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1.
Oncotarget ; 9(79): 34919-34934, 2018 Oct 09.
Article in English | MEDLINE | ID: mdl-30405884

ABSTRACT

Survivin, a member of the inhibitor of apoptosis (IAP) protein family plays a significant role in cell fate and function. It is significantly overexpressed in tumor cells and has been identified in most cancer cell types. A novel extracellular population has recently been identified and its function is still unknown. Emerging evidence continues to shed light on the important role the tumor microenvironment (TME) has on tumor survival and progression. This new population of survivin has been seen to enhance the tumor phenotype when internalized by recipient cells. In this paper, we sought to better understand the mechanism by which survivin is taken up by cancer cells and the possible role it plays in this phenomenon. We isolated the exosomal carriers of extracellular survivin and using a lipophilic stain, PKH67, we tracked their uptake with immunofluorescence and flow cytometry. We found that by blocking exosomal survivin, exosome internalization is reduced, signifying a novel function for this protein. We also discovered that the common membrane receptors, transferrin receptor, endothelin B receptor, insulin receptor alpha, and membrane glucocorticoid receptor all facilitate exosomal internalization. This understanding further clarifies the protein-protein interactions in the TME that may influence tumor progression and identifies additional potential chemotherapeutic targets.

2.
Cancer Invest ; 34(1): 1-11, 2016.
Article in English | MEDLINE | ID: mdl-26536157

ABSTRACT

Prostate cancer (PCa) remains the most frequently diagnosed male malignancy in Western countries and the second most common cause of male cancer death in the United States. The relatively elevated PCa incidence and mortality among African American men makes this cancer type a challenging health disparity disease. To increase the chance for successful trea tment, earlier detection and prediction of tumor aggress iveness will be important and need to be resolved. This study demonstrates that small membrane-bound vesicles shed from the tumor called exosomes contain ethnically and tumor-specific biomarkers, and could be exploited for their diagnostic and therapeutic potential.


Subject(s)
Ethnicity , Exosomes/metabolism , Prostatic Neoplasms/metabolism , Proteome , Proteomics , Biomarkers, Tumor , Case-Control Studies , Computational Biology , Humans , Male , Prostatic Neoplasms/blood , Proteomics/methods
3.
Onco Targets Ther ; 8: 495-507, 2015.
Article in English | MEDLINE | ID: mdl-25767396

ABSTRACT

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma.

4.
Article in English | MEDLINE | ID: mdl-26161298

ABSTRACT

Pancreatic cancer is a deadly and aggressive disease. Less than 1% of diagnosed patients survive 5 years with an average survival time of only 4-8 months. The only option for metastatic pancreatic cancer is chemotherapy where only the antimetabolites gemcitabine and 5-fluorouracil are used clinically. Unfortunately, efforts to improve chemotherapy regimens by combining, 5-fluorouracil or gemcitabine with other drugs, such as cisplatin or oxaliplatin, have not increased cell killing or improved patient survival. The novel antimetabolite zebularine shows promise, inducing apoptosis and arresting cellular growth in various pancreatic cancer cell lines. However, resistance to these antimetabolites remains a problem highlighting the need to discover and develop new antimetabolites that will improve a patient's overall survival.

5.
Chem Res Toxicol ; 24(3): 418-28, 2011 Mar 21.
Article in English | MEDLINE | ID: mdl-21319831

ABSTRACT

The persistent activation of innate immune cells in chronic inflammation is gaining recognition as a contributing factor in a number of human diseases. A distinguishing feature of activated leukocytes at sites of inflammation is their production of reactive species such as hypochlorous acid (HOCl). Investigating the role of reactive molecules such as HOCl in inflammation and human disease requires appropriate biomarkers. The preferred biomarker for HOCl, and by extension its synthesizing enzyme myeloperoxidase, is 3-chlorotyrosine. 3-Chlorotyrosine is a chemically stable product formed when HOCl, or an HOCl-generated chloramine, reacts with the tyrosine side chain and is readily measured by sensitive mass spectrometry methods. However, Whiteman and Spencer ((2008) Biochem. Biophys. Res. Commun., 371, 50 - 53.) noted that 3-chlorotyrosine is degraded by HOCl, calling into question its use as a biomarker. The kinetic rate constants for the reaction of 3-chlorotyrosine with HOCl, histidine chloramine, or lysine chloramine to form 3,5-dichlorotyrosine are reported. The kinetics of tyrosine chlorination in the context of a peptide with a nearby lysine residue was also determined and further supports the role of chloramines in the chlorination of protein-bound tyrosine residues. The likelihood of free and protein-bound 3,5-dichlorotyrosine occurring in vivo, given the reported rate constants, is discussed.


Subject(s)
Chloramines/chemistry , Hypochlorous Acid/chemistry , Tyrosine/analogs & derivatives , Biomarkers/analysis , Chlorides/chemistry , Chromatography, High Pressure Liquid , Humans , Hydrogen-Ion Concentration , Kinetics , Spectrophotometry, Ultraviolet , Tyrosine/chemistry , Tyrosine/isolation & purification
6.
Apoptosis ; 16(1): 1-12, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20717727

ABSTRACT

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.


Subject(s)
Cysteine Proteinase Inhibitors/metabolism , Exosomes/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Uterine Cervical Neoplasms/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western , Cell Proliferation , Cysteine Proteinase Inhibitors/genetics , Cytoskeleton/metabolism , Exocytosis/radiation effects , Exosomes/genetics , Extracellular Space/metabolism , Female , Gene Expression/radiation effects , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins/genetics , Protons , Radioisotopes , Secretory Pathway/radiation effects , Survivin , Up-Regulation , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/therapy
7.
J Biol Chem ; 285(28): 21868-76, 2010 Jul 09.
Article in English | MEDLINE | ID: mdl-20442396

ABSTRACT

Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the cross-talk relationship between H2B ubiquitination and H3 Lys-79 methylation.


Subject(s)
Histones/chemistry , Lysine/chemistry , Ubiquitin/chemistry , Chromatography, Liquid/methods , DNA Methylation , Electrophoresis, Polyacrylamide Gel , Gene Deletion , HL-60 Cells , Humans , Mass Spectrometry/methods , Models, Biological , Mutation , Proteomics/methods , U937 Cells
8.
Biochemistry ; 48(50): 11994-2004, 2009 Dec 22.
Article in English | MEDLINE | ID: mdl-19899814

ABSTRACT

The selection of nucleoside triphosphates by a polymerase is controlled by several energetic and structural features, including base pairing geometry as well as sugar structure and conformation. Whereas base pairing has been considered exhaustively, substantially less is known about the role of sugar modifications for both nucleotide incorporation and primer extension. In this study, we synthesized oligonucleotides containing 2'-fluoro-modified nucleosides with constrained sugar pucker in an internucleotide position and, for the first time, at a primer 3'-end. The thermodynamic stability of these duplexes was examined. The nucleoside 2'-deoxy-2'-fluoroarabinofuranosyluracil [U(2'F(ara))] favors the 2'-endo conformation (DNA-like), while 2'-deoxy-2'-fluororibofuranosyluracil [U(2'F(ribo))] favors the 3'-endo conformation (RNA-like). Oligonucleotides containing U(2'F(ara)) have slightly higher melting temperatures (T(m)) than those containing U(2'F(ribo)) when located in internucleotide positions or at the 3'-end and when correctly paired with adenine or mispaired with guanine. However, both modifications decrease the magnitude of DeltaH degrees and DeltaS degrees for duplex formation in all sequence contexts. In examining the thermodynamic properties for this set of oligonucleotides, we find entropy-enthalpy compensation is apparent. Our thermodynamic findings led to a series of experiments with DNA ligase that reveal, contrary to expectation based upon observed T(m) values, that the duplex containing the U(2'F(ribo)) analogue is more easily ligated. The 2'-fluoro-2'-deoxynucleosides examined here are valuable probes of the impact of sugar constraint and are also members of an important class of antitumor and antiviral agents. The data reported here may facilitate an understanding of the biological properties of these agents, as well as the contribution of sugar conformation to replication fidelity.


Subject(s)
Base Pair Mismatch , Base Pairing , Carbohydrates/chemistry , Thermodynamics , Carbohydrate Conformation , Carbohydrate Sequence , DNA Ligase ATP , DNA Ligases/chemistry , Deoxyuridine/analogs & derivatives , Deoxyuridine/chemistry , Escherichia coli Proteins/chemistry , Humans , Molecular Sequence Data , Nucleic Acid Heteroduplexes/chemistry , Oligonucleotides/chemical synthesis , Poly-ADP-Ribose Binding Proteins , Xenopus Proteins
9.
Biochemistry ; 48(47): 11312-8, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19863157

ABSTRACT

Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G). Melting studies indicate that oligonucleotide duplexes containing the ClU-G mispair are substantially less stable than those containing a ClU-A base pair. The melting temperature of the ClU-G mispair is not experimentally distinguishable from that of a T-G pair. NMR studies indicate that the ClU-G base pair adopts a wobble geometry at neutral pH, similar to a T-G mispair. The exchangeable protons of the ClU-G mispair broaden rapidly with an increase in temperature, indicating that the ClU-G mispair is less stable and opens more easily than the surrounding adjacent base pairs. Unlike the ClU-A base pair studied previously [Theruvathu, J. A., et al. (2009) Biochemistry 48, 7539-7546], the ClU-G mispair undergoes a pH-dependent structural change, assuming an ionized base pair configuration that approximates a Watson-Crick base pair at higher pH. Ionization of ClU in a DNA template could promote mispair formation and mutation, in accord with previous studies on other 5-halouracil analogues. The electron-withdrawing 5-chloro substituent facilitates ionization of the ClU N3 proton, promoting mispair formation, but it also renders the glycosidic bond susceptible to base cleavage by DNA repair glycosylases.


Subject(s)
Base Pairing , Guanine/chemistry , Hydrogen-Ion Concentration , Uracil/analogs & derivatives , Binding Sites , DNA Repair , Guanine/metabolism , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Protons , Temperature , Thermodynamics , Uracil/chemistry , Uracil/metabolism
10.
Biochemistry ; 48(31): 7539-46, 2009 Aug 11.
Article in English | MEDLINE | ID: mdl-19618901

ABSTRACT

Inflammation-mediated reactive molecules can damage DNA by oxidation and chlorination. The biological consequences of this damage are as yet incompletely understood. In this paper, we have constructed oligonucleotides containing 5-chlorouracil (ClU), one of the known inflammation damage products. The thermodynamic stability, base pairing configuration, and duplex conformation of oligonucleotides containing ClU paired opposite adenine have been examined. NMR spectra reveal that the ClU-A base pair adopts a geometry similar to that of the T-A base pair, and the ClU-A base pair-containing duplex adopts a normal B-form conformation. The line width of the imino proton of the ClU residue is substantially greater than that of the corresponding T imino proton; however, this difference is not attributed to a reduced thermal or thermodynamic stability or to an increased level of proton exchange with solvent. While the NMR studies reveal an increased level of chemical exchange for the ClU imino proton of the ClU-A base pair, the ClU residue is not a target for removal by the Escherichia coli mispaired uracil glycosylase, which senses damage-related helix instability. The results of this study are consistent with previous reports indicating that the DNA of replicating cells can tolerate substantial substitution with ClU. The fraudulent, pseudo-Watson-Crick ClU-A base pair is sufficiently stable to avoid glycosylase removal and, therefore, might constitute a persistent form of cellular DNA damage.


Subject(s)
Adenine/chemistry , Base Pairing , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/chemical synthesis , Oligonucleotides/chemical synthesis , Uracil/analogs & derivatives , Base Pair Mismatch/genetics , Base Pairing/genetics , Deoxyuridine/chemistry , Nucleic Acid Heteroduplexes/genetics , RNA Stability/genetics , Thermodynamics , Uracil/chemistry
11.
Chem Res Toxicol ; 22(6): 1194-204, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19480391

ABSTRACT

The nucleoside analogue 5-aza-2'-deoxycytidine (Decitabine, DAC) is one of several drugs in clinical use that inhibit DNA methyltransferases, leading to a decrease of 5-methylcytosine in newly replicated DNA and subsequent transcriptional activation of genes silenced by cytosine methylation. In addition to methyltransferase inhibition, DAC has demonstrated toxicity and potential mutagenicity, and can induce a DNA-repair response. The mechanisms accounting for these events are not well understood. DAC is chemically unstable in aqueous solutions, but there is little consensus between previous reports as to its half-life and corresponding products of decomposition at physiological temperature and pH, potentially confounding studies on its mechanism of action and long-term use in humans. Here, we have employed a battery of analytical methods to estimate kinetic rates and to characterize DAC decomposition products under conditions of physiological temperature and pH. Our results indicate that DAC decomposes into a plethora of products, formed by hydrolytic opening and deformylation of the triazine ring, in addition to anomerization and possibly other changes in the sugar ring structure. We also discuss the advantages and problems associated with each analytical method used. The results reported here will facilitate ongoing studies and clinical trials aimed at understanding the mechanisms of action, toxicity, and possible mutagenicity of DAC and related analogues.


Subject(s)
Azacitidine/analogs & derivatives , Enzyme Inhibitors/chemistry , Azacitidine/chemistry , Chromatography, High Pressure Liquid , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/metabolism , Decitabine , Gas Chromatography-Mass Spectrometry , Half-Life , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Spectrophotometry, Ultraviolet , Temperature , Time Factors
12.
Chem Res Toxicol ; 22(5): 885-93, 2009 May.
Article in English | MEDLINE | ID: mdl-19341313

ABSTRACT

Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH. However, the minimal reported activity of this enzyme, coupled with inconsistencies with previously published mass spectrometry data, compelled us to reexamine TH. The sequence reported here differs from the previously reported sequence at two amino acid positions and is consistent with previously reported mass spectrometry data. The cloned enzyme characterized in this report displayed substantial activity, indicating that the sequence differences are critical for activity. The substrate selectivity of TH against a series of pyrimidine analogues is consistent with that reported for the wild-type enzyme and, in part, explains the mode of selection of uracil analogues. A preliminary model of the active site has been constructed for the purposes of comparing TH with other members of this family. TH and alkB share in common the capacity to oxidize N-methyl groups. However, TH has the added capacity to oxidize the 5-methyl group of thymine, a property that is potentially important for enzymes that could act on DNA and modify DNA-protein interactions.


Subject(s)
Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Rhodotorula/genetics , Amino Acid Sequence , Cloning, Molecular , Ketoglutaric Acids/metabolism , Molecular Sequence Data , Rhodotorula/chemistry , Sequence Alignment , Thymine/metabolism
13.
J Virol ; 82(19): 9600-14, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18632871

ABSTRACT

High-risk strains of human papillomavirus, such as types 16 and 18, have been etiologically linked to cervical cancer. Most cervical cancer tissues are positive for both the E6 and E7 oncoproteins, since it is their cooperation that results in successful transformation and immortalization of infected cells. We have reported that E6 binds to tumor necrosis factor receptor 1 and to Fas-associated death domain (FADD) and, in doing so, prevents E6-expressing cells from responding to apoptotic stimuli. The binding site of E6 to FADD localizes to the first 23 amino acids of FADD and has now been further characterized by the use of deletion and site-directed mutants of FADD in pull-down and functional assays. The results from these experiments revealed that mutations of serine 16, serine 18, and leucine 20 obstruct FADD binding to E6, suggesting that these residues are part of the E6 binding domain on FADD. Because FADD does not contain the two previously identified E6 binding motifs, the LxxphiLsh motif, and the PDZ motif, a novel binding domain for E6 has been identified on FADD. Furthermore, peptides that correspond to this region can block E6/FADD binding in vitro and can resensitize E6-expressing cells to apoptotic stimuli in vivo. These results demonstrate the existence of a novel E6 binding domain.


Subject(s)
Fas-Associated Death Domain Protein/metabolism , Human papillomavirus 16/metabolism , Amino Acid Motifs , Amino Acid Sequence , Apoptosis , Cell Line, Tumor , Female , Humans , Molecular Conformation , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Receptors, Tumor Necrosis Factor, Type I/chemistry , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/therapy , Uterine Cervical Neoplasms/virology
14.
Chem Res Toxicol ; 21(5): 1028-38, 2008 May.
Article in English | MEDLINE | ID: mdl-18452314

ABSTRACT

While the last 30 years chronicles an extensive effort to understand the damage to DNA caused by reactive oxygen species (ROS), little research has examined the chemical damage to the histone proteins found in chromatin. Hypochlorous acid (HOCl), the primary product of activated neutrophils, is known to damage both DNA and proteins. This article describes the use of mass spectrometry to quantitate the formation of 3-chlorotyrosine and 3,5-dichlorotyrosine, stable and unique markers of protein damage caused by HOCl, in the core histone proteins. Our results indicate that up to 25% of the tyrosine in histone proteins become chlorinated by excess HOCl. We also observe significant formation of 3-chlorotyrosine and 3,5-dichlorotyrosine at low HOCl concentrations and short reaction times. We use mass spectrometry to identify the tyrosine residues on each histone protein that are chlorinated based on the observation of chlorine-containing peptides following protease digestion of histone proteins exposed to HOCl. The tyrosine residues preferentially chlorinated by HOCl are generally within three residues of a lysine or histidine residue, further implicating the initial formation of chloramines in the efficient chlorination of tyrosine residues. The methods and results described here should further our understanding of how HOCl produced at sites of inflammation might damage chromatin.


Subject(s)
Histones/metabolism , Hypochlorous Acid/pharmacology , Tyrosine/analogs & derivatives , Amino Acid Sequence , Chlorine/chemistry , Histones/chemistry , Histones/isolation & purification , Mass Spectrometry , Molecular Sequence Data , Tyrosine/chemistry , Tyrosine/metabolism
15.
Protein Eng Des Sel ; 21(3): 171-85, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18203802

ABSTRACT

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).


Subject(s)
Protein Structure, Secondary , Tryptophan/chemistry , Amino Acid Sequence , Circular Dichroism , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Proline/chemistry , Protein Folding , Thermodynamics , Tyrosine/chemistry
16.
Chem Res Toxicol ; 20(12): 1787-96, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17914883

ABSTRACT

The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2'-deoxyuridine (HmdU), has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analogue. This derivatized compound can be detected by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from the incorporation of exogenous HmdU. We further demonstrate that the addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.


Subject(s)
DNA Damage , DNA Repair , Gas Chromatography-Mass Spectrometry/methods , Thymidine/analogs & derivatives , Biomarkers/analysis , Cell Line, Tumor , Cell Survival/drug effects , Formates/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Hydrolysis , Sensitivity and Specificity , Thymidine/analysis , Thymidine/pharmacology , Thymine/metabolism
17.
Mol Cancer Ther ; 6(1): 163-72, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17218638

ABSTRACT

The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable, small-molecule inhibitor of the pim-1 kinase to foster basic and translational studies of the enzyme. We used an ELISA-based kinase assay to screen a diversity library of potential kinase inhibitors. The flavonol quercetagetin (3,3',4',5,6,7-hydroxyflavone) was identified as a moderately potent, ATP-competitive inhibitor (IC(50), 0.34 micromol/L). Resolution of the crystal structure of PIM1 in complex with quercetagetin or two other flavonoids revealed a spectrum of binding poses and hydrogen-bonding patterns in spite of strong similarity of the ligands. Quercetagetin was a highly selective inhibitor of PIM1 compared with PIM2 and seven other serine-threonine kinases. Quercetagetin was able to inhibit PIM1 activity in intact RWPE2 prostate cancer cells in a dose-dependent manner (ED(50), 5.5 micromol/L). RWPE2 cells treated with quercetagetin showed pronounced growth inhibition at inhibitor concentrations that blocked PIM1 kinase activity. Furthermore, the ability of quercetagetin to inhibit the growth of other prostate epithelial cell lines varied in proportion to their levels of PIM1 protein. Quercetagetin can function as a moderately potent and selective, cell-permeable inhibitor of the pim-1 kinase, and may be useful for proof-of-concept studies to support the development of clinically useful PIM1 inhibitors.


Subject(s)
Chromones/analysis , Chromones/pharmacology , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Chromones/chemistry , Crystallography, X-Ray , Flavones , Flavonoids/chemistry , Flavonoids/pharmacology , Humans , Male , Phenotype , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/chemistry , Protein Structure, Secondary , Proto-Oncogene Proteins c-pim-1/chemistry , Proto-Oncogene Proteins c-pim-1/deficiency , Sensitivity and Specificity , Substrate Specificity
18.
Chem Res Toxicol ; 19(4): 556-62, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16608167

ABSTRACT

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine-containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 A from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.


Subject(s)
Cytosine/chemistry , DNA Restriction Enzymes/chemistry , DNA/chemistry , Halogens/chemistry , Methyltransferases/chemistry
19.
Chem Res Toxicol ; 19(3): 414-20, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16544946

ABSTRACT

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.


Subject(s)
Aldehydes/analysis , Aldehydes/toxicity , Animals , Cattle , Chromatography, High Pressure Liquid , DNA/chemistry , DNA/drug effects , DNA Adducts/analysis , Indicators and Reagents , Magnetic Resonance Spectroscopy , Reference Standards , Salmonella typhimurium/drug effects , Salmonella typhimurium/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet
20.
Biopolymers ; 65(5): 354-61, 2002 Dec 05.
Article in English | MEDLINE | ID: mdl-12389215

ABSTRACT

Sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles are often used to mimic the membrane- or receptor-bound states of peptides in NMR studies. From the present examination of a 26-residue analog of exendin-4 (TrEX4) by NMR and CD in water, aqueous 30% trifluoroethanol (TFE), and bound to both SDS and DPC micelles, it is clear that these two lipid micelles can yield very different peptide structures. The Trp-cage fold (also observed in 30% TFE) is present when TrEX4 is bound to SDS micelles; however, tertiary structure is absent in the presence of DPC micelles. The loss of tertiary structure is attributed to an energetically favorable interaction (estimated as 2-3 kcal/mol) of the tryptophan side chain with the phosphocholine head groups. These dramatic structural differences suggest that care must be taken when using either SDS or DPC to mimic the membrane- or receptor-bound states.


Subject(s)
Peptides/chemistry , Tryptophan/chemistry , Venoms , Amino Acid Sequence , Circular Dichroism , Exenatide , In Vitro Techniques , Micelles , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Phosphorylcholine/chemistry , Protein Conformation , Solutions
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