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Annals of Neurology May 2017Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the...
OBJECTIVE
Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2 ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy.
METHODS
To test these hypotheses, we assessed two therapies in Tk2 mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP.
RESULTS
We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2 animals compared to dCMP+dTMP.
INTERPRETATION
Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.
Topics: Animals; Antimetabolites; DNA, Mitochondrial; Deoxycytidine Monophosphate; Disease Models, Animal; Drug Therapy, Combination; Metabolism, Inborn Errors; Mice; Mice, Transgenic; Mitochondrial Diseases; Tetrahydrouridine; Thymidine; Thymidine Kinase
PubMed: 28318037
DOI: 10.1002/ana.24922 -
Cell Death & Disease Feb 2024Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has...
Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.
Topics: Humans; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Deoxycytidine; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Gemcitabine; Paclitaxel; Pancreatic Neoplasms; Reactive Oxygen Species
PubMed: 38346958
DOI: 10.1038/s41419-024-06531-x -
International Journal of Clinical and... 2013Rheumatoid arthritis (RA) is a complex, multi-system disease whose primary site of inflammatory tissue damage is the joint. The increasing evidences indicate that...
Rheumatoid arthritis (RA) is a complex, multi-system disease whose primary site of inflammatory tissue damage is the joint. The increasing evidences indicate that activated RA fibroblast-like synoviocytes (FLS) play a critical role in the development of pannus by migrating into cartilage and bone. Furthermore FLS and T cells can activate each other in vitro and in vivo, which is crucial for the progress of RA. Deoxycytidine kinase (DCK) has been linked to peripheral T cell homeostatic proliferation and survival, which is very important for RA. Yet, the function of DCK in FLS is still unknown. Here, we present a story that DCK could regulate the migration and invasion of FLS through AKT pathway in RA patients. Moreover, DCK seems to be the upstream of AKT and FAK, and AKT inhibitor exerted the similar effect on FLS motility. In summary, our study characterized the new role of DCK in human primary FLS cells, and figured out the possible pathway DCK involved in, and these findings might propose DCK as a novel target for controlling joint destruction of RA.
Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Movement; Cells, Cultured; Cytoskeleton; Deoxycytidine Kinase; Female; Fibroblasts; Focal Adhesion Kinase 1; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Middle Aged; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Synovial Membrane; Tissue Inhibitor of Metalloproteinase-2; Transfection
PubMed: 24294360
DOI: No ID Found -
PloS One 2014Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA...
Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.
Topics: Animals; Ataxia Telangiectasia Mutated Proteins; B-Lymphocytes; Cell Line, Tumor; DNA Damage; DNA Repair; Deoxycytidine; Deoxycytidine Kinase; Deoxyribonucleosides; Genomic Instability; Hematopoiesis; Humans; Mice; Mice, Inbred BALB C; Mice, Knockout; Mutagenesis, Site-Directed; Phosphorylation; Protein Processing, Post-Translational; Substrate Specificity; T-Lymphocytes
PubMed: 25101980
DOI: 10.1371/journal.pone.0104125 -
Asian Journal of Pharmaceutical Sciences Jan 2022Gemcitabine has been extensively applied in treating various solid tumors. Nonetheless, the clinical performance of gemcitabine is severely restricted by its... (Review)
Review
Gemcitabine has been extensively applied in treating various solid tumors. Nonetheless, the clinical performance of gemcitabine is severely restricted by its unsatisfactory pharmacokinetic parameters and easy deactivation mainly because of its rapid deamination, deficiencies in deoxycytidine kinase (DCK), and alterations in nucleoside transporter. On this account, repeated injections with a high concentration of gemcitabine are adopted, leading to severe systemic toxicity to healthy cells. Accordingly, it is highly crucial to fabricate efficient gemcitabine delivery systems to obtain improved therapeutic efficacy of gemcitabine. A large number of gemcitabine pro-drugs were synthesized by chemical modification of gemcitabine to improve its biostability and bioavailability. Besides, gemcitabine-loaded nano-drugs were prepared to improve the delivery efficiency. In this review article, we introduced different strategies for improving the therapeutic performance of gemcitabine by the fabrication of pro-drugs and nano-drugs. We hope this review will provide new insight into the rational design of gemcitabine-based delivery strategies for enhanced cancer therapy.
PubMed: 35261643
DOI: 10.1016/j.ajps.2021.06.001 -
Multiple Sclerosis and Related Disorders Feb 2024Neutropenia serves as a risk factor for severe infection and is a consequence of some immune-depleting immunotherapies. This occurs in people with multiple sclerosis... (Review)
Review
Neutropenia serves as a risk factor for severe infection and is a consequence of some immune-depleting immunotherapies. This occurs in people with multiple sclerosis following chemotherapy-conditioning in haematopoietic stem cell transplantation and potent B cell targeting agents. Whilst CD52 is expressed by neutrophils and may contribute to early-onset neutropenia following alemtuzumab treatment, deoxycytidine kinase and CD20 antigen required for activity of cladribine tablets, off-label rituximab, ocrelizumab, ofatumumab and ublituximab are not or only weakly expressed by neutrophils. Therefore, alternative explanations are needed for the rare occurrence of early and late-onset neutropenia following such treatments. This probably occurs due to alterations in the balance of granulopoiesis and neutrophil removal. Neutrophils are short-lived, and their removal may be influenced by drug-associated infections, the killing mechanisms of the therapies and amplified by immune dyscrasia due to influences on neutropoiesis following growth factor rerouting for B cell recovery and cytokine deficits following lymphocyte depletion. This highlights the small but evident neutropenia risks following sustained B cell depletion with some treatments.
Topics: Humans; Multiple Sclerosis; Alemtuzumab; Rituximab; Immunologic Factors; Neutropenia; Antigens, CD20
PubMed: 38181696
DOI: 10.1016/j.msard.2023.105400 -
Journal of Neuroimmune Pharmacology :... Jun 2022Deoxycytidine kinase (dCK) and 5' deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by...
Deoxycytidine kinase (dCK) and 5' deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA.
Topics: Cladribine; Deoxycytidine Kinase
PubMed: 33851318
DOI: 10.1007/s11481-021-09994-3 -
Drug Metabolism and Disposition: the... Sep 2008Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK)...
Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) catalyze these reactions. We have applied a genotype-to-phenotype strategy to study DCK and CMPK pharmacogenomics. Specifically, we resequenced DCK and CMPK using 240 DNA samples, 60 each from African-American, Caucasian-American, Han Chinese-American, and Mexican-American subjects. We observed 28 DCK polymorphisms and 28 polymorphisms in CMPK, 33 of which were novel. Expression in COS-1 cells showed that variant allozyme enzyme activities ranged from 32 to 105% of the wild type (WT) for DCK and from 78 to 112% of WT for CMPK--with no significant differences in apparent K(m) values for either enzyme except for a DCK Val24/Ser122 double variant allozyme. Relative levels of DCK and CMPK immunoreactive protein in the COS-1 cells paralleled relative levels of enzyme activity and were significantly correlated for DCK (R(p) = 0.89, P = 0.0004) but not for CMPK (R(p) = 0.82, P = 0.095). The results of an analysis of DCK and CMPK structural models were compatible with the observed functional consequences of sequence alterations in variant allozymes. We also confirmed that the CMPK protein expressed in COS-1 cells and in a rabbit reticulocyte lysate was 196 rather than 228 amino acids in length. In summary, we determined common sequence variations in DCK and CMPK and systematically evaluated their functional implications. These gene sequence differences may contribute to variations in the metabolic activation of gemcitabine and other cytidine antimetabolites.
Topics: Antineoplastic Agents; Deoxycytidine; Deoxycytidine Kinase; Haplotypes; Humans; Kinetics; Linkage Disequilibrium; Models, Molecular; Nucleoside-Phosphate Kinase; Pharmacogenetics; Gemcitabine
PubMed: 18556440
DOI: 10.1124/dmd.108.020925 -
Cells Dec 2021Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action...
Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.
Topics: Apoptosis; Cell Differentiation; Cell Proliferation; Cladribine; Deoxycytidine Kinase; Humans; Immunomodulation; Leukocytes, Mononuclear; Lipopolysaccharides; Monocytes; Prodrugs
PubMed: 34943995
DOI: 10.3390/cells10123488