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1.
Nucleic Acids Res ; 46(2): 661-676, 2018 01 25.
Article in English | MEDLINE | ID: mdl-29165690

ABSTRACT

KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.


Subject(s)
G-Quadruplexes , Guanine/analogs & derivatives , Proto-Oncogene Proteins p21(ras)/chemistry , Transcription Initiation Site , Transcription, Genetic , Cell Line, Tumor , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Guanine/chemistry , Guanine/metabolism , HEK293 Cells , Heterogeneous Nuclear Ribonucleoprotein A1/genetics , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Humans , Oxidation-Reduction , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
2.
J Med Chem ; 60(23): 9448-9461, 2017 12 14.
Article in English | MEDLINE | ID: mdl-29140695

ABSTRACT

The human KRAS transcript contains a G-rich 5'-UTR sequence (77% GC) harboring several G4 motifs capable to form stable RNA G-quadruplex (RG4) structures that can serve as targets for small molecules. A biotin-streptavidin pull-down assay showed that 4,11-bis(2-aminoethylamino)anthra[2,3-b]furan-5,10-dione (2a) binds to RG4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays demonstrated that 2a and its analogue 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione (2b) repress translation in a dose-dependent manner. The effect of the G4-ligands on Panc-1 cancer cells has also been examined. Both 2a and 2b efficiently penetrate the cells, suppressing protein p21KRAS to <10% of the control. The KRAS down-regulation induces apoptosis together with a dramatic reduction of cell growth and colony formation. In summary, we report a strategy to suppress the KRAS oncogene in pancreatic cancer cells by means of small molecules binding to RG4s in the 5'-UTR of mRNA.


Subject(s)
5' Untranslated Regions/drug effects , G-Quadruplexes/drug effects , Protein Biosynthesis/drug effects , Proto-Oncogene Proteins p21(ras)/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Discovery , Humans , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Thiophenes/chemistry , Thiophenes/pharmacology
3.
Biochim Biophys Acta Gen Subj ; 1861(5 Pt B): 1389-1398, 2017 May.
Article in English | MEDLINE | ID: mdl-27888145

ABSTRACT

KRAS is one of the most mutated genes in human cancer. Its crucial role in the tumourigenesis of pancreatic ductal adenocarcinoma (PDAC) has been widely demonstrated. As this deadly cancer does not sufficiently respond to conventional chemotherapies, it is important to increase our knowledge of pancreatic cancer biology, in particular how oncogenic KRAS is regulated. The promoter of KRAS contains a GA-element composed of runs of guanines that fold into a G4 structure. This unusual DNA conformation is recognized by several nuclear proteins, including MAZ and hnRNP A1. Recent data have revealed that KRAS is interconnected to ILK and hnRNP A1 in a circuitry that enables pancreatic cancer cells to maintain an aggressive phenotype. The present review illustrates recent advances on how KRAS is regulated in pancreatic cancer cells, focusing on the formation of G4 structures in the KRAS promoter and their interaction with hnRNP A1. The newly discovered KRAS-ILK-hnRNP A1 regulatory loop is discussed, emphasizing its potential as a therapeutic target for PDAC-specific molecules. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.


Subject(s)
DNA, Neoplasm/genetics , G-Quadruplexes , Guanosine/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Transcription, Genetic , Binding Sites , DNA, Neoplasm/chemistry , DNA, Neoplasm/metabolism , Gene Expression Regulation, Neoplastic , Guanosine/chemistry , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/chemistry , Humans , Ligands , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Polymorphism, Genetic , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins p21(ras)/metabolism , Structure-Activity Relationship , Transcriptional Activation
4.
Biochim Biophys Acta ; 1859(4): 663-74, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26855080

ABSTRACT

It is now well established that in the human genome the canonical double helix coexists with folded G-quadruplex structures that are known to have important biological functions. In this review we summarize the current knowledge on quadruplex formation in the promoters of the ras genes that are mutated in about 30% of all human cancers. We describe the nuclear proteins that recognize these unusual DNA structures and discuss their function in transcription. We also examine the formation of G-quadruplexes in the 5'-untranslated region of the ras transcripts and conclude this review by reporting strategies that use either ras G-quadruplexes or proteins recognizing the ras G-quadruplexes as targets of anticancer small molecules.


Subject(s)
G-Quadruplexes , Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Transcription, Genetic , 5' Untranslated Regions/genetics , Genome, Human , Humans , Neoplasms/therapy , Promoter Regions, Genetic , Small Molecule Libraries/therapeutic use
5.
Future Med Chem ; 8(2): 179-94, 2016.
Article in English | MEDLINE | ID: mdl-26807879

ABSTRACT

Cationic porphyrins (Prs) and phthalocyanines (Pcs) are strong photosensitizers that have drawn much attention for their potential in photodynamic therapy. These compounds have the interesting property of binding to nucleic acids, in particular G-rich quadruplex-forming sequences in DNA and RNA. In this review, we highlight their potential as anticancer drugs.


Subject(s)
DNA/metabolism , Photosensitizing Agents/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , DNA/chemistry , G-Quadruplexes , Humans , Indoles/chemistry , Indoles/metabolism , Isoindoles , Neoplasms/drug therapy , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic use , Porphyrins/chemistry , Porphyrins/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , ras Proteins/genetics , ras Proteins/metabolism
6.
ACS Med Chem Lett ; 6(12): 1179-83, 2015 Dec 10.
Article in English | MEDLINE | ID: mdl-26713101

ABSTRACT

In a previous study we have demonstrated that two neighboring G-quadruplexes, hras-1 and hras-2, located immediately upstream of the major transcription start site of HRAS, bind MAZ, a nuclear factor that activates transcription (Cogoi, S.; et al. Nucl. Acid Res. 2014, 42, 8379). For the present study we have designed G4 oligonucleotides with anthraquinone insertions and locked nucleic acids (LNA) modifications mimicking quadruplex hras-1. Luciferase, qRT-PCR, and Western blot data demonstrate that these constructs efficiently down regulate HRAS in T24 bladder cancer cells. The inhibitory efficiency of the G4 oligonucleotides correlates with their nuclease resistance in the cell environment. By chromatin immunoprecipitation we show that the association of MAZ to the HRAS promoter is strongly attenuated by the designed G4 oligonucleotides, thus suggesting that these constructs behave with a decoy mechanism.

7.
Sci Rep ; 5: 18097, 2015 Dec 17.
Article in English | MEDLINE | ID: mdl-26674223

ABSTRACT

HRAS is regulated by two neighbouring quadruplex-forming GC-elements (hras-1 and hras-2), located upstream of the major transcription start sites (doi: 10.1093/nar/gku 5784). In this study we demonstrate that the C-rich strands of hras-1 and hras-2 fold into i-motif conformations (iMs) characterized under crowding conditions (PEG-300, 40% w/v) by semi-transitions at pH 6.3 and 6.7, respectively. Nondenaturing PAGE shows that the HRAS C-rich sequences migrate at both pH 5 and 7 as folded intramolecular structures. Chromatin immunoprecipitation shows that hnRNP A1 is associated under in vivo conditions to the GC-elements, while EMSA proves that hnRNP A1 binds tightly to the iMs. FRET and CD show that hnRNP A1 unfolds the iM structures upon binding. Furthermore, when hnRNP A1 is knocked out in T24 bladder cancer cells by a specific shRNA, the HRAS transcript level drops to 44 ± 5% of the control, suggesting that hnRNP A1 is necessary for gene activation. The sequestration by decoy oligonucleotides of the proteins (hnRNP A1 and others) binding to the HRAS iMs causes a significant inhibition of HRAS transcription. All these outcomes suggest that HRAS is regulated by a G-quadruplex/i-motif switch interacting with proteins that recognize non B-DNA conformations.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Protein Unfolding , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Base Sequence , Cell Line, Tumor , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Fluorescence Resonance Energy Transfer , GC Rich Sequence/genetics , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , Nucleotide Motifs/genetics , Protein Binding , Proto-Oncogene Proteins p21(ras)/genetics , RNA Interference , Regulatory Sequences, Nucleic Acid/genetics , Thermodynamics , Transition Temperature
8.
J Med Chem ; 58(14): 5476-85, 2015 Jul 23.
Article in English | MEDLINE | ID: mdl-26057859

ABSTRACT

We previously found that two neighboring G-quadruplexes behave as a molecular switch controlling the expression of HRAS (Cogoi, S.; Schekotikhin, A. E.; Xodo, L. E. Nucl. Acids Res. 2014, DOI: 10.1093/nar/gku574). In this study we have designed anthrathiophenediones with two chloroacetamidine-containing side chains (CATDs) as G-quadruplex binders and have examined their anticancer activity in T24 bladder cancer cells bearing mutant HRAS and in T24 xenografts. The designed CATDs (3a-e), bearing alkyl side chains of different length, penetrate T24 cancer cells more than their analogues with guanidine-containing side chains. The lead compounds 3a and 3c inhibit HRAS expression, metabolic activity, and colony formation in T24 cancer cells. They also activate a strong apoptotic response, as indicated by PARP-1, caspases 3/7, and annexin V/propidium iodide assays. Apoptosis occurs under conditions where cyclin D1 is down-regulated and the cell cycle arrested in G2 phase. Finally, compound 3a inhibits the growth of T24 xenografts and increases the median survival time of nude mice.


Subject(s)
Acetamides/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Thiophenes/chemistry , Thiophenes/pharmacology , Urinary Bladder Neoplasms/pathology , Alleles , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Biological Transport , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/metabolism , Drug Design , G-Quadruplexes/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , Genes, ras/genetics , Humans , Mice , Substrate Specificity , Survival Analysis , Thiophenes/chemical synthesis , Thiophenes/metabolism , Xenograft Model Antitumor Assays
9.
Nucleic Acids Res ; 42(13): 8379-88, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25013182

ABSTRACT

The HRAS promoter contains immediately upstream of the transcription start site two neighboring G-elements, each capable of folding into a G-quadruplex structure. We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1. In the present study we have examined the interaction between the HRAS promoter and MAZ, demonstrating for the first time that the protein unfolds the G-quadruplex structures. We also demonstrate that MAZ-GST, in the presence of the complementary strands, promotes a rapid transformation of the two HRAS quadruplexes into duplexes. By a mutational analysis of the HRAS G-elements, we dissected the MAZ-binding sites from the quadruplex-forming motifs, finding that the two neighboring G-quadruplexes bring about a dramatic repression of transcription, in a synergistic manner. We also discovered that the two G-quadruplexes are strong targets for small anticancer molecules. We found that a cell-penetrating anthratiophenedione (ATPD-1), which binds tightly to the G-quadruplexes (ΔT > 15°C), promotes the total extinction of HRAS transcription. In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%. Our study provides relevant information for the rationale design of targeted therapy drugs specific for the HRAS oncogene.


Subject(s)
DNA-Binding Proteins/metabolism , G-Quadruplexes , Gene Expression Regulation, Neoplastic , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/genetics , Transcription Factors/metabolism , Cell Line, Tumor , DNA/chemistry , DNA/metabolism , Gene Silencing , Humans , Transcriptional Activation , Urinary Bladder Neoplasms/genetics
10.
Mol Cancer ; 13: 75, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24684778

ABSTRACT

BACKGROUND: Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity. METHODS: Uptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots. RESULTS: Thanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5'-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis and necrosis. C14 also showed an intrinsic light-independent anticancer activity, as recently reported for G4-RNA binders. CONCLUSIONS: Porphyrins P4 and C14 impair the clonogenic growth and migration of B78-H1 melanoma cells and inhibit melanoma tumour growth in vivo. Evidence is provided that C14 acts through light-dependent (mRNA photocleavage) and light-independent (translation inhibition) mechanisms.


Subject(s)
Photochemotherapy , Photosensitizing Agents/administration & dosage , Porphyrins/administration & dosage , Skin Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Cell Line, Tumor , G-Quadruplexes/drug effects , Humans , MAP Kinase Signaling System/drug effects , Mice , Porphyrins/chemistry
11.
J Med Chem ; 56(7): 2764-78, 2013 Apr 11.
Article in English | MEDLINE | ID: mdl-23458775

ABSTRACT

We prepared a series of anthrathiophenediones (ATPDs) with guanidino-alkyl side chains of different length (compounds 1, 10-13). The aim was to investigate their interaction with DNA and RNA G-quadruplexes, their uptake in malignant and nonmalignant cells, and their capacity to modulate gene expression and inhibit cell growth. Flow cytometry showed that the ATPDs enter more efficiently in malignant T24 bladder cells than in nonmalignant embryonic kidney 293 or fibroblast NIH 3T3 cells. In T24 malignant cells, compound 1, with two ethyl side chains, is taken up by endocytosis, while 12 and 13, with respectively propyl and butyl side chains, are transported by passive diffusion. The designed ATPDs localize in the cytoplasm and nucleus and tightly bind to DNA and RNA G-quadruplexes. They also decrease HRAS expression, increase the cell population in G0/G1, and strongly inhibit proliferation in malignant T24 bladder cells, but not in nonmalignant 293 or NIH 3T3 cells.


Subject(s)
G-Quadruplexes , Genes, ras , Thiophenes/pharmacology , Urinary Bladder Neoplasms/metabolism , Animals , Cell Division , Cell Line, Tumor , Endocytosis , Flow Cytometry , Humans , Mice , NIH 3T3 Cells , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology
12.
Nucleic Acids Res ; 41(7): 4049-64, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23471001

ABSTRACT

KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3'-end, whereas to enhance the folding and stability, two polycyclic aromatic hydrocarbon units (TINA or AMANY) were inserted internally, to cap the quadruplex. The most active G4-decoy (2998), which had two para-TINAs, strongly suppressed KRAS expression in Panc-1 cells. It also repressed their metabolic activity (IC50 = 520 nM), and it inhibited cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased the Kaplan-Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy.


Subject(s)
Antineoplastic Agents/therapeutic use , DNA-Binding Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Proto-Oncogene Proteins/antagonists & inhibitors , Transcription Factors/metabolism , ras Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis , Binding Sites , Binding, Competitive , Cell Line, Tumor , DNA/chemistry , G-Quadruplexes , Humans , Mice , Mice, SCID , Oligonucleotides/chemistry , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins p21(ras) , Transcriptional Activation , ras Proteins/genetics , ras Proteins/metabolism
13.
Chem Commun (Camb) ; 48(6): 874-6, 2012 Jan 21.
Article in English | MEDLINE | ID: mdl-22127206

ABSTRACT

The KRAS transcript is characterized by a 192-nt 5'-UTR containing repetitive runs of two-guanines which can fold in several G-quadruplexes. These folded structures have a high affinity for the cationic porphyrin tri-meso(N-methyl-4-pyridyl), meso(N-tetradecyl-4-pyridyl) porphine (TMPyP4-C14), which efficiently penetrates cell membranes. Upon photoactivation TMPyP4-C14 induces a dramatic down-regulation of oncogenic KRAS and cell growth arrest in pancreatic cancer cells.


Subject(s)
Genes, ras/genetics , Porphyrins/chemistry , Porphyrins/pharmacology , Proto-Oncogene Proteins p21(ras)/metabolism , RNA/chemistry , 5' Untranslated Regions/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Circular Dichroism , Down-Regulation , Flow Cytometry , Fluorescence Resonance Energy Transfer , Guanine/chemistry , Humans , Photochemistry , Protein Biosynthesis/drug effects
14.
PLoS One ; 6(9): e24421, 2011.
Article in English | MEDLINE | ID: mdl-21931711

ABSTRACT

HRAS is a proto-oncogene involved in the tumorigenesis of urinary bladder cancer. In the HRAS promoter we identified two G-rich elements, hras-1 and hras-2, that fold, respectively, into an antiparallel and a parallel quadruplex (qhras-1, qhras-2). When we introduced in sequence hras-1 or hras-2 two point mutations that block quadruplex formation, transcription increased 5-fold, but when we stabilized the G-quadruplexes by guanidinium phthalocyanines, transcription decreased to 20% of control. By ChIP we found that sequence hras-1 is bound only by MAZ, while hras-2 is bound by MAZ and Sp1: two transcription factors recognizing guanine boxes. We also discovered by EMSA that recombinant MAZ-GST binds to both HRAS quadruplexes, while Sp1-GST only binds to qhras-1. The over-expression of MAZ and Sp1 synergistically activates HRAS transcription, while silencing each gene by RNAi results in a strong down-regulation of transcription. All these data indicate that the HRAS G-quadruplexes behave as transcription repressors. Finally, we designed decoy oligonucleotides mimicking the HRAS quadruplexes, bearing (R)-1-O-[4-(1-Pyrenylethynyl) phenylmethyl] glycerol and LNA modifications to increase their stability and nuclease resistance (G4-decoys). The G4-decoys repressed HRAS transcription and caused a strong antiproliferative effect, mediated by apoptosis, in T24 bladder cancer cells where HRAS is mutated.


Subject(s)
Drug Design , G-Quadruplexes , Oligonucleotides/therapeutic use , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/genetics , Urinary Bladder Neoplasms/drug therapy , Base Sequence , Cell Proliferation , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Indoles/metabolism , Isoindoles , Molecular Sequence Data , Point Mutation/genetics , Polymorphism, Genetic , Protein Binding , Proto-Oncogene Mas , Sp1 Transcription Factor/metabolism , Transcription Factors/metabolism , Transcription Initiation Site , Transcription, Genetic , Up-Regulation/genetics , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology
15.
Chem Commun (Camb) ; 47(17): 4965-7, 2011 May 07.
Article in English | MEDLINE | ID: mdl-21424008

ABSTRACT

We employed primer extension reactions to uncover folding motifs in a nuclease hypersensitive element (NHE) with a complex guanine pattern, located in the human KRAS promoter. We also identified and characterized a new G-rich motif of 21 nt capable of forming a parallel G-quadruplex that is disrupted by protein UP1.


Subject(s)
DNA Primers/metabolism , G-Quadruplexes , Genes, ras , Guanine/analysis , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Promoter Regions, Genetic , Amino Acid Motifs , Binding Sites , DNA Primers/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Guanine/metabolism , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Spectroscopy, Fourier Transform Infrared , Transcription, Genetic
16.
Chem Commun (Camb) ; 46(39): 7364-6, 2010 Oct 21.
Article in English | MEDLINE | ID: mdl-20820515

ABSTRACT

The cationic porphyrin TPrPyP4, with four propyl substituents in the pyridinium rings, is evaluated in vitro by spectroscopic and polymerase-stop assays as a G4-DNA ligand and in cultured cells as a modulator of gene transcription. A comparison with the widely used TMPyP4 is presented.


Subject(s)
DNA/chemistry , Porphyrins/chemistry , Animals , Base Sequence , G-Quadruplexes , Inhibitory Concentration 50 , Mice , Spectrophotometry, Ultraviolet
17.
J Biol Chem ; 285(29): 22003-16, 2010 Jul 16.
Article in English | MEDLINE | ID: mdl-20457603

ABSTRACT

The murine KRAS promoter contains a G-rich nuclease hypersensitive element (GA-element) upstream of the transcription start site that is essential for transcription. Pulldown and chromatin immunoprecipitation assays demonstrate that this GA-element is bound by the Myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 (PARP-1) proteins. These proteins are crucial for transcription, because when they are knocked down by short hairpin RNA, transcription is down-regulated. This is also the case when the poly(ADP-ribosyl)ation activity of PARP-1 is inhibited by 3,4-dihydro-5-[4-(1-piperidinyl) butoxyl]-1(2H) isoquinolinone. We found that MAZ specifically binds to the duplex and quadruplex conformations of the GA-element, whereas PARP-1 shows specificity only for the G-quadruplex. On the basis of fluorescence resonance energy transfer melting and polymerase stop assays we saw that MAZ stabilizes the KRAS quadruplex. When the capacity of folding in the GA-element is abrogated by specific G --> T or G --> A point mutations, KRAS transcription is down-regulated. Conversely, guanidine-modified phthalocyanines, which specifically interact with and stabilize the KRAS G-quadruplex, push the promoter activity up to more than double. Collectively, our data support a transcription mechanism for murine KRAS that involves MAZ, PARP-1 and duplex-quadruplex conformational changes in the promoter GA-element.


Subject(s)
DNA-Binding Proteins/metabolism , G-Quadruplexes , Guanine/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/genetics , Transcription Factors/metabolism , Animals , Base Sequence , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Fluorescence Resonance Energy Transfer , Indoles/chemistry , Isoindoles , Ligands , Mice , Molecular Sequence Data , Mutant Proteins/metabolism , NIH 3T3 Cells , Nucleic Acid Conformation , Protein Binding , Protein Stability , Transcription, Genetic
19.
Nucleic Acids Res ; 37(9): 2841-53, 2009 May.
Article in English | MEDLINE | ID: mdl-19282454

ABSTRACT

The promoter of the human KRAS proto-oncogene contains a structurally polymorphic nuclease hypersensitive element (NHE) whose purine strand forms a parallel G-quadruplex structure (called 32R). In a previous work we reported that quadruplex 32R is recognized by three nuclear proteins: PARP-1, Ku70 and hnRNP A1. In this study we describe the interaction of recombinant hnRNP A1 (A1) and its derivative Up1 with the KRAS G-quadruplex. Mobility-shift experiments show that A1/Up1 binds specifically, and also with a high affinity, to quadruplex 32R, while CD demonstrates that the proteins strongly reduce the intensity of the 260 nm-ellipticity-the hallmark for parallel G4-DNA-and unfold the G-quadruplex. Fluorescence resonance energy transfer melting experiments reveal that A1/Up1 completely abrogates the cooperative quadruplex-to-ssDNA transition that characterizes the KRAS quadruplex and facilitates the association between quadruplex 32R and its complementary polypyrimidine strand. When quadruplex 32R is stabilized by TMPyP4, A1/Up1 brings about only a partial destabilization of the G4-DNA structure. The possible role played by hnRNP A1 in the mechanism of KRAS transcription is discussed.


Subject(s)
G-Quadruplexes , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Binding Sites , Circular Dichroism , DNA/chemistry , Electrophoretic Mobility Shift Assay , Fluorescence Resonance Energy Transfer , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , Nucleic Acid Denaturation , Proto-Oncogene Mas , Proto-Oncogene Proteins p21(ras) , Transcription, Genetic
20.
J Med Chem ; 52(2): 564-8, 2009 Jan 22.
Article in English | MEDLINE | ID: mdl-19099510

ABSTRACT

A new quadruplex motif located in the promoter of the human KRAS gene, within a nuclease hypersensitive element (NHE), has been characterized. Oligonucleotides mimicking this quadruplex are found to compete with a DNA-protein complex between NHE and a nuclear extract from pancreatic cancer cells. When modified with (R)-1-O-[4-1-(1-pyrenylethynyl) phenylmethyl]glycerol insertions (TINA), the quadruplex oligonucleotides showed a dramatic increase of the T(m) (deltaT(m) from 22 to 32 degrees C) and a strong antiproliferative effects in Panc-1 cells.


Subject(s)
Cell Proliferation/drug effects , G-Quadruplexes , Genes, ras , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , Pyrenes/pharmacology , Base Sequence , Cell Line, Tumor , Electrophoretic Mobility Shift Assay , Glycerol/analogs & derivatives , Glycerol/chemistry , Humans , Pyrenes/chemistry
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