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Autophagy May 2021Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal...
Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by DNA methyltransferase DNMT3A of loci upon autophagy stimulation leads to the observed long-term decrease of isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions. ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2'-deoxycytidine; BafA1: bafilomycin A; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle's balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.
Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; DNA; DNA Methyltransferase 3A; Fibroblasts; Humans; Lysosomes; Memory, Long-Term; Methyltransferases; Mice; Zebrafish
PubMed: 32876528
DOI: 10.1080/15548627.2020.1816664 -
Clinical and Experimental Immunology Sep 2020Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier,...
Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. We examined if CdA modifies the phenotype and function of naive and activated primary mouse microglia, when applied in the concentrations 0·1-1 μM that putatively overlap human cerebrospinal fluid (CSF) concentrations. Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory interleukin (IL)-4 were treated with different concentrations of CdA for 24 h. Viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Phagocytotic ability and morphology were examined by flow cytometry and random migration using IncuCyte Zoom and TrackMate. Change in gene expression was examined by quantitative polymerase chain reaction (qPCR) and protein secretion by Meso Scale Discovery. We found that LPS and IL-4 up-regulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0·1-1 μM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 μM increased the IL-4-induced gene expression of arginase 1 (Arg1) and LPS-induced expression of IL-1β, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS) and Arg1, but protein secretion remained unaffected. CdA 10 μM potentiated the increased expression of anti-inflammatory TNF receptor 2 (TNF-R2) but not TNF-R1 induced by LPS. This suggests that microglia acquire a less activated phenotype when treated with 0·1-1 μM CdA that putatively overlaps human CSF concentrations. This may be related to the up-regulated gene expression of DCK upon activation, and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.
Topics: Animals; Anti-Inflammatory Agents; Blood-Brain Barrier; Cell Movement; Cells, Cultured; Cladribine; Gene Expression Regulation; Humans; Lymphocyte Depletion; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Phagocytosis; Receptors, Tumor Necrosis Factor, Type II
PubMed: 32492189
DOI: 10.1111/cei.13473 -
Nature Communications May 2023Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools....
Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.
Topics: Mice; Humans; Animals; Drug Repositioning; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Nucleotides; Drug Design; Disease Models, Animal
PubMed: 37248212
DOI: 10.1038/s41467-023-38668-2 -
Multiple Sclerosis and Related Disorders Jul 2021Cladribine is an approved selective immune reconstitution therapy for relapsing-remitting MS (RRMS). It was first developed and used to treat various forms of cancer,... (Review)
Review
Cladribine is an approved selective immune reconstitution therapy for relapsing-remitting MS (RRMS). It was first developed and used to treat various forms of cancer, particularly leukemia via parenteral administration. The oral tablet version of cladribine was later developed to treat RRMS, an autoimmune disorder of the central nervous system (CNS) with periods of relapse and remission. Cladribine is found to selectively deplete adaptive immune cell types, and its role on innate immune cells is largely unknown. Among the lymphocyte populations and subtypes, the magnitude and kinetics of depletion by cladribine vary substantially. The current consensus on the selective cytotoxic effect of cladribine is that it is dependent on the deoxycytidine kinase (DCK) to 5'nucleotidase (5-NT) ratio of the immune cell type. Nonetheless, there are some discrepancies that cannot be fully elucidated by the DCK:5-NT ratio paradigm. This review aims to delineate the development and pharmacological properties of cladribine, and elucidate its influence on lymphoid and myeloid cells in MS.
Topics: Cladribine; Humans; Immunosuppressive Agents; Multiple Sclerosis; Multiple Sclerosis, Relapsing-Remitting; Myeloid Cells
PubMed: 33901971
DOI: 10.1016/j.msard.2021.102962 -
Annals of Neurology Oct 2021Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants...
OBJECTIVE
Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2 ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2 mice.
METHODS
We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2 , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides.
RESULTS
AAV9 delivery of human TK2 cDNA to Tk2 mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2 mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2 mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan.
INTERPRETATION
Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2 mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.
Topics: Animals; Compassionate Use Trials; DNA, Mitochondrial; Genetic Therapy; Humans; Mice; Mitochondria; Mitochondrial Myopathies; Mutation; Thymidine; Thymidine Kinase
PubMed: 34338329
DOI: 10.1002/ana.26185 -
Molecular Cancer Therapeutics May 2024Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a...
Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared with FDA-approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a preclinical nucleoside prodrug candidate for DLBCL and ALL.
Topics: Humans; Animals; Deoxycytidine; Mice; Cell Line, Tumor; Lymphoma; Xenograft Model Antitumor Assays; Leukemia; Deoxycytidine Kinase
PubMed: 38064712
DOI: 10.1158/1535-7163.MCT-23-0487 -
Journal of Neuromuscular Diseases 2022Defects in the replication, maintenance, and repair of mitochondrial DNA (mtDNA) constitute a growing and genetically heterogeneous group of mitochondrial disorders.... (Review)
Review
Defects in the replication, maintenance, and repair of mitochondrial DNA (mtDNA) constitute a growing and genetically heterogeneous group of mitochondrial disorders. Multiple genes participate in these processes, including thymidine kinase 2 (TK2) encoding the mitochondrial matrix protein TK2, a critical component of the mitochondrial nucleotide salvage pathway. TK2 deficiency (TK2d) causes mtDNA depletion, multiple deletions, or both, which manifest predominantly as mitochondrial myopathy. A wide clinical spectrum phenotype includes a severe, rapidly progressive, early onset form (median survival: < 2 years); a less severe childhood-onset form; and a late-onset form with a variably slower rate of progression. Clinical presentation typically includes progressive weakness of limb, neck, facial, oropharyngeal, and respiratory muscle, whereas limb myopathy with ptosis, ophthalmoparesis, and respiratory involvement is more common in the late-onset form. Deoxynucleoside monophosphates and deoxynucleosides that can bypass the TK2 enzyme defect have been assessed in a mouse model, as well as under open-label compassionate use (expanded access) in TK2d patients, indicating clinical efficacy with a favorable side-effect profile. This treatment is currently undergoing testing in clinical trials intended to support approval in the US and European Union (EU). In the early expanded access program, growth differentiation factor 15 (GDF-15) appears to be a useful biomarker that correlates with therapeutic response. With the advent of a specific treatment and given the high morbidity and mortality associated with TK2d, clinicians need to know how to recognize and diagnose this disorder. Here, we summarize translational research about this rare condition emphasizing clinical aspects.
Topics: Animals; Child; DNA, Mitochondrial; Humans; Mice; Mitochondrial Myopathies; Muscular Diseases; Thymidine Kinase
PubMed: 35094997
DOI: 10.3233/JND-210786 -
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 Molecular... Apr 2023Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival...
Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.
Topics: Male; Humans; Deoxycytidine; Epithelial Cell Adhesion Molecule; Clinical Relevance; Gemcitabine; Pancreatic Neoplasms; Drug Resistance, Multiple; Neoplastic Stem Cells; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition
PubMed: 37108495
DOI: 10.3390/ijms24087331 -
Scientific Reports Oct 2019The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR,...
The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.
Topics: Adenosine; Amidines; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Caspases; Cell Line; Cell Line, Tumor; Cladribine; DNA Fragmentation; HL-60 Cells; Humans; Leukemia, Monocytic, Acute; Pyrazines; S Phase; Sulfones; THP-1 Cells
PubMed: 31575977
DOI: 10.1038/s41598-019-50509-1