively through interaction with Topo II, but not Topo I. In contrast, MRA and MMDX cause DNA breaks through interactions with both topoisomerases with a predominant inhibition of Topo I.
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PMID:Differential single- versus double-strand DNA breakage produced by doxorubicin and its morpholinyl analogues. 864 94

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFkappaB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFkappaB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IkappaB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IkappaB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFkappaB activation.
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PMID:Topoisomerase II is required for mitoxantrone to signal nuclear factor kappa B activation in HL60 cells. 1094 Mar 16

Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two alkaloids, which only differ by the respective orientation of their indole and quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a massive accumulation of P388 murine leukemia cells in the G2/M phase. With HL-60 human leukemia cells, the treatment with cryptolepine leads to the appearance of a hypo-diploid DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells, cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer drug mitoxantrone suggests that topoisomerase II may not represent the essential cellular target for the alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but both alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-caspases was found to be enhanced much more strongly with cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal cleavage of DNA in the HL-60 cells treated with the alkaloids. Altogether, the results shed light on the mechanism of action of these two plant alkaloids.
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PMID:Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis. 1109 95

Peroxisomicine A(1) (T-514) is a dimeric anthracenone first isolated from the plant Karwinskia humboldtiana. The compound presents a high and selective toxicity toward liver and skin cell cultures and is currently the subject of preclinical studies as an antitumor drug. To date, the molecular basis for its diverse biological effects remains poorly understood. To elucidate its mechanism of action, we studied its interaction with DNA and its effects on human DNA topoisomerases. Practically no interaction with DNA was detected. Peroxisomicine was found to inhibit topoisomerase II but not topoisomerase I. DNA relaxation and decatenation assays indicated that the drug interferes with the catalytic activity of topoisomerase II but does not stimulate DNA cleavage, in contrast to conventional topoisomerase poisons such as etoposide. Two human leukemia cell lines sensitive or resistant to mitoxantrone were used to assess the cytotoxicity of the toxin and its effect on the cell cycle. In both cases, peroxisomicine treatment was associated with a loss of cells from every phase of the cell cycle and was accompanied by a large increase in the sub-G1 region which is characteristic of apoptotic cells. The cell cycle changes were more pronounced with the sensitive HL-60 cells than with the resistant HL-60/MX2 cells (with reduced topoisomerase II activity), in agreement with the cytotoxicity measurements. Treatment of HL-60 cells with T-514 stimulated the cleavage of the poly(ADP-ribose) polymerase by intracellular proteases such as caspase-3. The cytometry and Western blot analyses reveal that peroxisomicine induces apoptosis in leukemia cells. In addition, we characterized a catabolite of peroxisomicine, named T-510R, in the form of a highly stable radical metabolite. The electron spin resonance and mass spectrometry data are consistent with the formation of an anionic semiquinonic radical. The oxidized product T-510R inhibits topoisomerase II with a reduced efficiency compared to the parent toxin and was found to be about 3-4 times less toxic to both the sensitive and resistant leukemia cell lines than T-514. Collectively, the results suggest that topoisomerase II inhibition plays a role in the cytotoxicity of the plant toxin peroxisomicine. Inhibition of topoisomerase II may serve as an inducing signal triggering the apoptotic cell death of leukemia cells exposed to the toxin. The dihydroxyanthracenone unit may represent a useful chemotype for the preparation of topoisomerase II-targeted anticancer agents.
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PMID:DNA topoisomerase II inhibition by peroxisomicine A(1) and its radical metabolite induces apoptotic cell death of HL-60 and HL-60/MX2 human leukemia cells. 1117 May 4

Absorption, melting temperature and linear dichroism measurements were performed to investigate the interaction with DNA of a series of 16 tricyclic and tetracyclic compounds related to the antiviral agent B-220. The relative DNA affinity of the test compounds containing an indolo[2,3-b]quinoxaline, pyridopyrazino[2,3-b]indoles or pyrazino[2,3-b]indole planar chromophore varies significantly depending on the nature of the side chain grafted onto the indole nitrogen. Compounds with a dimethylaminoethyl chain strongly bind to DNA and exhibit a preference for GC-rich DNA sequences, as revealed by DNase I footprinting. Weaker DNA interactions were detected with those bearing a morpholinoethyl side chain. The incorporation of a 2,3-dihydroxypropyl side chain does not reinforce the DNA interaction compared with the unsubstituted analogues. Both the DNA relaxation assay and cytotoxicity study using two human leukemia cell lines sensitive (HL-60) or resistant (HL-60/MX2) to the antitumor drug mitoxantrone, indicate that topoisomerase II is not a privileged target for the test compounds which only weakly interfere with the catalytic activity of the DNA cleaving enzyme. Cytometry studies showed that the most cytotoxic compounds induce a massive accumulation of cells in the G2/M phase of the cell cycle. Collectively, the data show a relationship between DNA binding and cytotoxicity in the indolo[2,3-b]quinoxaline series.
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PMID:DNA interaction and cytotoxicity of a new series of indolo[2,3-b]quinoxaline and pyridopyrazino[2,3-b]indole derivatives. 1164 Sep 15


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