14 research outputs found
Synthesis of Conformationally North-Locked Pyrimidine Nucleosides Built on an Oxabicyclo[3.1.0]hexane Scaffold
Beginning with a known 3-oxabicyclo[3.1.0]Âhexane scaffold
(I),
the relocation of the fused cyclopropane ring bond and the shifting
of the oxygen atom to an alternative location engendered a new 2-oxabicyclo[3.1.0]Âhexane
template (II) that mimics more closely the tetrahydrofuran ring of
conventional nucleosides. The synthesis of this new class of locked
nucleosides involved a novel approach that required the isocyanate
II (B = NCO) with a hydroxyl-protected scaffold as a pivotal intermediate
that was obtained in 11 steps from a known dihydrofuran precursor.
The completion of the nucleobases was successfully achieved by quenching
the isocyanate with the lithium salts of the corresponding acrylic
amides that led to the uracil and thymidine precursors in a single
step. Ring closure of these intermediates led to the target, locked
nucleosides. The anti-HIV activity of <b>29</b> (uridine analogue), <b>31</b> (thymidine analogue), and <b>34</b> (cytidine analogue)
was explored in human osteosarcoma (HOS) cells or modified HOS cells
(HOS-313) expressing the herpes simplex virus 1 thymidine kinase (HSV-1
TK). Only the cytidine analogue showed moderate activity in HOS-313
cells, which means that the compounds are not good substrates for
the cellular kinases
NMR spectra of the imino H region of the -MCT modified oligos at various temperatures
<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p> The assignments are as follows: filled diamond T8, filled square T7, filled triangle G2, filled circle G4, star G10 and double dagger, G12
1D NMR spectra of () T7 and () T7T8 imino regions at 200 μM strand concentration
<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p> Additional peaks that were not observed in 1–2 mM solutions are indicated with arrows
Changes in the chemical shift of the imino protons between the -MCT- and -MCT- modified ODN species and the native DD at 5°C () and at 25°C ()
<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p
Changes in the chemical shift of select non-exchangeable protons between the -MCT- () and -MCT- () modified ODN species and the native DD at 25°C
<p><b>Copyright information:</b></p><p>Taken from "Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′- (north) and 3′- (south) ‘locked’ templates"</p><p>Nucleic Acids Research 2007;35(6):1978-1991.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1895885.</p><p>© 2007 The Author(s)</p
A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota
Y-family DNA polymerases participate in replication stress
and
DNA damage tolerance mechanisms. The properties that allow these enzymes
to copy past bulky adducts or distorted template DNA can result in
a greater propensity for them to make mistakes. Of the four human
Y-family members, human DNA polymerase iota (hpol ι)
is the most error-prone. In the current study, we elucidate the molecular
basis for improving the fidelity of hpol ι through use
of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine
triphosphate (N-MC-dATP). Three crystal structures were solved of
hpol ι in complex with DNA containing a template 2′-deoxythymidine
(dT) paired with an incoming dNTP or modified nucleotide triphosphate.
The ternary complex of hpol ι inserting N-MC-dATP opposite
dT reveals that the adenine ring is stabilized in the <i>anti</i> orientation about the pseudo-glycosyl torsion angle, which mimics
precisely the mutagenic arrangement of dGTP:dT normally preferred
by hpol ι. The stabilized <i>anti</i> conformation
occurs without notable contacts from the protein but likely results
from constraints imposed by the bicyclo[3.1.0]Âhexane scaffold of the
modified nucleotide. Unmodified dATP and South-MC-dATP each adopt <i>syn</i> glycosyl orientations to form Hoogsteen base pairs with
dT. The Hoogsteen orientation exhibits weaker base-stacking interactions
and is less catalytically favorable than <i>anti</i> N-MC-dATP.
Thus, N-MC-dATP corrects the error-prone nature of hpol ι
by preventing the Hoogsteen base-pairing mode normally observed for
hpol ι-catalyzed insertion of dATP opposite dT. These
results provide a previously unrecognized means of altering the efficiency
and the fidelity of a human translesion DNA polymerase
Promotion of Glioblastoma Cell Motility by Enhancer of Zeste Homolog 2 (EZH2) Is Mediated by AXL Receptor Kinase
<div><p>Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that epigenetically silences gene transcription through histone H3 lysine trimethylation (H3K27me3). EZH2 has been implicated in stem cell maintenance and is overexpressed in hematological and solid malignancie`s including malignant glioma. EZH2 is thought to promote tumor progression by silencing tumor suppressor genes. Hence pharmacological disruption of the PRC2 is an attractive therapeutic strategy for cancer treatment. Here we show that EZH2 is expressed in human glioma and correlates with malignancy. Silencing of EZH2 reduced glioma cell proliferation and invasiveness. While we did not observe induction of cell cycle-associated tumor suppressor genes by silencing or pharmacological inhibition of EZH2, microarray analyses demonstrated a strong transcriptional reduction of the AXL receptor kinase. Neither histone nor DNA methylation appeared to be involved in the positive regulation of AXL by EZH2. Silencing AXL mimicked the antiinvasive effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas with high EZH2 expression. Collectively these data suggest that EZH2 drives glioma invasiveness via transcriptional control of AXL independent of histone or DNA methylation.</p> </div
Methylation independent regulation of AXL.
<p><b>A </b><i>AXL</i> mRNA expression of U87MG human glioma cells treated with 5 µM DZNep for 120 h in comparison to control. <b>B</b> Analysis of AXL protein expression in U87MG glioma cells untreated (solid line) or treated with 5 µM DZNep for 120 h (dashed line) stained with an AXL specific antibody (blue) or an isotype control antibody (red). <b>C</b> Comparison of genes with decreased (upper Venn-diagram) or increased (lower Venn diagram) expression after 120 h of <i>EZH2</i> knockdown (purple) or DZNep treatment (blue) of U87MG glioma cells. Cutoff: 1.5 fold change. <b>D </b><i>AXL</i> mRNA expression of U87MG human malignant glioma cells after 96 h treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza) (black bars) or DMSO (white bar). <b>E </b><i>EZH2</i> and <i>AXL</i> mRNA expression of U87MG glioma cells after the stimulation with indicated concentrations of the histone deacetylase inhibitors suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA) (black bars) or DMSO (white bars) for 24 h. Asterisk indicates * (p<0.05). Error bars indicate s.e.m.</p
EZH2-knockdown inhibits proliferation and invasion of human malignant glioma cells.
<p><b>A </b><i>EZH2</i> transcript expression was decreased 24 h after si<i>EZH2</i> treatment (left). Western blot showing EZH2 protein expression, 120 h after knockdown by siRNA (right). Tubulin served as loading control. <b>B</b> Cell cycle analysis of U87MG glioma cells 120 h after specific knockdown of <i>EZH2</i> (si<i>EZH2</i>, right) or scrambled control (siC, left). <b>C</b> Invasion of U87MG glioma cells with transient <i>EZH2</i> knockdown (lower panel, black bar) through a matrigel-coated boyden chamber in comparison to control (upper panel, white bar). <b>D</b> Representative dot plots and corresponding analysis of Annexin-V-FITC/DAPI co-staining of U87MG glioma cells untreated or treated for 120 h with 10 µM DZNep. The lower left quadrants represent the living cells (low Annexin-V-FITC-/DAPI-signal), the lower right quadrants represents early apoptosis (low DAPI- and strong Annexin-V-FITC-signal) and the upper right late apoptotic/necrotic cells (double-stained cells). <b>E</b> H3K27me3 methylation was strongly decreased in whole cell lysates of U87MG glioma cells after treatment with 5 µM DZNep or after specific knockdown of <i>EZH2</i> for 120 h. Tubulin served as loading control. <b>F</b> Cell cycle analysis of U87MG glioma cells untreated or treated for 120 h with 500 nM and 5 µM DZNep. <b>G</b> Analysis of nestin expression in S24 glioma-initiating cells untreated (left) or treated with 5 µM DZNep for 120 h (right) by flow cytometry. <b>H</b> Matrigel boyden chamber assay of U87MG glioma cells untreated (upper panel, white bar) and treated (lower panel, black bar) with 5 µM DZNep. Asterisk indicates * (p<0.05). Error bars indicate s.e.m.</p