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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)

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.
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PMID:Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair. 950 Apr 59

The metabolic fate of covalently linked DNA-protein complexes (cross-links) is not clearly understood. Our aim was to investigate the processing of protein-DNA cross-links by cellular enzymes. As an example of a DNA-protein cross-link, we have constructed frozen topoisomerase-DNA conjugates and investigated their processing by human cell-free extracts. A suicide DNA substrate was constructed that upon reaction with vaccinia type I topoisomerase yielded a highly stable covalent DNA-protein cross-link. When this conjugate was treated with human nuclear or whole cell extracts, two sites of DNA breakpoints were detected: one set of double-stranded breaks occurred close to the 3' side of the topoisomerase (topo) conjugation site, and there was another set of nicks about 30 nucleotides 3' to the topo site. The double-stranded breaks were not made by extracts from xeroderma pigmentosum group A mutant cells, suggesting that the xeroderma pigmentosum group A damage recognition protein may be required for the occurrence of DNA breakage. In addition to these DNA breakage reactions, there was an activity that resulted in the delinking of the frozen topoisomerase (or proteolytic fragments thereof) from the DNA substrate, which was followed by a ligation step that restored the continuity of the broken DNA strand at the erstwhile topo attachment site. We suggest that frozen topoisomerase-DNA conjugates (and perhaps other types of covalent DNA-protein complexes) are processed by multiple pathways that may involve the cleavage of the DNA in the covalent protein-DNA complex and/or enzymatic delinking followed by ligation of the broken DNA ends. These processes may represent the "repair" of DNA-protein cross-links.
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PMID:Mechanisms for the processing of a frozen topoisomerase-DNA conjugate by human cell-free extracts. 954 38

DNA topoisomerase IIalpha was monitored with the monoclonal antibody Ki-S1 in human fibroblasts after irradiation of cells with gamma rays from a 137Cs source or treatment with the DNA topoisomerase II inhibitor doxorubicin. DNA topoisomerase IIalpha was localized immunohistochemically as bright fluorescent dots in the karyoplasm. The fibroblasts investigated originated from normal human donors and a xeroderma pigmentosum (XP) patient (XP12BE). All cell lines examined showed a time- and dose-dependent increase in DNA topoisomerase IIalpha abundance after irradiation or treatment with doxorubicin. No principal difference in response was seen between normal and XP fibroblasts towards either treatment alone. After irradiation with 9 Gy, the effect was detectable after as little as 30 min and lasted for at least 6 h. After doxorubicin treatment, topoisomerase II overexpression occurred within less than 2 h. It passed through a maximum and began to decrease after approximately 6 h. In principle, the increase in DNA topoisomerase IIalpha may result from (i) architectural changes of interphase chromatin leading to enhanced accessibility of preformed enzyme to the antibody, (ii) enhanced gene expression, or (iii) enhanced stabilization of mRNA or protein molecules. The increase in enzyme levels may be part of the well-known DNA damage responses that operate in cell-protective or DNA-reparative pathways. Thus, the action of DNA topoisomerase II would serve to catalyze preparatory steps in DNA repair. We also found overexpression of the Bax protein and p16 predominantly in treated XP cells, suggesting that the DNA-damaging protocols elicited signals for apoptosis and cell-cycle arrest. From the simultaneous increase in DNA topoisomerase IIalpha and Bax, one may conclude that DNA topoisomerase IIalpha also plays role in apoptosis.
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PMID:Doxorubicin and gamma rays increase the level of DNA topoisomerase IIalpha in nuclei of normal and xeroderma pigmentosum fibroblasts. 971 98

Immunohistochemical methods were used to determine abundance and subnuclear distribution of DNA topoisomerase I and the Bax protein in normal and excision-repair-deficient xeroderma pigmentosum (XP) fibroblasts after irradiation of cells with gamma rays or UV light, or exposure to the topoisomerase I inhibitor topotecan. DNA topoisomerase I and Bax were monitored using antisera raised against the human proteins. In addition, topoisomerases IIalpha and IIbeta were made visible with specific antibodies. In untreated cells, DNA topoisomerase I was found to occur in the cytoplasm and in nucleoli. Irradiation with gamma rays (2-12 Gy) or UV light (0.3-1.2 mW/cm2) changed the staining pattern in nuclei such that a multitude of small topoisomerase-I-rich centers occurred, which were evenly distributed over the karyoplasm. Simultaneously nucleoli disintegrated. Treatment of fibroblasts with topotecan (6-100 microM concentrations) resulted in similar alterations although the changes were much more pronounced. Combinations of topotecan and gamma irradiation caused additive effects. We conclude that the increase in the number of topoisomerase-I-positive spots and the high fluorescence intensity of the latter may reflect three biological processes: (i) enhanced transcriptional activity (e.g. of DNA damage response genes), (ii) tagging of damaged DNA sites for repair, or (iii) initiation of apoptosis. In separate assays using normal and XP cells, a dose-dependent increase in protein reacting with Bax antibody was observed in nuclei, following treatment with gamma rays or topotecan. In addition, topotecan induced a netlike arrangement of this Bax protein in nuclei. The meshes of the net structure resembled vesicles. DNA staining with 4',6-diamidino-2-phenylindole dihydrochloride revealed that the vesicle-type structures contained DNA. Upon further incubation with topotecan, cells showing the netlike Bax arrangement eventually died. We conclude that topotecan-induced changes made visible by nuclear Bax protein are associated with apoptosis. XP cells, when treated with topotecan, responded more readily than normal cells with both an increase in nuclear Bax protein and rearrangement of Bax, indicating that UV repair functions may be required to process DNA damage inflicted by topotecan. Monitoring of DNA topoisomerases IIalpha and IIbeta in gamma-irradiated cells with antibodies revealed a dramatic increase in the IIalpha form and a redistribution of the IIbeta form representing fragmentation of nucleoli.
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PMID:Subnuclear distribution of DNA topoisomerase I and Bax protein in normal and xeroderma pigmentosum fibroblasts after irradiation with UV light and gamma rays or treatment with topotecan. 1023 74

The tumor suppressor gene product p53 can bind to and inhibit the helicase activity of the multisubunit transcription-repair factor TFIIH. We previously reported that p53-mediated apoptosis is attenuated in primary human fibroblasts from individuals with Xeroderma Pigmentosum (XP) that harbor mutations in the TFIIH DNA helicases XPD or XPB. In this study we show that apoptosis is reduced and delayed in three XPD lymphoblastoid cell lines (LCLs), but not in an XPD heterozygote LCL, after exposure to doxorubicin, a DNA-damaging agent and topoisomerase II inhibitor frequently used in cancer therapy. Apoptosis was assessed by quantitation of Annexin V binding to exposed phosphatidylserine residues and by caspase-mediated cleavage of Poly(ADP)Ribose Polymerase (PARP). Apoptosis induced by doxorubicin was suppressed in LCLs retrovirally transduced with the Human Papillomavirus 16 E6 oncoprotein, consistent with the hypothesis that this is a p53-dependent process. PARP cleavage was not delayed in XPD LCLs in response to anti-Fas (CD95) antibody-mediated apoptosis, thus, the defect in the apoptotic pathway in these cells lies upstream of caspase activation. Similar changes in the expression of apoptosis-effector genes, p53, and p53-responsive genes p21Cip1/WAF-1/Sid1 (p21), gadd45, bcl-2 and bax were observed in normal and XPD LCLs after treatment with doxorubicin, indicating that delayed apoptosis was not a consequence of defective transcription of these genes. Thus, our studies provide further support to the hypothesis that XPD and p53 can functionally interact in a p53-mediated apoptotic pathway.
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PMID:Drug-induced apoptosis is delayed and reduced in XPD lymphoblastoid cell lines: possible role of TFIIH in p53-mediated apoptotic cell death. 1046 15

Cisplatin or carboplatin is commonly used with gemcitabine, docetaxel, paclitaxel or vinorelbine as chemotherapy doublets in the treatment of advanced non-small cell lung cancer. Several randomized trials have failed to identify major differences in survival between any of these doublets. This lack of evidence for improvement in survival with any chemotherapy regimen has created a tabula rasa in which no more large randomized trials should be conducted with out including a genetic analysis. Patients see survival as their major concern, and other considerations, such as cost of treatment and qualify of life, are relegated to lower positions. Genetic alterations related to the transcription-coupled repair pathway of the nucleotide excision repair system (TC-NER) have revealed the subset of patients who are resistant to cisplatin. TC-NER involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. For a long time, ERCC1 mRNA levels have been known to correlate with DNA repair capacity in various tissues. Levels of DNA cisplatin adducts in peripheral blood and buccal mucosa cells predict chemotherapy response, and high ERCC1 mRNA levels have been related to chemoresistance in ovarian cancer and in malignant lymphocytes from chronic lymphocytic leukemia. Moreover , in some instances, mRNA expression has been correlated with polymorphisms. Overexpression of ERCC1 correlates with poor survival gemcitabine/cisplatin-treated non-small cell lung cancer patients. An ongoing customized ERCC1-based chemotherapy trial has been established on this knowledge. Patients are randomized to the control arm of cisplatin/docetaxel is combined with cisplatin or gemcitabine according to ERCC1 levels. To date, 80 patients have been included. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. Some XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to disease progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; p = 0.03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biological redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings indicate that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival. This highlights the possibilities of individually tailored chemotherapy. However, in patients treated with cisplatin/vinorelbine, the opposite effect has been observed. Patients with Lys751Lys had a longer time to progression. When docetaxel was added to gemcitabine/cisplatin, patients with Lys751Lys also had better survival. Our findings indicate that TC-NER status can help to decide between cisplatin/gemcitabine and docetaxel/ cisplatin. TC-NER-dependent activity is similar to other anticancer agents that cause DNA-binding enzymes to kill cells (topoisomerase inhibitors). At least 50% of non-small cell lung cancer patients harbor Lys751Lys and can benefit from docetaxel/ cisplatin treatment. Genes involved in spindle formation, centrosome functions and mRNA transport along the microtubule tracks should provide further information on potential markers of docetaxel resistance.
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PMID:Influence of genetic markers on survival in non-small cell lung cancer. 1466 33

Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.
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PMID:FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks. 2658 Dec 12

Etoposide (VP16) is a topoisomerase II inhibitor and has been used for the treatment of non-small cell lung cancer (NSCLC). Xeroderma pigmentosum complementation group C (XPC) protein is a DNA damage recognition factor in nucleotide excision repair and involved in regulating NSCLC cell proliferation and viability. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) enhanced etoposide-induced cytotoxicity in NSCLC cells through modulating the XPC expression. We found that etoposide increased XPC expression in an AKT activation manner in 2 squamous cell carcinoma H1703 and H520 cells. Knockdown of XPC using siRNA or inactivation of AKT by pharmacological inhibitor PI3K inhibitor (LY294002) enhanced the cytotoxic effects of etoposide. In contrast, enforced expression of XPC cDNA or AKT-CA (a constitutively active form of AKT) reduced the cytotoxicity and cell growth inhibition of etoposide. Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of etoposide in NSCLC cells, which were associated with the downregulation of XPC expression and inactivation of AKT. Our findings suggested that the Hsp90 inhibition induced XPC downregulation involved in enhancing the etoposide-induced cytotoxicity in H1703 and H520 cells.
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PMID:17-(Allylamino)-17-Demethoxygeldanamycin Enhances Etoposide-Induced Cytotoxicity via the Downregulation of Xeroderma Pigmentosum Complementation Group C Expression in Human Lung Squamous Cell Carcinoma Cells. 2995 87


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