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)

Melanoma has proven to be resistant to conventional chemotherapy; however,the mechanism of chemoresistance is still unclear. Recent reports show that the transcription factor, E2F-1, may play a role in mediating cytotoxicity of certain chemotherapeutic agents. We have shown in a previous study that adenovirus-mediated overexpression of E2F-1 can efficiently induce apoptosis in melanoma cells. In the present study, the effect of E2F-1 expression on drug sensitivity of melanoma cells was evaluated. Two human melanoma cell lines, SK-MEL-28 and SK-MEL-2, were treated with drugs (etoposide, Adriamycin, roscovitine, cisplatin, 5-fluorouracil, or cycloheximide), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1) at a multiplicity of infection of 1 in vitro. E2F-1 expression was confirmed by Western blot analysis. Sublethal concentrations of each drug alone or infection with Ad-E2F-1 alone produced <5% apoptosis by 3 days posttreatment. Conversely, cotreatment with Ad-E2F-1 and low concentrations of etoposide or Adriamycin markedly sensitized melanoma cells to apoptotic cell death. A slight enhancement of the cytotoxicity of roscovitine was demonstrated in combination with E2F-1 overexpression, but not to cisplatin, 5-fluorouracil, or cycloheximide. Ad-LacZ infection showed no obvious effects on drug sensitivity. Overexpression of p21 can block apoptosis induced by the combination chemogene therapy of Ad-E2F-1 and topoisomerase II poisons and does not require its proliferating cell nuclear antigen-binding ability. The protein synthesis inhibitor cycloheximide also has a cytotoxicity-protective effect against topoisomerase II inhibitor/E2F-1-induced apoptosis and suggests that new protein synthesis is required for this process. Topoisomerase II inhibitors also cooperated with Ad-E2F-1 to enhance antitumor activity in an in vivo model using xenografts in nude mice. When combined with Adriamycin or etoposide, E2F-1 adenovirus therapy resulted in an 87% or 91% decrease in tumor size, respectively, compared with controls (P < 0.002). Our results show that adenovirus-mediated E2F-1 gene transfer can sensitize melanoma cells to some chemotherapeutic agents, particularly topoisomerase II poisons, in vitro and in vivo. These results suggest a new chemosensitization strategy for melanoma gene therapy.
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PMID:Adenovirus-mediated E2F-1 gene transfer sensitizes melanoma cells to apoptosis induced by topoisomerase II inhibitors. 1191 54

Hybridization with cDNA arrays was used to obtain expression profiles of 214 protein-tyrosine kinase, protein-tyrosine phosphatase, dual-specific phosphatase, and other genes for kidney carcinomas (KC) and normal kidney tissues of 34 patients and for seven carcinoma cell lines. Computer analysis revealed three clusters of genes coexpressed in KC. A proliferating-cell gene cluster included MET, VIM, MYC, TOP2A, PCNA, etc. A neoangiogenesis and blood-cell gene cluster included LCK, HCK, FGR, MMP9, CSFR1, VEGF, FLT1, and KDR. A cluster corresponding to normal, differentiated kidney cells included ERBB2 (HER2) for receptor protein-tyrosine kinase, several phosphatase genes (PTPRE, PTPRB, DUSP9), and EGF. The results suggested that MET, DUSP9, PCNA, TOP2A, and VIM may serve as diagnostic and prognostic markers in KC. Tubulin and topoisomerase II were assumed to be promising targets for cell proliferation inhibitors in KC.
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PMID:[Molecular portrait of human kidney carcinomas: the gene expression profiling of protein-tyrosine kinases and tyrosine phosphatases which controlled regulatory signals in the cells]. 1206 34

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.
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PMID:Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication. 1239 88

The expression of mitosin, a novel proliferation-associated molecule was evaluated immunohistochemically in a consecutive series of 47 patients with primary intracranial benign and atypical meningiomas. Mitosin expression was correlated with proliferation markers Ki-67 (MIB-1), proliferating cell nuclear antigen (PCNA), topoisomerase IIalpha (TopoIIalpha) and mitotic index, as well as with standard clinicopathological parameters and patient outcome. Seven tumors recurred (14.8%) following gross total resection, within a follow-up period ranging from 21 to 108 months (median 60 months). The higher proliferation indices were obtained with mitosin and PCNA and the lower ones with TopoIIalpha. Mitosin labeling index (LI) ranged from 0.1 to 57% (median 3%), with a significant overlapping of values between grades. A significant positive correlation was shown between mitosin LI on the one hand and Ki-67 LI (p < 0.001), or the mitotic index (p = 0.027) on the other. The incidence of recurrence was higher in cases with a mitosin LI higher than 3% (p = 0.048). Univariate analysis disclosed mitosin LI (p = 0.033) along with the mitotic index (p = 0.024) and tumor size (p = 0.028) as significant predictors of shortened recurrence-free survival. In multivariate analysis, the labeling indices of mitosin (p = 0.035) and Ki-67 (p = 0.032), along with tumor size, were shown to provide independent prognostic information, beyond that obtained by standard clinical and pathological parameters. However, as indicated by factor analysis, the prognostic information yielded by mitosin was superior to that provided by the remaining proliferation markers (p = 0.041). We conclude that mitosin immunohistochemical expression, although failing to discriminate between benign and atypical meningiomas, may be of use as a novel cell proliferation marker and as a predictor of tumor recurrence.
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PMID:Mitosin, a novel marker of cell proliferation and early recurrence in intracranial meningiomas. 1250 85

Proliferation markers are widely used in general surgical pathology and also in pituitary pathology. They should help for differing aggressive or rapidly growing tumors from those with slower growth, as cellular atypia is not helpful for identifying aggressive adenomas of the pituitary. Only the number of mitoses is important for prognosis. A lot of markers can be used: antibodies for cyclins A, B, D and E, for proliferating cell nuclear antigen, Ki-67/Mib-1, antibodies for the inhibitory proteins p16, p27, p53, and for DNA topoisomerase IIalpha. A marker for apoptosis and its inhibitors may be also important. From our experiences, Mib-1 is the most reliable marker. The recommendation of this marker in the WHO classification of pituitary adenomas is fully justified.
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PMID:Proliferation markers and cell cycle inhibitors in pituitary adenomas. 1528 42

DNA topoisomerase inhibitors induce a specific signaling cascade that promotes an active apoptotic caspase-dependent cell death process. However, little is known about the initial signals elicited by these agents. In the present study, we compared apoptosis in HL-60 cells treated either with the chemotherapeutic drug etoposide (VP16) alone or combined with the broad caspase inhibitor ZVAD.fmk. Apoptosis was assessed by changes in cell morphology and agarose gel electrophoresis of extracted cell DNA. We found that ZVAD.fmk prevents VP16-induced DNA fragmentation and the appearance of an increased number of apoptotic cells in the culture. We also compared the effects of etoposide alone or together with the pan-caspase inhibitor ZVAD.fmk on proliferating cell nuclear antigen, Bcl-2, and actin expression in human promyelocytic leukemia HL-60 cells. In addition, we screened for proteins that were initially upregulated in a caspase-dependent manner. Indeed, some proteins were induced in the cytoplasm and subsequently accumulated in the nuclei after etoposide treatment. This process was slightly inhibited by the caspase inhibitor ZVAD.fmk. We suggest that these proteins are associated with the induction of specific signaling cascades that characterize the apoptotic cell death process.
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PMID:Alterations in protein expression in HL-60 cells during etoposide-induced apoptosis modulated by the caspase inhibitor ZVAD.fmk. 1565 22

Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288) is a candidate for possible advancement to phase I clinical trial. Aminoflavone has a unique activity profile in the NCI 60 cell lines (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/dtp_search.html), and exhibits potent cellular and animal antitumor activity. To elucidate the mechanism of action of aminoflavone, we studied DNA damage in MCF-7 cells. Aminoflavone induced DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB). Aminoflavone induced high levels of DPC and much lower level of SSB than camptothecin, which induces equal levels of DPC and SSB due to the trapping topoisomerase I-DNA complexes. Accordingly, neither topoisomerase I nor topoisomerase II were detectable in the aminoflavone-induced DPC. Aminoflavone also induced dose- and time-dependent histone H2AX phosphorylation (gamma-H2AX). Gamma-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented gamma-H2AX formation, suggesting that gamma-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no gamma-H2AX foci were found in proliferating cell nuclear antigen-negative or in mitotic cells. Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analyses showed DNA synthesis inhibition uniformly throughout the S phase after exposure to aminoflavone. Aminoflavone also induced RPA2 and p53 phosphorylation, and induced p21(Waf1/Cip1) and MDM2, demonstrating S-phase checkpoint activation. These studies suggest that aminoflavone produces replication-dependent DNA lesions and S-phase checkpoint activation following DPC formation. Gamma-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies.
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PMID:DNA-protein cross-links and replication-dependent histone H2AX phosphorylation induced by aminoflavone (NSC 686288), a novel anticancer agent active against human breast cancer cells. 1595 81

In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended single-stranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposide-induced inhibition of DNA replication in early S phase.
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PMID:The dispersal of replication proteins after Etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway. 1645 27

Caspase-3 is the ultimate executioner caspase that is essential for the nuclear changes associated with apoptosis. We investigated caspase-3 immunohistochemical expression in 58 primary intracranial meningiomas, using one monoclonal antibody detecting both precursor and cleaved caspase-3 (CPP32) and a second recognizing only the cleaved activated form (ASP175). Caspase-3 expression was analyzed in relation to baseline apoptosis-as illustrated by the expression of anti-single stranded DNA (ss-DNA), the antiapoptotic protein bcl-2, proliferation indices (Ki-67, PCNA, topoisomerase IIa, mitosin C), hormonal status (estrogen, progesterone, androgen receptors), standard clinicopathological parameters and patients' disease-free survival. Caspase-3 immunostaining was observed in 62% of cases for CPP32 and in 24% for ASP175. In both instances, the labeling index (LI) was significantly correlated with ss-DNA LI (p=0.038 and p=0.018). CPP32 but not ASP175 LI positively correlated with the mitotic index (p=0.001) and PCNA LI (p=0.004). Both CPP32 and ASP175 LIs were increased in nonbenign meningiomas (p<0.0001 and p=0.0035 respectively). In univariate and multivariate survival analyses, caspase-3 predicted meningioma recurrence, independently affecting disease-free survival (p=0.011 and p=0.047 respectively for CPP32; p<0.0001 and p=0.012 respectively for ASP175). Caspase-3 may prove to be a useful predictor of early recurrence in a group of neoplasms characterized by the frequent discordance between histology and clinical behavior.
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PMID:Caspase-3 immunohistochemical expression is a marker of apoptosis, increased grade and early recurrence in intracranial meningiomas. 1714 87

Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18-/-) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18-/- cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18-/- cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18-/- cells was also demonstrated by alkaline comet assay.
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PMID:Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination. 1715 48


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