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The Journal of Biological Chemistry Nov 2007In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a...
In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.
Topics: Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cytoplasm; Cytosol; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Hydroxyurea; Mitochondria; Models, Biological; Models, Genetic; RNA Interference; Thymidine Kinase; Thymine Nucleotides
PubMed: 17913703
DOI: 10.1074/jbc.M705923200 -
Thymidine kinase 1 as a tumor biomarker: technical advances offer new potential to an old biomarker.Biomarkers in Medicine Sep 2018Thymidine kinase 1 (TK1) is a key enzyme in DNA precursor synthesis. It is upregulated during the S phase of the cell cycle and its presence in cells is an indicator of... (Review)
Review
Thymidine kinase 1 (TK1) is a key enzyme in DNA precursor synthesis. It is upregulated during the S phase of the cell cycle and its presence in cells is an indicator of active cell proliferation. In studies since the 1980s, TK1 has been shown as a clinically valuable biomarker for the management of hematological malignancies. However, TK1 activity assays may underestimate serum TK1 in subjects with solid tumors limiting its sensitivity. The development of TK1 immunoassays has made the assay of TK1 more widely available and increased its applicability to solid tumor diseases. This paper will review TK1 as a tumor biomarker with emphasis on recent studies and technologies plus highlight its potential in drug discovery and as a therapeutic target.
Topics: Biomarkers, Tumor; Hematologic Neoplasms; Humans; Neoplasm Proteins; Thymidine Kinase
PubMed: 30039979
DOI: 10.2217/bmm-2018-0157 -
Analytica Chimica Acta Feb 2019Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on...
Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry.
Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.
Topics: Enzymes, Immobilized; Flow Injection Analysis; Humans; Kinetics; Mass Spectrometry; Nanoparticles; Nucleoside-Diphosphate Kinase; Nucleoside-Phosphate Kinase; Phosphorylation; Thymidine; Thymidine Kinase
PubMed: 30612642
DOI: 10.1016/j.aca.2018.10.032 -
PloS One 2022Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for both hematological and solid malignancies. However, recent...
Selection of human single domain antibodies (sdAb) against thymidine kinase 1 and their incorporation into sdAb-Fc antibody constructs for potential use in cancer therapy.
Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for both hematological and solid malignancies. However, recent studies targeting TK1 at protein and mRNA levels have shown that TK1 may be useful as a therapeutic target. In order to examine the use of TK1 as a therapeutic target, it is necessary to develop therapeutics specific for it. Single domain antibodies (sdAbs), represent an exciting approach for the development of immunotherapeutics due to their cost-effective production and higher tumor penetration than conventional antibodies. In this study, we isolated sdAb fragments specific to human TK1 from a human sdAb library. A total of 400 sdAbs were screened through 5 rounds of selection by monoclonal phage ELISA. The most sensitive sdAb fragments were selected as candidates for preclinical testing. The sdAb fragments showed specificity for human TK1 in phage ELISA, Western blot analysis and had an estimated limit of detection of 3.9 ng/ml for the antibody fragments 4-H-TK1_A1 and 4-H-TK1_D1. The antibody fragments were successfully expressed and used for detection of membrane associated TK1 (mTK1) through flow cytometry on cancer cells [lung (~95%), colon (~87%), breast (~53%)] and healthy human mononuclear cells (MNC). The most sensitive antibody fragments, 4-H-TK1_A1 and 4-H-TK1_D1 were fused to an engineered IgG1 Fc fragment. When added to cancer cells expressing mTK1 co-cultured with human MNCs, the anti-TK1-sdAb-IgG1_A1 and D1 were able to elicit a significant antibody-dependent cell-mediated cytotoxicity (ADCC) response against lung cancer cells compared to isotype controls (P<0.0267 and P<0.0265, respectively). To our knowledge this is the first time that the isolation and evaluation of human anti-TK1 single domain antibodies using phage display technology has been reported. The antibody fragments isolated here may represent a valuable resource for the detection and the targeting of TK1 on tumor cells.
Topics: Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Neoplasms; Single-Domain Antibodies; Thymidine Kinase
PubMed: 35239730
DOI: 10.1371/journal.pone.0264822 -
Genes Feb 2023Short tandem DNA repeats are drivers of genome instability. To identify suppressors of break-induced mutagenesis human cells, unbiased genetic screens were conducted...
Short tandem DNA repeats are drivers of genome instability. To identify suppressors of break-induced mutagenesis human cells, unbiased genetic screens were conducted using a lentiviral shRNA library. The recipient cells possessed fragile non-B DNA that could induce DNA double-strand breaks (DSBs), integrated at an ectopic chromosomal site adjacent to a thymidine kinase marker gene. Mutagenesis of the thymidine kinase gene rendered cells resistant to the nucleoside analog ganciclovir (GCV). The screen identified genes that have established roles in DNA replication and repair, chromatin modification, responses to ionizing radiation, and genes encoding proteins enriched at replication forks. Novel loci implicated in BIR included olfactory receptors, the G0S2 oncogene/tumor suppressor axis, the EIF3H-METTL3 translational regulator, and the SUDS3 subunit of the Sin3A corepressor. Consistent with a role in suppressing BIR, siRNA knockdown of selected candidates increased the frequency of the GCV phenotype and increased DNA rearrangements near the ectopic non-B DNA. Inverse PCR and DNA sequence analyses showed that hits identified in the screen increased genome instability. Further analysis quantitated repeat-induced hypermutagenesis at the ectopic site and showed that knockdown of a primary hit, COPS2, induced mutagenic hotspots, remodeled the replication fork, and increased nonallelic chromosome template switches.
Topics: Humans; Thymidine Kinase; DNA Repair; DNA Replication; Recombination, Genetic; Genomic Instability; Methyltransferases
PubMed: 36833325
DOI: 10.3390/genes14020398 -
Medicinal Research Reviews Sep 2008Human mitochondrial thymidine kinase (TK2) is a pyrimidine deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of pyrimidine deoxynucleosides to their... (Review)
Review
Human mitochondrial thymidine kinase (TK2) is a pyrimidine deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of pyrimidine deoxynucleosides to their corresponding deoxynucleoside 5'-monophosphates by gamma-phosphoryl transfer from ATP. In resting cells, TK2 is suggested to play a key role in the mitochondrial salvage pathway to provide pyrimidine nucleotides for mitochondrial DNA (mtDNA) synthesis and maintenance. However, recently the physiological role of TK2turned out to have direct clinical relevance as well. Point mutations in the gene encoding TK2 have been correlated to mtDNA disorders in a heterogeneous group of patients suffering from the so-called mtDNA depletion syndrome (MDS). TK2 activity could also be involved in mitochondrial toxicity associated to prolonged treatment with antiviral nucleoside analogues like AZT and FIAU. Therefore, TK2 inhibitors can be considered as valuable tools to unravel the role of TK2 in the maintenance and homeostasis of mitochondrial nucleotide pools and mtDNA, and to clarify the contribution of TK2 activity to mitochondrial toxicity of certain antivirals. Highly selective TK-2 inhibitors having an acyclic nucleoside structure and efficiently discriminating between TK-2 and the closely related TK-1 have already been reported. It is actually unclear whether these agents efficiently reach the inner mitochondrial compartment. In the present review article,structural features of TK2, MDS-related mutations observed in TK2 and their role in MDS will be discussed. Also, an update on novel and selective TK2 inhibitors will be provided.
Topics: Amino Acid Sequence; DNA, Mitochondrial; Enzyme Inhibitors; Humans; Mitochondria; Molecular Sequence Data; Sequence Alignment; Thymidine Kinase; Zidovudine
PubMed: 18459168
DOI: 10.1002/med.20124 -
International Journal of Molecular... Aug 2021Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the... (Review)
Review
Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM-CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT-GM-CSF-danger signal system by means of artificial cancer specific promoters or a modified delivery system.
Topics: Animals; Cancer Vaccines; Gene Transfer Techniques; Genes, Transgenic, Suicide; Genetic Therapy; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy; Neoplasms; Oncolytic Virotherapy; Prodrugs; Thymidine Kinase
PubMed: 34502287
DOI: 10.3390/ijms22179376 -
Applied and Environmental Microbiology May 1990One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA....
One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.
Topics: Autoradiography; Bacteria; Escherichia coli; Simplexvirus; Thymidine; Thymidine Kinase; Water Microbiology
PubMed: 2160223
DOI: 10.1128/aem.56.5.1367-1372.1990 -
PloS One 2022Thymidine Kinase 1 (TK1) plays an important role in DNA precursor synthesis and serum TK1 activity has been used as a biomarker for prognosis and therapy monitoring of...
Thymidine Kinase 1 (TK1) plays an important role in DNA precursor synthesis and serum TK1 activity has been used as a biomarker for prognosis and therapy monitoring of different malignancies. AroCell has developed a dual monoclonal antibody ELISA for determination of TK1 protein in clinical samples. The purpose of the study is to validate the ELISA analytically in relation to the gold standard, [3H]-deoxythymidine (dThd) phosphorylation assay for TK1 activity using sera from patients with different malignancies. The colorimetric TK 210 ELISA was validated analytically by assessment of precision, linearity, interfering substances, and stability. For the clinical validation, serum samples from patients with hematological malignancies (n = 100), breast cancer (n = 56), prostate cancer (n = 70) and blood donors (n = 159) were analyzed using TK 210 ELISA and TK1 activity by [3H]-deoxythymidine (dThd) phosphorylation assay. The sandwich TK 210 ELISA was highly specific for TK1 protein having a detection limit of 0.12 ng/mL, with a functional sensitivity of 0.25 ng/mL. Within-run CVs ranged from 5.5% to 10% and between-run CVs ranged from 5% to 15%. The ratio of observed to expected dilutional parallelism of 5 serum samples was in the range of 80-120%. Samples exhibited stability through four freeze/thaw cycles and 5 days at 4°C. Further, the ROC curve analysis showed that TK 210 ELISA and [3H]-dThd phosphorylation assay had similar sensitivity (62% vs 59%) in hematological malignancies. However, in the case of breast and prostate cancer sera, TK 210 ELISA had higher sensitivity (59% and 44%) compared to [3H]-dThd phosphorylation assay (47% and 25%) at a specificity of 98%. These data demonstrate that the dual monoclonal antibody based AroCell TK 210 ELISA is a robust, accurate and precise tool for measuring TK1 protein in different malignancies that can improve the clinical applications of TK1 as a biomarker in cancer management.
Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Breast Neoplasms; DNA; Enzyme-Linked Immunosorbent Assay; Hematologic Neoplasms; Humans; Male; Prostatic Neoplasms; Thymidine; Thymidine Kinase
PubMed: 36201558
DOI: 10.1371/journal.pone.0275444 -
Journal of Chemical Information and... Jan 2022Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains...
Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains elusive. We perform microsecond molecular dynamics simulations of the inactive enzyme form bound to a bisubstrate inhibitor that was shown experimentally to activate another TK1-like kinase, TK (TK). Our results are in excellent agreement with the experimental findings for the TK closed-to-open state transition. We show that the inhibitor induces an increase of the enzyme radius of gyration due to the expansion on one of the dimer interfaces; the structural changes observed, including the active site pocket volume increase and the decrease in the monomer-monomer buried surface area and of the number of hydrogen bonds (as compared to the inactive enzyme control simulation), indicate that the catalytically competent (open) conformation of hTK1 can be assumed in the presence of an activating ligand.
Topics: Catalytic Domain; Humans; Molecular Dynamics Simulation; Protein Conformation; Thymidine Kinase
PubMed: 34919400
DOI: 10.1021/acs.jcim.1c01157