Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A study was made of geno- and phenotypic changes, associated with of multidrug resistance development in murine 1F7 hybridoma cells, selected for adriamycin and ethidium bromide resistance (1F7-EBR and 1F7-ADR). In both cell lines overexpression of mdr1 gene was observed, while amplification of mdr1 gene was detected only in 1F7-ADR cells. Karyotypic analysis revealed in resistant cells the presence of a specific marker M45 absent from parental cells, thus suggesting its selective importance. The M45 length instability, as well as the presence of a distal typical homogeneously stained region in it provide an evidence for a link between M45 and mdr1 gene amplification. The frequency of double minute chromosomes in parental lines was the same as in multidrug resistant lines. In 1F7-ADR cells, in contrast with 1F7-EBR cells, the enhancement of immunoglobulin production and the increase in immunoglobulin gamma 2b heavy chain gene expression were observed, which correlated with a decline in DNA-topoisomerase II activity.
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PMID:[The genome structure and phenotypic characteristics of murine hybridoma 1F7 cells selected in the presence of adriamycin and ethidium bromide]. 949 May 14

Previous studies have demonstrated decreased levels of DNA topoisomerase II alpha protein and messenger RNA in the Adriamycin-resistant P388 murine leukemia cell line P388/ADR/7 compared to the sensitive P388/4 cell line. An allelic fusion event involving the topoisomerase II alpha and the retinoic acid receptor a genes has been identified in these cells that probably contributes to the decreased topoisomerase II activity in P388/ADR/7 cells. However, this allelic mutation may be a minor contributor or even incidental to the resistance phenotype, since these cells display other candidate mechanisms of resistance, including increased P-glycoprotein, increased glutathione-S-transferase activity and an increased onset of DNA repair. To establish a role for topoisomerase II alpha in mediating the Adriamycin resistance phenotype, complementation of the mutant allele was attempted by transfecting the murine P388/ADR/7 cells with a human topoisomerase II alpha expression construct under the control of the human metallothionein IIA promoter. The majority of transfected cell lines that were obtained by selection in hygromycin B contained copies of the integrated expression construct that were rearranged. Only two of thirty-two transfected cell lines were found to contain a single, unrearranged copy of the human topoisomerase II alpha cDNA. P388/ADR/7 cell lines carrying an integrated, intact human topoisomerase II alpha expression vector were more sensitive to Adriamycin, daunorubicin, mitoxantrone, and etoposide, but not to actinomycin D and vincristine compared to control cells transfected with vector alone or cell lines with rearranged topoisomerase II alpha expression constructs. These findings suggest that topoisomerase II alpha is a selective and significant contributor to multifactorial resistance.
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PMID:Selective sensitization of adriamycin-resistant P388 murine leukemia cells to antineoplastic agents following transfection with human DNA topoisomerase II alpha. 949 16

A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIalpha or IIbeta showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIalpha and topo IIbeta mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIalpha in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIbeta levels were similar in both lines. Moreover, the amount of in vivo topo IIalpha-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIalpha gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIalpha polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIalpha may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.
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PMID:Reduced activity of topoisomerase II in an Adriamycin-resistant human stomach-adenocarcinoma cell line. 952 30

Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vinca, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.
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PMID:Vinflunine (20',20'-difluoro-3',4'-dihydrovinorelbine), a novel Vinca alkaloid, which participates in P-glycoprotein (Pgp)-mediated multidrug resistance in vivo and in vitro. 974 May 39

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonal form of vitamin D, has anticancer activity in vivo and in vitro. Doxorubicin exerts its cytotoxic effect on tumor cells mainly by two mechanisms: (a) generation of reactive oxygen species (ROS); and (b) inhibition of topoisomerase II. We studied the combined cytotoxic action of 1,25(OH)2D3 and doxorubicin on MCF-7 breast cancer cells. Pretreatement with 1,25(OH)2D3 resulted in enhanced cytotoxicity of doxorubicin. The average enhancing effect after a 72-h pretreatment with 1,25(OH)2D3 (10 nM) followed by a 24-h treatment with 1 microg/ml doxorubicin was 74+/-9% (mean +/- SE). Under these experimental conditions, 1,25(OH)2D3 on its own did not affect cell number or viability. 1,25(OH)2D3 also enhanced the cytotoxic activity of another ROS generating quinone, menadione, but did not affect cytotoxicity induced by the topoisomerase inhibitor etoposide. The antioxidant N-acetylcysteine slightly reduced the cytotoxic activity of doxorubicin but had a marked protective effect against the combined action of 1,25(OH)2D3 and doxorubicin. These results indicate that ROS are involved in the interaction between 1,25(OH)2D3 and doxorubicin. 1,25(OH)2D3 also increased doxorubicin cytotoxicity in primary cultures of rat cardiomyocytes. Treatment of MCF-7 cells with 1,25(OH)2D3 alone markedly reduced the activity, protein, and mRNA levels of the cytoplasmic antioxidant enzyme Cu/Zn superoxide dismutase, which indicated that the hormone inhibits its biosynthesis. This reduction in the antioxidant capacity of the cells could account for the synergistic interaction between 1,25(OH)2D3 and doxorubicin and may also suggest increased efficacy of 1,25(OH)2D3 or its analogues in combination with other ROS-generating anticancer therapeutic modalities.
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PMID:1,25-Dihydroxyvitamin D3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage. 1002 76

Doxorubicin has been a constituent of antitumor drug protocols for a broad spectrum of cancers for more than two decades. Side effects and resistance continue to be important limitations. Drug targets responsible for both side effects and anti-tumor activity are cell membrane receptors, cell membrane lipids, nucleic acids and topoisomerase. Induction of oxidative stress is responsible for most if not all biological activity. An important consequence of oxidative stress is the production of formaldehyde which can subsequently be utilized by the drug for covalent bonding to nucleic acids and other targets as shown by in vitro experiments. Multidrug resistance mechanisms inhibit drug-induced DNA damage, drug uptake, and drug-induced oxidative stress. Synthetic anthracyclines conjugated to formaldehyde circumvent some if not all of the resistance mechanisms. Consequently, anthracycline-formaldehyde conjugates have potential for the treatment of resistant cancer.
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PMID:A redox pathway leading to the alkylation of nucleic acids by doxorubicin and related anthracyclines: application to the design of antitumor drugs for resistant cancer. 1019 40

Doxorubicin induces DNA breakage by stabilizing a cleavable topoisomerase II-DNA complex. In contrast, topoisomerase II catalytic inhibitor ICRF-193 and uncoupling inhibitor aclarubicin interfere with the cleavable complex formation. We analysed combination effects of these drugs using two-dimensional flow cytometry of DNA content and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling assay. Both ICRF-193 and aclarubicin attenuated the cytotoxic effect of doxorubicin on HL-60 cells (85% and 46% maximum reduction, respectively), which suggested that doxorubicin exerts its cytotoxic effect at least partially through the topoisomerase II-dependent DNA cleavage. Doxorubicin and ICRF-193 both induced G2 arrest in HL-60 cells, by which they may have reduced the cytotoxic effect of vincristine. Indeed, although ICRF-193 inhibited doxorubicin-induced apoptosis, ICRF-193 and doxorubicin cooperated in arresting HL-60 cells at G2 phase. These results indicated that G2 arrest was caused not only by DNA damage but also through a DNA damage-free, topoisomerase II inactivation-induced pathway. Western blot analysis showed that both types of G2 arrest were mediated by the inhibition of p34cdc2 dephosphorylation.
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PMID:Inhibition of p34cdc2 dephosphorylation in DNA damage- and topoisomerase II inactivation-induced G2 arrests in HL-60 cells. 1035 37

Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I.
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PMID:Topoisomerase poisoning activity of novel disaccharide anthracyclines. 1038 86

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.
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PMID:Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines. 1050 97

S 16020 is a new olivacine derivative which has been shown to intercalate into DNA and to stabilize the cleavable complex formed by DNA and purified topoisomerase II. The aim of the present study was to estimate the impact of time exposure on the in vitro activity of S 16020. This was done on seven cancer cell lines of human origin (head and neck, kidney, and ovary). Doxorubicin was used as a reference drug. The cytotoxic activity of S 16020 remained stable during at least 3 h. A loss of activity of about 30% was apparent after 6 or 24 h preincubation. This relative loss of activity reached about 50% after 72 h preincubation. Considering all tested cell lines, the average IC50 decrease was 89+/-8% for S 16020 with incubation times between 1 and 72 h. An exposure index (El) was calculated to evaluate the effect of time on the cytotoxic efficacy. The reference time was 1 h exposure. The El values were corrected to take into account the loss of drug activity. For the majority of cell lines EI values were greater than 1 for both drugs, particularly after a 6 h exposure time. This means that, in this case as compared to the shorter exposure (1 h), increasing time has a relative detrimental effect on drug efficacy. For the two cancer cell lines of ovarian origin, El values remained close to 1 for both drugs whatever the total exposure time. This means that, in this case, time and concentration have symmetrical effects on cell survival. The pharmacological information provided by the present study may be useful in designing future clinical trials on this potentially interesting new topoisomerase II inhibitor. As a consequence of these data, 1 and 3 h drug administration schedules are currently tested during phase I trials.
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PMID:Time-schedule dependency of S 16020, a new topoisomerase II inhibitor. 1088 8


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