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)

The retinoblastoma tumor suppressor protein (RB) is targeted for inactivation in the majority of human tumors, underscoring its critical role in attenuating cellular proliferation. RB inhibits proliferation by repressing the transcription of genes that are essential for cell cycle progression. To repress transcription, RB assembles multiprotein complexes containing chromatin-modifying enzymes, including histone deacetylases (HDACs). However, the extent to which HDACs participate in transcriptional repression and are required for RB-mediated repression has not been established. Here, we investigated the role of HDACs in RB-dependent cell cycle inhibition and transcriptional repression. We find that active RB mediates histone deacetylation on cyclin A, Cdc2, topoisomerase IIalpha, and thymidylate synthase promoters. We also demonstrate that this deacetylation is HDAC dependent, since the HDAC inhibitor trichostatin A (TSA) prevented histone deacetylation at each promoter. However, TSA treatment blocked RB repression of only a specific subset of genes, thereby demonstrating that the requirement of HDACs for RB-mediated transcriptional repression is promoter specific. The HDAC-independent repression was not associated with DNA methylation or gene silencing but was readily reversible. We show that this form of repression resulted in altered chromatin structure and was dependent on SWI/SNF chromatin remodeling activity. Importantly, we find that cell cycle inhibitory action of RB is not intrinsically dependent on the ability to recruit HDAC activity. Thus, while HDACs do play a major role in RB-mediated repression, they are dispensable for the repression of critical targets leading to cell cycle arrest.
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PMID:Histone deacetylation of RB-responsive promoters: requisite for specific gene repression but dispensable for cell cycle inhibition. 1456 17

Several anticancer drugs target DNA or enzymes acting on the DNA. Because chromatin DNA is tightly compacted, accessibility to the drug target may reduce the efficiency of these anticancer drugs. We thus treated four human cancer cell lines and two normal epithelial cell lines with either trichostatin A (TSA) or SAHA, two histone deacetylase inhibitors, before exposing the cells to VP-16, ellipticine, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, or cyclophosmamide. Pretreatment with TSA or SAHA increased the killing efficiency of VP-16, ellipticine, doxorubicin, and cisplatin. The magnitude of sensitization is cell type specific and is >10-fold for VP-16 in D54, a brain tumor cell line intrinsically resistant to topoisomerase II inhibitors. Topoisomerase II levels and activity were not affected by this treatment, but p53, p21, and Gadd45 protein levels were markedly induced. Moreover, pretreatment with TSA also increased VP-16-induced apoptosis in a p53-dependent and -independent manner. Treating the cells in the reverse order (anticancer drug first, followed by TSA or SAHA) had no more cytotoxic effect than the drug alone. These data suggest that loosening-up the chromatin structure by histone acetylation can increase the efficiency of several anticancer drugs targeting DNA. This may be advantageous for treating tumors intrinsically resistant to these drugs.
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PMID:Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. 1461 26

Pancreatic cancer is a common, highly lethal disease that is rising in incidence. Chemotherapy based on 5-fluorouracil (5-FU) has been shown to prolong survival in advanced pancreatic cancer. Gemcitabine improves major symptoms and survival outcomes compared with bolus 5-FU. Many novel small molecules are being widely and actively researched. These compounds are based on classical mechanisms of action as well as biological therapies targeting novel cellular survival pathways, and include fluoropyrimidines, nucleoside cytidine analogues, platinum analogues, topoisomerase-inhibitors, antimicrotubule agents, proteasome inhibitors, vitamin D analogues, arachidonic acid pathway inhibitors, histone deacytylator inhibitors, farnesyltransferase inhibitors and epidermal growth factor receptor therapies. Adjuvant chemotherapy has also demonstrated the best survival outcomes following resection compared to other adjuvant or neo-adjuvant strategies such as radiation-based treatments. These benefits are superimposed on the dramatic increase in resectability rates and reduction in post-operative mortality achieved by centralisation of treatment in high-volume speciality centres. Newer 'small-molecule' drugs as well as the latest 'large-molecule' biological agents hold considerable promise for the future. Real advances are anticipated over the next five years but are dependent on large randomised controlled trials for success.
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PMID:Review article: chemotherapy for pancreatic cancer. 1465 25

The organization of the human dystrophin gene into loop domains has been studied using two different experimental approaches: excision of DNA loops mediated by nuclear matrix-bound topoisomerase II and in situ hybridization of different probes with histone-depleted nuclei (nuclear halos). Our objective was to examine if the DNA loops mapped by this biochemical approach coincide with loops visualized by microscopy. The results obtained using both approaches were in good agreement. Eight loops separated by attachment regions of different length were mapped in the upstream part (up to exon 54) of the gene by topoisomerase II-mediated excision. One of these loops was then directly visualized by in situ hybridization of the corresponding bacmid clone with nuclear halos. This is the first direct demonstration that a DNA domain mapped as a loop using a biochemical approach corresponds to a loop visible on cytological preparations. The validity of this result and of the whole map of loop domains was confirmed by in situ hybridization using probes derived from other attachment regions or loops mapped by topoisomerase II-mediated cleavage; these probes hybridized on the core or halo region, respectively, of nuclear halos. Our results demonstrate that a single transcription unit may be organized into several loops and that DNA loop attachment regions may be fairly long. Three out of four replication origins mapped in this gene co-localize with loop attachment regions, and the major deletion hot spot is harbored in an attachment region. These results strongly suggest that partitioning of genomic DNA into specific loops attached to a skeletal structure is a characteristic feature of eukaryotic chromosome organization in interphase.
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PMID:Visualization of individual DNA loops and a map of loop domains in the human dystrophin gene. 1508 87

We reported recently that exposure of hamster V79 fibroblasts to 6 drugs that varied in their ability to produce DNA double-strand breaks stimulated formation of phosphorylated histone H2AX (serine 139 phosphorylated histone H2AX; gammaH2AX). Using flow cytometry to analyze gammaH2AX antibody-stained cells 1 h after a 30-min drug treatment, the fraction of cells that showed the control levels of gammaH2AX correlated well with the fraction of cells that survived to form colonies. This observation is now extended to V79 and SiHa human cervical carcinoma cells grown as multicell spheroids and SiHa xenografts and SCCVII tumors in mice. Animals were injected with etoposide, a topoisomerase-II inhibitor that targets proliferating cells or 3-amino-1,2,4-benzotriazine-1,3-dioxide (tirapazamine), a bioreductive cytotoxin that targets hypoxic cells. For spheroids, gammaH2AX intensity predicted clonogenic cell survival for cells recovered 90 min after drug injection, regardless of position of the cells within the spheroid. Similar results were obtained for etoposide in tumors; however, the gammaH2AX signal for tirapazamine was smaller than expected for the observed amount of cell killing. Frozen sections of tumors confirmed the greater intensity of gammaH2AX staining in cells close to blood vessels of tumors soon after treatment with etoposide and the opposite pattern for tumors exposed to tirapazamine. Analysis of cells or frozen sections from mouse spleen and kidney suggests that information can also be obtained on initial damage in normal tissues. These results support the possibility of using gammaH2AX antibody staining as a method to aid in prediction of tumor and normal tissue response to treatment.
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PMID:Phosphorylated histone H2AX in spheroids, tumors, and tissues of mice exposed to etoposide and 3-amino-1,2,4-benzotriazine-1,3-dioxide. 1528 43

Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.
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PMID:Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks. 1530 27

DNA damage by double-strand breaks induces arrest during interphase in mammalian cells. It is not clear whether DNA damage can arrest cells in mitosis. We show here that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin. Durable arrest at metaphase occurs, however, with ICRF-193, a topoisomerase II inhibitor that does not damage DNA. Arrest with ICRF-193 is not accompanied by recruitment of Mad2 or Bub1 to kinetochores, nor by phosphorylation of the histone H2AX, indicating arrest by ICRF-193 is not due to activation of the spindle assembly checkpoint, nor is it a response to DNA damage. VP-16, another decatenation inhibitor, induces metaphase arrest only at concentrations well above those that induce DNA damage. We conclude that decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.
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PMID:Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase. 1538 86

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser(343)), SMC1 (Ser(957)), Chk1 (Ser(317) and Ser(345)), and Chk2 (Ser(33/35) and Thr(68)). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.
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PMID:Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species. 1548 21

In this study, we examined alterations in the apoptotic response of tamoxifen (TAM)-resistant breast cancer cells. We used an in vitro selection approach for TAM resistance by means of long-term culture of MCF-7 breast cancer cells with increasing concentrations of TAM. The apoptotic response to TAM was determined by means of ELISA measurement of apoptotic DNA-histone complexes in cytoplasm and by Annexin-V staining. MCF-7(LT) cells isolated after 5 months of long-term treatment with TAM exhibited a significantly reduced apoptotic response to this drug, even if administered in high concentrations up to 20 microM. This reduced apoptotic response was also observed after treatment with the topoisomerase II inhibitor etoposide, a pro-apoptotic antineoplastic drug. Microarray experiments comparing the transcriptome of MCF-7(LT) and wild-type cells revealed both the down-regulated expression of several genes coding for pro-apoptotic proteins and the up-regulation of genes coding for apoptosis inhibitors. Further experiments to determine expression changes of the receptor tyrosine kinases HER2 and epidermal growth factor receptor did not reveal any alterations in MCF-7(LT) if compared to wild-type cells. Our findings suggest that long-term treatment with TAM in vitro does not necessarily change the expression of receptor tyrosine kinases, but can modulate the expression of apoptotic key genes impairing the apoptotic response of MCF-7 breast cancer cells.
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PMID:Tamoxifen long-term treatment in vitro alters the apoptotic response of MCF-7 breast cancer cells. 1549 41

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.
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PMID:On the nature and origin of DNA strand breaks in elongating spermatids. 1577 60


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