Promoter Region Of Oncogene Research Articles

DNA G-quadruplex and its potential as anticancer drug target.

G-quadruplex secondary structures are four-stranded globular nucleic acid structures form in the specific DNA and RNA G-rich sequences with biological significance such as human telomeres, oncogene-promoter regions, replication initiation sites, and 5' and 3'-untranslated (UTR) regions. The non-canonical G-quadruplex secondary structures can readily form under physiologically relevant ionic conditions and are considered to be new molecular target for cancer therapeutics. This review discusses the essential progress in our lab related to the structures and functions of biologically relevant DNA G-quadruplexes in human gene promoters and telomeres, and the opportunities presented for the development of G-quadruplex-targeted small- molecule drugs.

Abstract 3811: G-quadruplex-forming genomic sequences homologous to Pu27 interact with c-Myc promoter and regulate c-Myc transcription

Abstract Introduction: The transcription factor c-Myc regulates a large array of genes, plays an important role in cell growth/differentiation, cell cycle progression and apoptosis hence is tightly regulated both at the transcriptional and protein level. The NHEIII1 upstream of P1 promoter regulates up to 80% of c-Myc transcription, and comprises a C/G rich region able to form G-quadruplex structure (Pu27). G-quadruplex forming motifs seem to be abundant in the genome, located in the promoter region of oncogenes. We identify 13 new potential G-quadruplex forming motifs with more than 90% identity with Pu27, located in different chromosomes of the human genome and none near gene promoter regions. Methods: BLAT search was used to identify the homologous sequences of Pu27. The G-quadruplex formation was evaluated by Circular Dichroism Spectrometry (CD), the specific interaction of each of the homologous sequences with the double strand DNA containing NHEIII1/Pu27 was documented by electrophoretic mobility shift assay (EMSA). We investigate 3 members of the Pu27 oligonucleotides family: Pu5- (42nu), Pu9- (20nu) and Pu27 (27nu) in: 1) a dose response (1, 3, 5 or 10µM)-time course (24, 48, 72 , 96 hrs) study on cell growth inhibition in 4 leukemia cell lines measured by MTT. 2) the effect on c-Myc expression in U937 measured by QRT-PCR, Flow-cytometry and Western Blot. 3) global gene expression evaluated in a gene array study in U937 exposed for 24h, 48h and 72h. Results: Thirteen oligonucleotide sequences almost identical to Pu27 were identified spread-out in 12 different chromosomes. Except for Pu27 (c-Myc promoter) none of the sequences were within a promoter region. All Pu27 homologous sequences contain the 5 runs of 3 to 4 Guanines, form G-quadruplex, and specifically bind to the dsDNA containing Pu27 sequence. The evaluation of a dose response/time course for Pu27 (Chr8), Pu5- (Chr5) and Pu9- (Chr9) on growth inhibition of U937, K562, HL-60 and Raji show a difference in sensitivity of the cell lines to the oligonucleotide sequences (Raji (more sensitive than)>U937>K562>HL-60), and a difference in efficiency of the oligonucleotide sequences (Pu27>Pu5->Pu9-). Gene and protein expression of c-Myc were similarly down regulated by the 3 sequences in U937 exposed to Pu27, Pu5- or Pu9- for 3 days. The analysis of the global gene expression reveal an early effect (24h) of Pu27 followed by Pu5- (48h), by 72h exposure all three Pu oligonucleotides will have affected genes involved in cell cycle progression/regulation, DNA damage repair and histone modification . Conclusion: We identify the presence of numerous sequences homologous to Pu27 located in different chromosomes sharing Pu27 structure and function. The addition of exogenous Pu27, Pu5- or Pu9- down regulate c-Myc expression and inhibit leukemia cells growth making this G-quadruplex structure a key regulator in NHEIII1 and a potential target for cancer therapy. Citation Format: Francine Rezzoug, Shelia D. Thomas, Eric C. Rouchka, Donald M. Miller. G-quadruplex-forming genomic sequences homologous to Pu27 interact with c-Myc promoter and regulate c-Myc transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3811. doi:10.1158/1538-7445.AM2014-3811

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

BackgroundCytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively. MethodsSpectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present. ResultsThe cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson–Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3–7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson–Crick duplex across a pH range from approximately 3.0 to 6.5. ConclusionsTwo cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson–Crick duplex. General significanceWatson–Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures.

Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy

Existence of G-quadruplex DNA in vivo always attract widespread interest in the field of biology and biological chemistry. We reported our findings for the existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Probes for selective G-quadruplex cross-linking was designed and synthesized that show high selectivity for G-quadruplex cross-linking. Further biological studies demonstrated its good inhibition activity against murine melanoma cells. To further investigate if G-quadruplex DNA was formed in vivo and as the target, a derivative was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll 70 and PEG, was used to investigate the compound's pure cross-linking ability upon preformed G-quadruplex. Thus, as a potent G-quadruplex cross-linking agent, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy.

Reversible Conformational Switching of i‐Motif DNA Studied by Fluorescence Spectroscopy

Non-B DNAs, which can form unique structures other than double helix of B-DNA, have attracted considerable attention from scientists in various fields including biology, chemistry and physics etc. Among them, i-motif DNA, which is formed from cytosine (C)-rich sequences found in telomeric DNA and the promoter region of oncogenes, has been extensively investigated as a signpost and controller for the oncogene expression at the transcription level and as a promising material in nanotechnology. Fluorescence techniques such as fluorescence resonance energy transfer (FRET) and the fluorescence quenching are important for studying DNA and in particular for the visualization of reversible conformational switching of i-motif DNA that is triggered by the protonation. Here, we review the latest studies on the conformational dynamics of i-motif DNA as well as the application of FRET and fluorescence quenching techniques to the visualization of reversible conformational switching of i-motif DNA in nano-biotechnology.

48 NMR study of the interaction between G-quadruplexes and small molecules

Guanine-rich oligonucleotides are able to adopt secondary DNA structures, known as G-quadruplexes. Such G-rich sequences are found in human telomeres, promoter regions of oncogenes, 5′ untranslated regions (UTRs) of mRNAs and human intronic sequences. Studies have shown that small molecules can induce anti-cancer effect through stabilizing or promoting G-quadruplex formation. In order to design and develop a potent drug, structural details on the interaction between small molecules and G-quadruplexes are invaluable. In this study, we seek to understand the structural determinants involved in the interaction between G-quadruplexes and small molecules. NMR spectroscopy is employed to resolve the structures of two intramolecular G-quadruplexes bound to small molecules. These resolved complexes allow us to structurally design new potent drugs for their application in anti-cancer therapy.

Stabilization and Induction of Oligonucleotide i-Motif Structure via Graphene Quantum Dots

DNA i-motif structures have been found in telomeric, centromeric DNA and many in the promoter region of oncogenes; thus they might be attractive targets for gene-regulation processes and anticancer therapeutics. We demonstrate in this work that i-motif structures can be stabilized by graphene quantum dots (GQDs) under acidic conditions, and more importantly GQDs can promote the formation of the i-motif structure under alkaline or physiological conditions. We illustrate that the GQDs stabilize the i-motif structure through end-stacking of the bases at its loop regions, thus reducing its solvent-accessible area. Under physiological or alkaline conditions, the end-stacking of GQDs on the unfolded structure shifts the equilibrium between the i-motif and unfolded structure toward the i-motif structure, thus promoting its formation. The possibility of fine-tuning the stability of the i-motif and inducing its formation would make GQDs useful in gene regulation and oligonucleotide-based therapeutics.

Mechanism of the antiproliferative activity of some naphthalene diimide G-quadruplex ligands.

G-quadruplexes are higher-order nucleic acid structures that can form in G-rich telomeres and promoter regions of oncogenes. Telomeric quadruplex stabilization by small molecules can lead to telomere uncapping, followed by DNA damage response and senescence, as well as chromosomal fusions leading to deregulation of mitosis, followed by apoptosis and downregulation of oncogene expression. We report here on investigations into the mechanism of action of tetra-substituted naphthalene diimide ligands on the basis of cell biologic data together with a National Cancer Institute COMPARE study. We conclude that four principal mechanisms of action are implicated for these compounds: 1) telomere uncapping with subsequent DNA damage response and senescence; 2) inhibition of transcription/translation of oncogenes; 3) genomic instability through telomeric DNA end fusions, resulting in mitotic catastrophe and apoptosis; and 4) induction of chromosomal instability by telomere aggregate formation.

An In Silico Study of the Differential Effect of Oxidation on Two Biologically Relevant G-Quadruplexes: Possible Implications in Oncogene Expression

G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential “hot-spots” for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.

Methylation pattern of oncogene HRAS gene promoter region and its clinical relevance to urocystic tumorigenesis

The HRAS gene encodes a protein with a molecular weight of 21 kDa (P21) called H-Ras that is involved primarily in regulating cell growth, division and apoptosis. Through a process known as signal transduction, the H-Ras protein relays signals from outside of the cell to the cell's nucleus. These signals instruct the cell to grow or divide. HRAS is in the Ras family of oncogenes. When mutated, oncogenes have the potential to cause normal cells to become cancerous cells. Considering the upsurge of evidences that abnormality in CpG methylation of the oncogene promoter region can cause cancer, we want to understand the relationships between the methylation status of the HRAS promoter region and bladder cancer. To investigate the methylation pattern of HRAS gene transcriptional regulation region (TRR), bisulfite-sequencing PCR-based sequencing analysis was performed among 15 bladder cancer tissues and 5 normal bladder tissues. Analysis of HRAS gene TRR methylation showed that the methylation level of HRAS has clinical relevance (P = 0.0049, by unpaired Student's t test) with bladder cancer. Furthermore, the unpaired Student's t test analysis showed the extremely significant relationship between tumor and normal at CpG site of the 3rd (P < 0.0001), 28th (P = 0.0006), and significant association between tumor and methylation at CpG site of the 12th (P = 0.0024). Abnormal methylation of the HRAS gene may be an early event during urocystic tumorigenesis and may be further used as a cancer biomarker in bladder tissue for early diagnosis and a potential therapeutic target.

Experimental Methods for Studying the Interactions between G-Quadruplex Structures and Ligands

The present paper reviews the recent advances in and applications of experimental techniques used to study interactions between G-quadruplex structures and ligands that are potentially of pharmaceutical interest. Several instrumental techniques are used to study such interactions. The application of spectroscopic techniques such as molecular absorption, circular dichroism, molecular fluorescence, mass spectrometry and nuclear magnetic resonance are reviewed and we discuss the type of information (qualitative or quantitative) that can be obtained from the use of each technique. Additionally, the application of complementary techniques such as surface plasmon resonance, isothermal titration calorimetry and different methods based on biochemistry is considered. For each technique, the main applications are presented and they are classified according to the family of the ligand and the type of G-quadruplex forming sequence (human telomeric or promoter region of oncogenes) considered.

Study of STAT3 G-quadruplex folding patterns by CD spectroscopy and molecular modeling

Abstract The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising targets in the cancer therapy. A G-quadruplex in the downstream flanking region of the signal transducer and activator of transcription 3 (STAT3) gene was explored. Its folding patterns were proposed to be 3:2:2 and 3:3:1 loop isomers by the mutation analysis by CD spectroscopy. The structures were constructed and refined by molecular modeling method.

Structure-based optimization of FDA-approved drug methylene blue as a c-myc G-quadruplex DNA stabilizer

G-quadruplexes are non-canonical DNA secondary structures putatively present in the promoter regions of oncogenes in the human genome. The targeting of promoter G-quadruplex structures to repress oncogene transcription represents a potential anticancer strategy. Here, we have used high-throughput virtual screening to identify FDA-approved drug methylene blue (MB) as a promising scaffold for binding the c-myc oncogene G-quadruplex DNA. Based on molecular docking analysis of MB to the c-myc G-quadruplex, we designed and screened 50 MB derivatives containing side chains that could interact with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring compounds were synthesized and the interactions with the c-myc G-quadruplex were investigated using the FID assay. The results showed that the methylene blue derivatives 6a–c were able to bind to the c-myc G-quadruplex with greater binding affinity compared to the known G-quadruplex binding ligand, crystal violet. The activity of the most potent compound identified from the FID assay, 6b, as an inhibitor for polymerase-drive DNA extension was examined using a PCR-stop assay and compared against that of the parent compound methylene blue. The results of the PCR-stop assay showed that the addition of the side chain improved the activity of the derivatives as an inhibitor compared to the parent compound. The MB derivative 6b was shown to be highly selective towards c-myc G-quadruplex over double-stranded DNA and other biologically relevant G-quadruplexes using UV–visible spectroscopy and mass spectrometry, respectively. The MB derivative 6b could induce or stabilize c-myc G-quadruplex formation in both cell-free and cellular biological models, and displayed higher cytoxicity against human hepatocarcinoma cells compared to the parent compound, MB.

Rehybridization of Promoter DNA Targets in Chromatin to Discover Transcription Factors of Oncogenes

Identification of transcription factors (TF) assembled at promoter regions of oncogenes is vital to understand how gene expression is altered in cancer and in tumor microenvironments. Most TF identification methods are based on one‐on‐one DNA/protein interactions in vitro, which may not reflect DNA/protein interactions in vivo and may overlook protein complex information. Moreover, existing methods for chromatin pull down target pre‐identified proteins rather than DNA, which limits the ability to discover new proteins that may bind to specific genomic sequences. We are pursuing a new approach, chromatin rehybridization (CR), which preserves and detects DNA/protein and protein/protein interactions that occur in live cells and targets specific DNA sequences rather than associated proteins.The G‐quadruplex forming Pu27‐mer located upstream of the c‐MYC oncogene and its associated TFs including cellular nucleic acid binding protein (CNBP) serve as the model system for development of the CR technique. Preliminary results demonstrate that CNBP is pulled down with the targeted Pu27‐mer in MCF‐7 cells using CR. At least one unreported TF associated with c‐MYC was also detected and will be identified using mass spectrometry.The CR method addresses the need for full proteomic analysis of particular genomic regions. This work is supported by NIH through grant R21 CA134419‐01A1.

Structural Insights into G-Quadruplexes: Towards New Anticancer Drugs

DNA G-quadruplexes are DNA secondary structures formed in specific G-rich sequences. DNA sequences that can form G-quadruplexes have been found in regions with biological significance, such as human telomeres and oncogene-promoter regions. DNA G-quadruplexes have recently emerged as a new class of novel molecular targets for anticancer drugs. Recent progress on structural studies of the biologically relevant G-quadruplexes formed in human telomeres and in the promoter regions of human oncogenes will be discussed, as well as recent advances in the design and development of G-quadruplex-interactive drugs. DNA G-quadruplexes can readily form in solution under physiological conditions and are globularly folded nucleic acid structures. The molecular structures of intramolecular G-quadruplexes appear to differ from one another and, therefore, in principle may be differentially regulated and targeted by different proteins and drugs.

Characterization of SV40 T‐antigen G‐quadruplex Helicase Activity Using SPR

Simian Virus 40 (SV40) is a double stranded DNA virus helpful as a model system for eukaryotic cellular DNA replication. It encodes a Large T‐antigen (T‐ag) protein, which is essential for viral genome replication and regulation of the host cell cycle. Acting as a helicase enzyme, T‐ag is able to unwind various forms of DNA. Guanine rich sequences of DNA capable of forming G‐quadruplex knot‐like structures have been identified in several eukaryotic oncogene promoter regions, as well as in the SV40 virus genome, and may possibly influence transcription activity. T‐ag forms an active hexameric unit in the presence of ATP, and was demonstrated to bind single‐strand, duplex, and G‐quadruplex DNA using real time surface plasmon resonance (SPR). Probing with single strand binding protein (SSB) confirmed that T‐ag activity resulted in the unwinding of an intermolecular quadruplex.

Biophysical Studies of the c-MYC NHE III 1 Promoter: Model Quadruplex Interactions with a Cationic Porphyrin

Regulation of the structural equilibrium of G-quadruplex-forming sequences located in the promoter regions of oncogenes by the binding of small molecules has shown potential as a new avenue for cancer chemotherapy. In this study, microcalorimetry (isothermal titration calorimetry and differential scanning calorimetry), electronic spectroscopy (ultraviolet-visible and circular dichroism), and molecular modeling were used to probe the complex interactions between a cationic porphryin mesotetra (N-methyl-4-pyridyl) porphine (TMPyP4) and the c-MYC PU 27-mer quadruplex. The stoichiometry at saturation is 4:1 mol of TMPyP4/c-MYC PU 27-mer G-quadruplex as determined by isothermal titration calorimetry, circular dichroism, and ultraviolet-visible spectroscopy. The four independent TMPyP4 binding sites fall into one of two modes. The two binding modes are different with respect to affinity, enthalpy change, and entropy change for formation of the 1:1 and 2:1, or 3:1 and 4:1 complexes. Binding of TMPyP4, at or near physiologic ionic strength ([K +] = 0.13 M), is described by a “two-independent-sites model.” The two highest-affinity sites exhibit a K 1 of 1.6 × 10 7 M −1 and the two lowest-affinity sites exhibit a K 2 of 4.2 × 10 5 M −1. Dissection of the free-energy change into the enthalpy- and entropy-change contributions for the two modes is consistent with both “intercalative” and “exterior” binding mechanisms. An additional complexity is that there may be as many as six possible conformational quadruplex isomers based on the sequence. Differential scanning calorimetry experiments demonstrated two distinct melting events ( T m1 = 74.7°C and T m2 = 91.2°C) resulting from a mixture of at least two conformers for the c-MYC PU 27-mer in solution.

Activation of nuclear factor kappa B and oncogene expression by 12(R)-hydroxyeicosatrienoic acid, an angiogenic factor in microvessel endothelial cells.

12(R)-Hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid (12(R)-HETrE) is an arachidonic acid metabolite formed by the corneal epithelium of several species, porcine leukocytes, and human and rat epidermal cells. It is a potent, stereospecific proinflammatory and angiogenic factor and its synthesis is increased manyfold in inflamed tissues, e.g. cornea and skin. It is possible that the angiogenic activity of 12(R)-HETrE is due to a direct mitogenic effect on microvessel endothelial cells via yet to be elucidated cellular and molecular mechanisms. In the present study, we demonstrated the ability of 12(R)-HETrE to stimulate the growth of quiescent endothelial cells in a time- and concentration-dependent manner with a maximal effect at 0.1 nM. This effect was highly stereospecific since its enantiomer, 12(S)-HETrE, had no effect within the same concentration range. Northern blot analysis and transient transfection experiments with chloramphenicol acetyltransferase constructs of oncogene promoter regions demonstrated significant increases over control (0.5% fetal calf serum) in c-myc-, c-jun, and c-fos mRNA levels and expression in cells treated with 0.1 nM 12(R)-HETrE. Electrophoretic mobility shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE with specific radiolabeled oligonucleotides corresponding to known transcriptional binding sites, including AP-1, AP-2, SP1, TRE, NF kappa B, TFIID, OKT1, CREB, CTF/NF1, and GRE demonstrated a markedly rapid and specific increase in the binding activity of NF kappa B and to a lesser extent, AP-1. No significant increase was observed in the binding of other transcription factors assayed as compared to control (untreated) cells. Since the protooncogenes (c-fos, c-jun, and c-myc) are immediate early response genes that are implicated in the process of cell proliferation and differentiation, and activation of certain transcription factors, in particular NF kappa B, is associated with the immediate response of the cell to an injury, we propose that 12(R)HETrE's mitogenic and angiogenic activities are mediated, in part, via the activation of NF kappa B and expression of these protooncogenes.