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)

Using global gene expression analyses, multiple novel tumor markers overexpressed in infiltrating ductal adenocarcinomas of the pancreas have recently been identified. However, the expression of these markers in morphologically similar adenocarcinomas of the biliary tree has not been investigated. The purpose of the present study was 3-fold. First, we used 8 markers that have been shown to be overexpressed in whole tissue sections of pancreatic adenocarcinomas to validate tissue microarrays (TMAs) created from a series of pancreatic adenocarcinomas (n=68). The labeling patterns of 6 epithelial markers (fascin, mucin 4, 14-3-3sigma, prostate stem cell antigen, topoisomerase IIalpha, and cdc2/p34) were concordant with previously published studies on whole tissue sections, yet required far fewer slides and reagents. Mesothelin, an epithelial marker, and heat shock protein 47, a marker of peritumoral desmoplasia, showed lower levels of expression in the TMAs when compared with whole tissue sections. Second, we examined the previously unknown expression of the same 8 novel tumor proteins in cancers of the biliary tree by using TMAs created from a series of intrahepatic cholangiocarcinomas, gallbladder adenocarcinomas, and adenocarcinomas of the distal common bile duct (n=38). Each of the 8 markers was overexpressed in the biliary cancers, ranging from 14% demonstrating at least focal labeling with prostate stem cell antigen to 100% labeling with cdc2/p34. Most of the markers showed lower frequencies of expression in the biliary tract carcinomas in comparison to the pancreatic adenocarcinomas. In addition, expression patterns varied with location in the biliary system (intrahepatic versus gallbladder versus distal common bile duct). These differences were statistically significant (P<0.05) for mesothelin, mucin 4, and heat shock protein 47. Finally, the expression of selected markers in neoplastic progression of gallbladder cancer was examined. Two markers, fascin and mesothelin, showed up-regulation of expression with transition from carcinoma in situ to invasive adenocarcinoma, implicating a role for these markers in neoplastic progression. The results of this study indicate that TMA technology provides valid and cost-effective means to screen large numbers of novel tumor markers, even in tumors such as pancreatic and biliary adenocarcinomas that characteristically have abundant desmoplastic stroma. In addition, novel tumor markers of pancreatic adenocarcinomas show similar, yet not identical, expression patterns in biliary carcinomas. Therefore, these markers are potentially useful in developing diagnostic tests and treatment paradigms for tumors involving the biliary system.
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PMID:Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays. 1501 93

Doxorubicin (DOX) is a DNA topoisomerase II inhibitor widely used in anticancer treatment, however, it can lead to irreversible cardiac damage with severe debilitation. TBP-binding associated factor 1 (TAF1) is increased in DOX damaged hearts in vivo and in cardiomyocytes in vitro. To identify the functional role for TAF1 in DOX-treated heart we overexpressed wild type and mutant TAF1 in H9c2 cells. Overexpression of wild-type TAF1, but not N-terminal kinase domain mutants, increased tolerance to DOX in confluent cells. DOX treatment can cause prolonged G1 arrest. We found increased cdk2 activity coupled to increased cyclin E protein and decreased p21(waf1Cip1) and p27(Kip1) protein to correlate only with increased DOX tolerance and wild-type TAF1. DOX sensitivity was restored when the cdk2-inhibitor Roscovitine was co-administered with DOX. Overexpression of cdk2-alone increased resistance to DOX. Thus, TAF1 induced DOX tolerance in confluent cells through an increase in cdk2 activity is directed by the TAF1 N-terminal domain. These studies suggest new avenues for myocardial protection against DOX toxicity and suggest a role for cdk2 in chemorefractory cells.
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PMID:TBP-associated factor 1 overexpression induces tolerance to Doxorubicin in confluent H9c2 cells by an increase in cdk2 activity and cyclin E expression. 1512 10

Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.
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PMID:A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation. 1560 76

The infected-cell protein 22 (ICP22), a regulatory protein encoded by the alpha22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the U(S)11, U(L)38, and U(L)41 genes. ICP22 has two activities. Thus, ICP22 and the U(L)13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (U(L)42), bind topoisomerase IIalpha in an ICP22-dependent manner. In addition, ICP22 and U(L)13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral U(S)3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or deltaU(L)13 but not deltaU(S)3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the U(L)13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although U(S)3 kinase appears to play a role in a cell-type-dependent fashion.
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PMID:The carboxyl-terminal domain of RNA polymerase II is phosphorylated by a complex containing cdk9 and infected-cell protein 22 of herpes simplex virus 1. 1589 Sep 14

The vaccinia-related kinase (VRK) proteins are a new family with three members in the human kinome. The VRK1 protein phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. In normal squamous epithelium, VRK1 is expressed in the proliferation area. Because VRK1 can stabilize p53, the expression of the VRK1 protein was analyzed in the context of the p53 pathway and the proliferation phenotype in a series of 73 head and neck squamous cell carcinomas. VRK1 protein level positively correlated with p53 response proteins, particularly hdm2 and p21. The VRK1 protein also correlated positively with several proteins associated with proliferation, such as cyclin-dependent kinase 2 (CDK2), CDK6, cdc2, cyclins B1 and A, topoisomerase II, survivin, and Ki67. The level of VRK1 protein behaves like a proliferation marker in this series of head and neck squamous cell carcinomas. To identify a possible regulatory role for VRK1 and because it regulates gene transcription, the promoters of two genes were studied, CDK2 and SURVIVIN, whose proteins correlated positively with VRK1. VRK1 increases the activity of both the CDK2 and SURVIVIN gene promoters. The expression of VRK1 was analyzed in the context of regulators of the G1-S transition. VRK1 protein levels increase in response to E2F1 and are reduced by retinoblastoma and p16. These data suggest that VRK1 might play a role in cell cycle regulation and is likely to represent the beginning of a new control mechanism of cell cycle, particularly late in the G1-S phase.
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PMID:VRK1 signaling pathway in the context of the proliferation phenotype in head and neck squamous cell carcinoma. 1654 55

In addition to its function as a cyclin-dependent kinase (cdk) inhibitor, p21waf1 fulfills additional roles involved in DNA replication and transcriptional regulation that could also contribute to cell cycle arrest. In this study, we have shown that p21waf1 functions as a transcriptional repressor of the myc and cdc25A genes. Ectopic expression of the cell cycle inhibitor down-modulates myc and cdc25A transcription but has no effect on cdk4 levels. Using chromatin immunoprecipitation, we found that p21waf1 is recruited to the promoters of these two genes together with the STAT3 and E2F1 transcription factors. Its presence on DNA is associated with an inhibition of the recruitment of the p300 histone acetylase and with a down-regulation of histone H4 acetylation. The same effect was also observed following DNA damage because topoisomerase inhibitors such as sn38 or doxorubicin also induce the association of p21waf1 with DNA. Following transcriptional repression of the myc and cdc25A genes, cells were arrested in the fraction with 4 N DNA content. By contrast, the expression of these two genes remains elevated in the absence of the cell cycle inhibitor, and p21waf1-/- cells re-replicate their DNA and become polyploid. In light of these results, we propose that p21waf1 simultaneously targets cdk and transcriptional regulators to prevent the expression of oncogenic pathways upon DNA damage.
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PMID:The cell cycle inhibitor p21waf1 binds to the myc and cdc25A promoters upon DNA damage and induces transcriptional repression. 1692 15

The prolyl isomerase Pin1 plays important roles in numerous cellular processes. Here we provide evidence that Pin1 has an important function in chromosome condensation during mitosis. We first demonstrate that the interaction of Pin1 with chromatin is greatly elevated in G2/M phase and that this correlates with the presence on chromosomes of several mitotic phosphoproteins, especially topoisomerase (Topo) IIalpha. Inducible overexpression of Pin1 was shown to result in higher M phase-specific phosphorylation, while downregulation of Pin1 by siRNA treatment reduced phosphorylation of TopoIIalpha and other mitotic proteins. Furthermore, immunodepletion of Pin1 from mitotic cell extracts prevented such extracts from inducing chromosome condensation when added to S phase nuclei. Indeed, purified Pin1 and cdc2/cyclin B kinase were by themselves sufficient to induce condensation. This reflects the ability of Pin1 to increase TopoIIalpha phosphorylation by cdc2/cyclin B in vitro, which in turn dramatically increased formation of a TopoIIalpha/Pin1/DNA complex.
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PMID:The prolyl isomerase Pin1 functions in mitotic chromosome condensation. 1746 29

Earlier studies have shown that ICP22 and the U(L)13 protein kinase but not the U(S)3 kinase are required for optimal expression of a subset of late (gamma(2)) genes exemplified by U(L)38, U(L)41, and U(S)11. In primate cells, ICP22 mediates the disappearance of inactive isoforms of cdc2 and degradation of cyclins A and B1. Active cdc2 acquires a new partner, the viral DNA synthesis processivity factor U(L)42. The cdc2-U(L)42 complex recruits and phosphorylates topoisomerase IIalpha for efficient expression of the gamma(2) genes listed above. In uninfected cells, the cdc25C phosphatase activates cdc2 by removing two inhibitory phosphates. The accompanying report shows that in the absence of cdc25C, the rate of degradation of cyclin B1 is similar to that occurring in infected wild-type mouse embryo fibroblast cells but the levels of cdc2 increase, and the accumulation of a subset of late proteins and virus yields are reduced. This report links ICP22 with cdc25C. We show that in infected cells, ICP22 and U(S)3 protein kinase mediate the phosphorylation of cdc25C at its C-terminal domain. In in vitro assays with purified components, both U(L)13 and U(S)3 viral kinases phosphorylate cdc25C and ICP22. cdc25C also interacts with cdc2. However, in infected cells, the ability of cdc25C to activate cdc2 by dephosphorylation of the inactive cdc2 protein is reduced. Coupled with the phosphorylation of cdc25C by the U(S)3 kinase, the results raise the possibility that herpes simplex virus 1 diverts cdc25C to perform functions other than those performed in uninfected cells.
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PMID:The interaction of herpes simplex virus 1 regulatory protein ICP22 with the cdc25C phosphatase is enabled in vitro by viral protein kinases US3 and UL13. 1827 72

Earlier studies have shown that in herpes simplex virus 1-infected cells, ICP22 upregulates the accumulation of a subset of gamma(2) proteins exemplified by the products of the U(L)38, U(L)41, and U(S)11 genes. The ICP22-dependent process involves degradation of cyclins A and B1, the stabilization and activation of cdc2, physical interaction of activated cdc2 with the U(L)42 DNA synthesis processivity factor, and recruitment and phosphorylation of topoisomerase IIalpha by the cdc2/U(L)42 complex. Activation of cdc2, the first step in the process, is a key function of the mitotic phosphatase cdc25C. To define the role of cdc25C, we probed some features of the ICP22-dependent pathway of upregulation of gamma(2) genes in cdc25C(-/-) cells and in cdc25C(+/+) cells derived from sibling mice. We report that cyclin B1 turned over in cdc25C(+/+) or cdc25C(-/-) cells at the same rate, that cdc2 increased in amount, and that U(S)11 and U(L)38 proteins and infectious virus accumulated in smaller amounts than in wild-type infected cells. The reduction in U(L)38 protein accumulation and virus was greater in cdc25C(-/-) cells infected with virus lacking ICP22 than in cells infected with wild-type virus. We conclude that cdc25C phosphatase plays a role in viral replication and that this role extends beyond its function of activating cdc2 for initiation of the ICP22-dependent cascade for upregulation of gamma(2) gene expression.
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PMID:Role of cellular phosphatase cdc25C in herpes simplex virus 1 replication. 1827 75

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-gamma(35)S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50 = 0.15 microM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90alpha inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.
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PMID:Binding of protein kinase inhibitors to synapsin I inferred from pair-wise binding site similarity measurements. 2080 48


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