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1.
Acta Neuropathol ; 136(2): 211-226, 2018 08.
Article in English | MEDLINE | ID: mdl-29909548

ABSTRACT

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.


Subject(s)
Ependymoma/genetics , Gene Expression Regulation, Neoplastic/genetics , Infratentorial Neoplasms/genetics , Mutation/genetics , Oncogene Proteins/genetics , DNA Methylation , Ependymoma/classification , Ependymoma/pathology , Female , Gene Expression Profiling , HEK293 Cells , Histones/genetics , Humans , Infratentorial Neoplasms/classification , Infratentorial Neoplasms/pathology , Male , Transfection
2.
Acta Neuropathol ; 131(6): 833-45, 2016 06.
Article in English | MEDLINE | ID: mdl-26810070

ABSTRACT

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.


Subject(s)
Brain Neoplasms/pathology , Genes, myb , Genetic Predisposition to Disease , Glioma/genetics , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Adolescent , Adult , Astrocytoma/genetics , Astrocytoma/pathology , Brain Neoplasms/genetics , Child , Child, Preschool , DNA-Binding Proteins , Female , Ganglioglioma/genetics , Ganglioglioma/pathology , Glioma/pathology , Humans , Infant , Male , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Proto-Oncogene Proteins/genetics , RNA-Binding Proteins , Trans-Activators/genetics , Transcription Factors , Young Adult
3.
Cancer Cell ; 27(5): 728-43, 2015 May 11.
Article in English | MEDLINE | ID: mdl-25965575

ABSTRACT

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.


Subject(s)
Age Factors , Central Nervous System Neoplasms/pathology , Ependymoma/pathology , Adaptor Proteins, Signal Transducing/genetics , Adolescent , Adult , Aged , Central Nervous System Neoplasms/classification , Central Nervous System Neoplasms/genetics , Child , Child, Preschool , DNA Methylation , Ependymoma/classification , Ependymoma/genetics , Female , Gene Dosage , Gene Expression Profiling , Gene Fusion , Humans , Infant , Male , Middle Aged , Phosphoproteins/genetics , Transcription Factors , Transcription, Genetic , YAP-Signaling Proteins , Young Adult
4.
Nature ; 506(7489): 451-5, 2014 Feb 27.
Article in English | MEDLINE | ID: mdl-24553141

ABSTRACT

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.


Subject(s)
Cell Transformation, Neoplastic , Ependymoma/genetics , Ependymoma/metabolism , NF-kappa B/metabolism , Proteins/metabolism , Signal Transduction , Transcription Factor RelA/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Base Sequence , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line , Cell Nucleus/metabolism , Cell Transformation, Neoplastic/genetics , Chromosomes, Human, Pair 11/genetics , Ependymoma/pathology , Female , Humans , Mice , Models, Genetic , Molecular Sequence Data , NF-kappa B/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/pathology , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Proteins/genetics , Transcription Factor RelA/genetics , Transcription Factors , Translocation, Genetic/genetics , YAP-Signaling Proteins
5.
Acta Neuropathol Commun ; 2: 18, 2014 Feb 14.
Article in English | MEDLINE | ID: mdl-24529209

ABSTRACT

BACKGROUND: Gangliogliomas are low-grade glioneuronal tumors of the central nervous system and the commonest cause of chronic intractable epilepsy. Most gangliogliomas (>70%) arise in the temporal lobe, and infratentorial tumors account for less than 10%. Posterior fossa gangliogliomas can have the features of a classic supratentorial tumor or a pilocytic astrocytoma with focal gangliocytic differentiation, and this observation led to the hypothesis tested in this study - gangliogliomas of the posterior fossa and spinal cord consist of two morphologic types that can be distinguished by specific genetic alterations. RESULTS: Histological review of 27 pediatric gangliogliomas from the posterior fossa and spinal cord indicated that they could be readily placed into two groups: classic gangliogliomas (group I; n = 16) and tumors that appeared largely as a pilocytic astrocytoma, but with foci of gangliocytic differentiation (group II; n = 11). Detailed radiological review, which was blind to morphologic assignment, identified a triad of features, hemorrhage, midline location, and the presence of cysts or necrosis, that distinguished the two morphological groups with a sensitivity of 91% and specificity of 100%. Molecular genetic analysis revealed BRAF duplication and a KIAA1549-BRAF fusion gene in 82% of group II tumors, but in none of the group I tumors, and a BRAF:p.V600E mutation in 43% of group I tumors, but in none of the group II tumors. CONCLUSIONS: Our study provides support for a classification that would divide infratentorial gangliogliomas into two categories, (classic) gangliogliomas and pilocytic astrocytomas with gangliocytic differentiation, which have distinct morphological, radiological, and molecular characteristics.


Subject(s)
Ganglioglioma , Infratentorial Neoplasms , Proto-Oncogene Proteins B-raf/genetics , Recombinant Fusion Proteins/genetics , Spinal Cord Neoplasms , Adolescent , Child , Child, Preschool , Female , Ganglioglioma/classification , Ganglioglioma/genetics , Ganglioglioma/pathology , Genetic Testing , Humans , Infant , Infratentorial Neoplasms/classification , Infratentorial Neoplasms/genetics , Infratentorial Neoplasms/pathology , Male , Mutation/genetics , Spinal Cord Neoplasms/classification , Spinal Cord Neoplasms/genetics , Spinal Cord Neoplasms/pathology , Young Adult
6.
Nat Genet ; 45(6): 602-12, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23583981

ABSTRACT

The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.


Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Adolescent , Animals , Base Sequence , Brain Neoplasms/pathology , Child , Child, Preschool , Female , Gene Duplication , Gene Rearrangement , Genes, myb , Genome-Wide Association Study , Glioma/pathology , Humans , Infant , Male , Mice , Mice, Nude , Molecular Sequence Data , Mutation , Neoplasm Grading , Neoplasm Transplantation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf , Receptor, Fibroblast Growth Factor, Type 1/genetics , Sequence Analysis, DNA , Signal Transduction , Trans-Activators/genetics , Transcriptome
7.
Bioorg Med Chem ; 21(7): 1972-7, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23395113

ABSTRACT

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5 µM inhibited cellular DNA replication at 10 µM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.


Subject(s)
DNA Replication/drug effects , DNA/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Interaction Maps/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , DNA Damage/drug effects , HeLa Cells , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Structure-Activity Relationship
8.
J Biol Chem ; 287(17): 14289-300, 2012 Apr 20.
Article in English | MEDLINE | ID: mdl-22383522

ABSTRACT

We have discovered that 3,3',5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC(50) of ~1 µm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser(139))histone H2AX induction and cell growth inhibition was observed.


Subject(s)
DNA Replication/physiology , Proliferating Cell Nuclear Antigen/metabolism , Chemistry, Pharmaceutical/methods , Chromatin/metabolism , Crystallography, X-Ray/methods , Drug Design , Flow Cytometry/methods , Genes, Reporter , HeLa Cells , Humans , Inhibitory Concentration 50 , Ligands , Microscopy, Fluorescence/methods , Molecular Conformation , Peptides/chemistry , Phosphorylation , Protein Interaction Mapping/methods , Recombinant Proteins/chemistry
9.
Biochem J ; 435(2): 451-62, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21299497

ABSTRACT

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Drug Delivery Systems/methods , Macromolecular Substances/antagonists & inhibitors , Animals , Cells, Cultured , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Drug Discovery/methods , Drug Evaluation, Preclinical , High-Throughput Screening Assays/methods , Humans , Indoles/pharmacology , Macromolecular Substances/metabolism , Models, Biological , Phenylpropionates/pharmacology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Phosphoproteins/physiology , Protein Binding/drug effects , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/metabolism , Receptors, Lysophosphatidic Acid/physiology , Small Molecule Libraries/pharmacology , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/metabolism , Sodium-Hydrogen Exchangers/physiology , Swine
10.
Biopolymers ; 95(1): 24-30, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20872873

ABSTRACT

We have previously reported ketoprofen amide compounds as inhibitors of GLI1-mediated transcription, an essential down-stream element of the Hedgehog (Hh) pathway. These compounds inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with GLI1 and in Rh30 cells that endogenously overexpress GLI1. Here we have designed new derivatives of these compounds aiming to explore the structure-activation relationship (SAR). By replacing the ketone carbonyl group of the ketoprofen moiety with an ether, amide, sulfonamide, or sulfone, we found several new compounds that are equipotent to the ketoprofen amide compounds. Among them, sulfone 30 inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with GLI1 and in Rh30 cells that endogenously overexpress GLI1.


Subject(s)
Biopolymers/chemistry , Transcription Factors/antagonists & inhibitors , Biopolymers/pharmacology , Cells, Cultured , Drug Design , Molecular Structure , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity Relationship , Transcription Factors/chemistry , Transcription, Genetic , Zinc Finger Protein GLI1
11.
Bioorg Med Chem ; 18(13): 4801-11, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20605720

ABSTRACT

We have previously reported small-molecule inhibitors of Gli1-mediated transcription, an essential down-stream element of the Hh pathway. We created new derivatives of the previous compounds aiming to improve the druggable properties. The new compounds, amide conjugates of ketoprofen and indole, showed inhibitory activity and membrane permeability, while also improving the microsome stability. Among them, 33 and 42 inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with Gli1 with 2.6 µM and 1.6 µM of IC50, respectively, and in Rh30 cells that endogenously overexpress Gli1, and were selective to Gli1 over Gli2.


Subject(s)
Amides/chemistry , Hedgehog Proteins/metabolism , Indoles/chemistry , Indoles/chemical synthesis , Ketoprofen/chemistry , Transcription Factors/antagonists & inhibitors , Amides/chemical synthesis , Amides/pharmacology , Cell Line , Cell Membrane Permeability/drug effects , Genes, Reporter , Humans , Indoles/pharmacology , Kruppel-Like Transcription Factors/antagonists & inhibitors , Kruppel-Like Transcription Factors/metabolism , Microsomes/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2
12.
J Med Chem ; 52(14): 4277-87, 2009 Jul 23.
Article in English | MEDLINE | ID: mdl-19545120

ABSTRACT

We report novel inhibitors of Gli1-mediated transcription as potential anticancer agents. Focused chemical libraries were designed and assessed for inhibition of functional cell-based Gli1-mediated transcription and selective toxicity toward cancer cells. The SAR was revealed, and the selectivity of the lead compounds' inhibition of Gli1-mediated transcription over that of Gli2 was determined. Compound 63 (NMDA298-1), which inhibited Gli1-mediated transcription in C3H10T1/2 cells with an IC(50) of 6.9 muM, showed 3-fold selectivity for inhibiting transcription mediated by Gli1 over that by Gli2. Cell-viability assays were performed to evaluate the chemical library in a normal cell line and a panel of cancer cell lines with or without up-regulated expression of the Gli1 gene. These compounds decreased the viability of several cancer cell lines but were less active in the noncancerous BJ-hTERT cells.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Neoplasms/pathology , Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Genes, Reporter , Humans , Inhibitory Concentration 50 , Oncogene Proteins/genetics , Pyrazoles/chemistry , Structure-Activity Relationship , Trans-Activators/genetics , Tyramine/chemistry , Zinc Finger Protein GLI1
13.
Protein Sci ; 18(6): 1326-31, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19472416

ABSTRACT

In probing the mechanism of inhibition of hypoxia inducible factor (HIF-1) by campothecins, we investigated the ability of human topoisomerase I to bind and cleave HIF-1 response element (HRE), which contains the known camptothecin-mediated topoisomerase I cleavage site 5'-TG. We observed that the selection of 5'-TG by human topoisomerase I and topotecan depends to a large extent on the specific flanking sequences, and that the presence of a G at the -2 position (where cleavage occurs between -1 and +1) prevents the HRE site from being a preferred site for such cleavage. Furthermore, the presence of -2 T/A can induce the cleavage at a less preferred TC or TA site. However, in the absence of a more preferred site, the HRE site is shown to be cleaved by human topoisomerase I in the presence of topotecan. Thus, it is implied that the -2 base has a significant influence on the selection of the camptothecin-mediated Topo I cleavage site, which can overcome the preference for +1G. While the cleavage site recognition has been known to be based on the concerted effect of several bases spanning the cleavage site, such a determining effect of an individual base has not been previously recognized. A possible base-specific interaction between DNA and topoisomerase I may be responsible for this sequence selectivity.


Subject(s)
Camptothecin/pharmacology , DNA Cleavage , DNA Topoisomerases, Type I/metabolism , Enzyme Inhibitors/pharmacology , Base Sequence , Humans , Hypoxia-Inducible Factor 1/genetics , Oligonucleotides/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Response Elements , Topoisomerase I Inhibitors
14.
Protein Sci ; 18(5): 994-1002, 2009 May.
Article in English | MEDLINE | ID: mdl-19388021

ABSTRACT

Members of the Wnt family of lipoglycoproteins initiate signaling by binding to Frizzled (Fz) receptors, and the signal is then relayed by Disheveled (Dvl). The Dvl PDZ domain is known to interact directly with a peptide derived from the KTXXXW motif of Fz7, which is conserved in all known Fz subtypes. We found that an extended region spanning the KTXXXW motif on both its N-terminal and C-terminal sides dramatically influences the affinity of peptides derived from Fz7 for Dvl PDZ. An alanine scanning study identified the specific residues external to the KTXXXW motif that are important for high-affinity binding. In a circular dichroism analysis, mutation of some of these critical residues resulted in peptide conformational changes, suggesting that the secondary structure of the peptides contributes to Fz-Dvl PDZ binding. Of the 10 known Fz subtypes, peptides derived from only Fz1, Fz2, Fz3, Fz4, and Fz7 directly bound to Dvl PDZ domain in our study. Other Fz subtypes, including some known to be involved in Wnt/beta-catenin signaling (Fz5, Fz9), did not bind to Dvl, suggesting that direct interaction with Dvl PDZ does not determine the subtype-specific functionality of Fz. Molecular modeling and circular dichroism studies indicated that the Fz peptides that bind to Dvl PDZ domain form specific conformations that are different from those of nonbinding peptides.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Frizzled Receptors/chemistry , Phosphoproteins/chemistry , Wnt Proteins/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Models, Molecular , Molecular Sequence Data , Monte Carlo Method , PDZ Domains/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Thermodynamics , Wnt Proteins/genetics , Wnt Proteins/metabolism
16.
Mol Cancer Ther ; 7(6): 1633-8, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18566234

ABSTRACT

We describe the rational generation of small-molecule agents that suppress tumor cell growth by down-regulating canonical Wnt signaling. We first produced a chemical library of the derivatives of indole-2-ketones and carbinols; we then screened them by using scalable assays of biochemical antagonism of Dishevelled-1 PDZ domain interactions and cell-based assays of Dishevelled-1-driven T-cell factor-mediated transcription. Compounds showing parallel effects in these assays were tested for selective induction of apoptosis in cancer cells. A new compound (24) that met the criteria for high biochemical antagonism, T-cell factor-mediated transcription, and induction of tumor-selective apoptosis was found to significantly suppress the growth of tumor xenografts in mice.


Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Neoplasms/pathology , Phosphoproteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , TCF Transcription Factors/metabolism , Transcription, Genetic/drug effects , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 7/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Dishevelled Proteins , Enzyme Activation/drug effects , Female , Humans , Inhibitory Concentration 50 , Mice , Mice, Nude , Neoplasms/enzymology , Neoplasms/genetics , Small Molecule Libraries/analysis , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Xenograft Model Antitumor Assays
17.
Bioorg Med Chem Lett ; 18(3): 946-9, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-18180158

ABSTRACT

We designed and synthesized a series of indole-2-amide-based compounds that antagonize interaction between the Dishevelled (Dvl) PDZ domain and a peptide derived from the natural PDZ ligand Frizzled-7 (Fz7). These compounds inhibit Tcf-mediated transcription activated by exogenous Dvl via the biochemical antagonism. We confirmed tumor cell-selective activation of caspases by these compounds.


Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Amides/chemical synthesis , Amides/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Models, Molecular , Phosphoproteins/antagonists & inhibitors , TCF Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amides/chemistry , Apoptosis/drug effects , Combinatorial Chemistry Techniques , Dishevelled Proteins , Humans , Indoles/chemistry , Molecular Structure , PDZ Domains/drug effects , Phosphoproteins/metabolism , Structure-Activity Relationship , TCF Transcription Factors/drug effects , Transcription, Genetic/drug effects , Wnt Proteins/physiology
18.
Nucleic Acids Res ; 35(15): 4927-40, 2007.
Article in English | MEDLINE | ID: mdl-17626043

ABSTRACT

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K(+) solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K(+) solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3'-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.


Subject(s)
DNA/chemistry , Models, Molecular , Telomere/chemistry , Base Sequence , DNA Mutational Analysis , G-Quadruplexes , Guanine/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Potassium/chemistry , Protons , Solutions
19.
J Struct Biol ; 159(1): 111-21, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17434318

ABSTRACT

Human topoisomerase I (topo I) is an essential cellular enzyme that relaxes DNA supercoiling. The 6.3 kDa C-terminal domain of topo I contains the active site tyrosine (Tyr723) but lacks enzymatic activity by itself. Activity can be fully reconstituted when the C-terminal domain is associated with the 56 kDa core domain. Even though several crystal structures of topo I/DNA complexes are available, crystal structures of the free topo I protein or its individual domain fragments have been difficult to obtain. In this report we analyze the human topo I C-terminal domain structure using a variety of biophysical methods. Our results indicate that this fragment protein (topo6.3) appears to be in a molten globule state. It appears to have a native-like tertiary fold that contains a large population of alpha-helix secondary structure and extensive surface hydrophobic regions. Topo6.3 is known to be readily activated with the association of the topo I core domain, and the molten globule state of topo6.3 is likely to be an energy-favorable conformation for the free topo I C-terminal domain protein. The structural fluctuation and plasticity may represent an efficient mechanism in the topo I functional pathway, where the flexibility aids in the complementary association with the core domain and in the formation of a fully productive topo I complex.


Subject(s)
DNA Topoisomerases, Type I/chemistry , Tyrosine , Binding Sites , Humans , Hydrophobic and Hydrophilic Interactions , Peptide Fragments/chemistry , Pliability , Protein Structure, Tertiary
20.
Nucleic Acids Res ; 35(7): 2440-50, 2007.
Article in English | MEDLINE | ID: mdl-17395643

ABSTRACT

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K(+). The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel-antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5'- and 3'-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K(+). Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K(+). The unique human telomeric G-quadruplex structure formed in K(+) suggests that it can be specifically targeted for anticancer drug design.


Subject(s)
Adenine/chemistry , DNA/chemistry , Guanine/chemistry , Models, Molecular , Potassium/chemistry , Telomere/chemistry , G-Quadruplexes , Humans , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Solutions
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