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1.
PLoS One ; 18(3): e0283428, 2023.
Article in English | MEDLINE | ID: mdl-36996070

ABSTRACT

Nicotinamide adenine dinucleotide (NAD) is a central molecule in cellular metabolism that has been implicated in human health, the aging process, and an array of human diseases. NAD is well known as an electron storage molecule, cycling between NAD and the reduced NADH. In addition, NAD is cleaved into nicotinamide and Adenine diphosphate ribose by NAD-consuming enzymes such as sirtuins, PARPs and CD38. There are numerous pathways for the biosynthesis of NAD to maintain a baseline concentration and thus avoid cellular death. The NAD salvage pathway, a two-step process to regenerate NAD after cleavage, is the predominant pathway for humans. Nicotinamide PhosphribosylTransferase (NAMPT) is the rate-limiting enzyme within the salvage path. Exposure to pharmacological modulators of NAMPT has been reported to either deplete or increase NAD levels. This study used a curated set of virtual compounds coupled with biochemical assays to identify novel activators of NAMPT. Autodock Vina generated a ranking of the National Cancer Institute's Diversity Set III molecular library. The library contains a set of organic molecules with diverse functional groups and carbon skeletons that can be used to identify lead compounds. The target NAMPT surface encompassed a novel binding location that included the NAMPT dimerization plane, the openings to the two active site channels, and a portion of the known binding location for NAMPT substrate and product. Ranked molecules were evaluated in a biochemical assay using purified recombinant NAMPT enzyme. Two novel carbon skeletons were confirmed to stimulate NAMPT activity. Compound 20 (NSC9037) is a polyphenolic xanthene derivative in the fluorescein family, while compound 2 (NSC19803) is the polyphenolic myricitrin nature product. Micromolar quantities of compound 20 or compound 2 can double NAMPT's product formation. In addition, natural products that contain high concentrations of polyphenolic flavonoids, similar to myricitrin, also stimulate NAMPT activity. Confirmation of a novel binding site for these compounds will further our understanding of the cellular mechanism leading to NAD homeostasis and better human health outcomes.


Subject(s)
Neoplasms , Sirtuins , Humans , NAD/metabolism , Cytokines/metabolism , Niacinamide , Sirtuins/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism
2.
Curr Mol Pharmacol ; 5(1): 68-87, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22122465

ABSTRACT

DNA damage plays a causal role in numerous disease processes. Hence, it is suggested that DNA repair proteins, which maintain the integrity of the nuclear and mitochondrial genomes, play a critical role in reducing the onset of multiple diseases, including cancer, diabetes and neurodegeneration. As the primary DNA polymerase involved in base excision repair, DNA polymerase ß (Polß) has been implicated in multiple cellular processes, including genome maintenance and telomere processing and is suggested to play a role in oncogenic transformation, cell viability following stress and the cellular response to radiation, chemotherapy and environmental genotoxicants. Therefore, Polß inhibitors may prove to be effective in cancer treatment. However, Polß has a complex and highly regulated role in DNA metabolism. This complicates the development of effective Polß-specific inhibitors useful for improving chemotherapy and radiation response without impacting normal cellular function. With multiple enzymatic activities, numerous binding partners and complex modes of regulation from post-translational modifications, there are many opportunities for Polß inhibition that have yet to be resolved. To shed light on the varying possibilities and approaches of targeting Polß for potential therapeutic intervention, we summarize the reported small molecule inhibitors of Polß and discuss the genetic, biochemical and chemical studies that implicate additional options for Polß inhibition. Further, we offer suggestions on possible inhibitor combinatorial approaches and the potential for tumor specificity for Polß-inhibitors.


Subject(s)
DNA Polymerase beta/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Neoplasms/drug therapy , Neoplasms/enzymology , Biological Products/chemistry , Biological Products/therapeutic use , DNA Polymerase beta/genetics , DNA Polymerase beta/metabolism , DNA Repair/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Mutation , Protein Processing, Post-Translational
3.
Protein J ; 30(1): 1-8, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21113733

ABSTRACT

The American Cancer Society's 2009 statistics estimate that 1 out of every 4 deaths is cancer related. Genomic instability is a common feature of cancerous states, and an increase in genomic instability is the diagnostic feature of Bloom Syndrome. Bloom Syndrome, a rare disorder characterized by a predisposition to cancer, is caused by mutations of the BLM gene. This study focuses on the partnerships of BLM protein to RAD51, a Homologous Recombination repair protein essential for survival. A systematic set of BLM deletion fragments were generated to refine the protein binding domains of BLM to RAD51 and determine interacting regions of BLM and ssDNA. Results show that RAD51 and ssDNA interact in overlapping regions; BLM100₋214 and BLM1317₋1367. The overlapping nature of these regions suggests a preferential binding for one partner that could function to regulate homologous recombination and therefore helps to clarify the role of BLM in maintaining genomic stability.


Subject(s)
Genomic Instability/genetics , Protein Interaction Domains and Motifs/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , RecQ Helicases/genetics , RecQ Helicases/metabolism , Binding, Competitive , Bloom Syndrome/genetics , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/genetics , Humans , RecQ Helicases/isolation & purification , Recombination, Genetic
4.
Antioxid Redox Signal ; 14(12): 2491-507, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-20649466

ABSTRACT

Nuclear and mitochondrial genomes are under continuous assault by a combination of environmentally and endogenously derived reactive oxygen species, inducing the formation and accumulation of mutagenic, toxic, and/or genome-destabilizing DNA lesions. Failure to resolve these lesions through one or more DNA-repair processes is associated with genome instability, mitochondrial dysfunction, neurodegeneration, inflammation, aging, and cancer, emphasizing the importance of characterizing the pathways and proteins involved in the repair of oxidative DNA damage. This review focuses on the repair of oxidative damage-induced lesions in nuclear and mitochondrial DNA mediated by the base excision repair (BER) pathway in mammalian cells. We discuss the multiple BER subpathways that are initiated by one of 11 different DNA glycosylases of three subtypes: (a) bifunctional with an associated ß-lyase activity; (b) monofunctional; and (c) bifunctional with an associated ß,δ-lyase activity. These three subtypes of DNA glycosylases all initiate BER but yield different chemical intermediates and hence different BER complexes to complete repair. Additionally, we briefly summarize alternate repair events mediated by BER proteins and the role of BER in the repair of mitochondrial DNA damage induced by ROS. Finally, we discuss the relation of BER and oxidative DNA damage in the onset of human disease.


Subject(s)
DNA Damage , DNA Repair , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Humans , Molecular Structure , Neoplasms/genetics , Oxidation-Reduction , Reactive Oxygen Species/metabolism
5.
Mutat Res ; 673(2): 141-8, 2009 Mar 17.
Article in English | MEDLINE | ID: mdl-19429515

ABSTRACT

Arylphosphonium salts (APS) are compounds that have both lipophilic and cationic character, allowing them facile transport through plasma membranes or cell walls to accumulate in the cytoplasm or mitochondria of cells. APS molecules preferentially accumulate in tumor cells and are therefore under investigation as tumor imaging agents and mitochondrial targeting molecules. We have generated a systematic set of APS to study their ability to associate with DNA. The chemical structure of the APS determines the extent of its interaction with DNA and therefore its ability to aggregate the DNA. Also, APS compounds blocked DNA amplification in vitro at concentrations below the aggregation threshold, corroborating the structure/interaction relationship. Furthermore, the extent of APS:DNA interaction strongly correlates with bacterial toxicity, implying that APS molecules may deter cellular metabolic DNA pathways. Finally, DNA repair deficient and DNA bypass polymerase deficient bacterial strains were screened for sensitivity to APS. Interestingly, no single pathway for the repair or tolerance of these compounds was solely responsible for APS mediated toxicity. Taken together, these findings suggest that APS compounds may be capable of targeting and regulating unchecked cell growth and therefore show potential applications as a chemotherapeutic agent.


Subject(s)
DNA, Bacterial/metabolism , Organophosphorus Compounds/metabolism , Organophosphorus Compounds/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/metabolism , Anti-Infective Agents/pharmacology , Cations/pharmacology , DNA/drug effects , DNA/metabolism , DNA, Bacterial/drug effects , Dose-Response Relationship, Drug , Escherichia coli/drug effects , Escherichia coli/genetics , Gene Amplification/drug effects , Microbial Sensitivity Tests , Microbial Viability/drug effects , Models, Biological , Molecular Conformation , Organophosphorus Compounds/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Salts/chemistry , Salts/metabolism , Salts/pharmacology , Structure-Activity Relationship
6.
Cytometry A ; 71(12): 991-1002, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17929292

ABSTRACT

Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm(3)/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10(5). This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.


Subject(s)
Image Cytometry/methods , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence, Multiphoton/methods , Animals , Cell Count/methods , Green Fluorescent Proteins/analysis , Humans , Image Cytometry/instrumentation , Luminescent Proteins/analysis , Mice , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton/instrumentation , NIH 3T3 Cells , Skin/cytology
7.
DNA Repair (Amst) ; 6(6): 695-711, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17337257

ABSTRACT

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neuro-degeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multi-protein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation.


Subject(s)
DNA Damage , DNA Repair , DNA/chemistry , Protein Processing, Post-Translational , Animals , DNA Glycosylases/metabolism , DNA Topoisomerases/metabolism , Humans , Models, Genetic , Phosphorylation
8.
Cancer Res ; 65(14): 6394-400, 2005 Jul 15.
Article in English | MEDLINE | ID: mdl-16024643

ABSTRACT

DNA-alkylating agents have a central role in the curative therapy of many human tumors; yet, resistance to these agents limits their effectiveness. The efficacy of the alkylating agent temozolomide has been attributed to the induction of O6-MeG, a DNA lesion repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT). Resistance to temozolomide has been ascribed to elevated levels of MGMT and/or reduced mismatch repair. However, >80% of the DNA lesions induced by temozolomide are N-methylated bases that are recognized by DNA glycosylases and not by MGMT, and so resistance to temozolomide may also be due, in part, to robust base excision repair (BER). We used isogenic cells deficient in the BER enzymes DNA polymerase-beta (pol-beta) and alkyladenine DNA glycosylase (Aag) to determine the role of BER in the cytotoxic effect of temozolomide. Pol-beta-deficient cells were significantly more susceptible to killing by temozolomide than wild-type or Aag-deficient cells, a hypersensitivity likely caused by accumulation of BER intermediates. RNA interference-mediated pol-beta suppression was sufficient to increase temozolomide efficacy, whereas a deficiency in pol-iota or pol-lambda did not increase temozolomide-mediated cytotoxicity. Overexpression of Aag (the initiating BER enzyme) triggered a further increase in temozolomide-induced cytotoxicity. Enhanced Aag expression, coupled with pol-beta knockdown, increased temozolomide efficacy up to 4-fold. Furthermore, loss of pol-beta coupled with temozolomide treatment triggered the phosphorylation of H2AX, indicating the activation of the DNA damage response pathway as a result of unrepaired lesions. Thus, the BER pathway is a major contributor to cellular resistance to temozolomide and its efficacy depends on specific BER gene expression and activity.


Subject(s)
DNA Repair/physiology , Dacarbazine/analogs & derivatives , Animals , Antineoplastic Agents, Alkylating/pharmacology , Cell Death/drug effects , Cell Death/physiology , Cell Line, Transformed , DNA Glycosylases/biosynthesis , DNA Glycosylases/deficiency , DNA Glycosylases/metabolism , DNA Polymerase beta/deficiency , DNA Polymerase beta/genetics , DNA Polymerase beta/metabolism , Dacarbazine/pharmacology , Drug Resistance, Neoplasm , Histones/metabolism , Mice , Phosphorylation , RNA, Small Interfering/genetics , Temozolomide , Transfection
9.
Oncogene ; 24(32): 5016-25, 2005 Jul 28.
Article in English | MEDLINE | ID: mdl-15856014

ABSTRACT

Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyper-recombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a million-fold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.


Subject(s)
Antineoplastic Agents/pharmacology , Mitomycin/pharmacology , Recombination, Genetic/drug effects , Stem Cells/physiology , Animals , Bystander Effect , Cell Communication , Mice , Stem Cells/cytology , Stem Cells/drug effects
10.
J Biol Chem ; 278(41): 39951-9, 2003 Oct 10.
Article in English | MEDLINE | ID: mdl-12882965

ABSTRACT

DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase beta (beta-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of beta-pol-mediated 5'-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of beta-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that beta-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery.


Subject(s)
DNA Repair/physiology , Alkylating Agents/toxicity , Animals , Base Sequence , Cells, Cultured , DNA/biosynthesis , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Glycosylases/deficiency , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , DNA Methylation , DNA Polymerase beta/deficiency , DNA Polymerase beta/genetics , DNA Polymerase beta/metabolism , DNA Repair/genetics , Methyl Methanesulfonate/toxicity , Methylnitronitrosoguanidine/toxicity , Mice , Mice, Knockout , Mutation , Phenotype , Recombination, Genetic , Tumor Suppressor Protein p53/metabolism
11.
Proc Natl Acad Sci U S A ; 100(11): 6325-30, 2003 May 27.
Article in English | MEDLINE | ID: mdl-12750464

ABSTRACT

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions. Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.


Subject(s)
Bacterial Proteins/genetics , Luminescent Proteins/genetics , Mitosis/genetics , Recombination, Genetic , Repetitive Sequences, Nucleic Acid , Animals , DNA Damage , DNA, Complementary , Mice , Mice, Transgenic
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