scholarly journals Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress

2012 ◽  
Vol 209 (12) ◽  
pp. 2215-2228 ◽  
Author(s):  
Wayne R. Austin ◽  
Amanda L. Armijo ◽  
Dean O. Campbell ◽  
Arun S. Singh ◽  
Terry Hsieh ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Stress ◽  
Nucleotide Metabolism ◽  
Salvage Pathway ◽  
Double Knockout ◽  
Functional Link ◽  
Dividing Cells ◽  
Nucleoside Salvage ◽  
B Lymphoid

Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1−/− erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK−/− counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK “tune” dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.

scholarly journals HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of β-catenin, MYC, and WT1

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1436 ◽  
Author(s):  
Mandy Beyer ◽  
Annette Romanski ◽  
Al-Hassan M. Mustafa ◽  
Miriam Pons ◽  
Iris Büchler ◽  
...  
Keyword(s):  
Dna Damage ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Wilms Tumor ◽  
Leukemic Cell ◽  
Replication Stress ◽  
Leukemic Cells ◽  
Hdac6 Inhibitor

Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors β-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of β-catenin. Indomethacin destabilizes β-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of β-catenin by WT1. In conclusion, reduced levels of β-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.

scholarly journals Werner Protein Protects Nonproliferating Cells from Oxidative DNA Damage

2005 ◽  
Vol 25 (23) ◽  
pp. 10492-10506 ◽  
Author(s):  
Anna M. Szekely ◽  
Franziska Bleichert ◽  
Astrid Nümann ◽  
Stephen Van Komen ◽  
Elisabeth Manasanch ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Oxidative Dna Damage ◽  
Werner Syndrome ◽  
Human Fibroblasts ◽  
Telomere Maintenance ◽  
Telomere Binding Protein ◽  
Damage Response ◽  
Dividing Cells

ABSTRACT Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of γH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.

scholarly journals Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252587
Author(s):  
Yuriko Inomata ◽  
Takuya Abe ◽  
Masataka Tsuda ◽  
Shunichi Takeda ◽  
Kouji Hirota
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Division Of Labor ◽  
Double Knockout ◽  
Translesion Dna Synthesis ◽  
Translesion Dna ◽  
Template Dna ◽  
Y Family ◽  
Higher Sensitivity

Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη−/−, POLι−/−, POLκ−/−, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη−/− cells, but not POLι−/− or POLκ−/− cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη−/− cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.

scholarly journals Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Bente Benedict ◽  
Tanja van Harn ◽  
Marleen Dekker ◽  
Simone Hermsen ◽  
Asli Kucukosmanoglu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Origin Firing ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Cycle Arrest

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.

scholarly journals Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

2014 ◽  
Vol 211 (3) ◽  
pp. 473-486 ◽  
Author(s):  
David A. Nathanson ◽  
Amanda L. Armijo ◽  
Michelle Tom ◽  
Zheng Li ◽  
Elizabeth Dimitrova ◽  
...  
Keyword(s):  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Nucleotide Metabolism ◽  
Leukemic Cells ◽  
Biosynthetic Pathways ◽  
Metabolic Switch ◽  
Positron Emission ◽  
Molecule Inhibitor ◽  
Nucleoside Salvage

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

scholarly journals Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

2020 ◽  
Vol 48 (21) ◽  
pp. 12234-12251
Author(s):  
Torkild Visnes ◽  
Carlos Benítez-Buelga ◽  
Armando Cázares-Körner ◽  
Kumar Sanjiv ◽  
Bishoy M F Hanna ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Therapeutic Index ◽  
Replication Stress ◽  
Transformed Cells ◽  
Wide Range

Abstract Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

scholarly journals Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dulama Richani ◽  
Cathy F. Lavea ◽  
Raji Kanakkaparambil ◽  
Angelique H. Riepsamen ◽  
Michael J. Bertoldo ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Adenine Nucleotide ◽  
Cumulus Cells ◽  
Nucleotide Metabolism ◽  
Salvage Pathway ◽  
Developmental Potential ◽  
Mouse Oocytes ◽  
Junctional Communication

AbstractA follicular spike in cyclic AMP (cAMP) and its subsequent degradation to AMP promotes oocyte maturation and ovulation. In vitro matured (IVM) oocytes do not receive the cAMP increase that occurs in vivo, and artificial elevation of cAMP in IVM cumulus-oocyte complexes improves oocyte developmental potential. This study examined whether mouse oocytes can use the cAMP degradation product AMP to generate ATP via the adenosine salvage pathway, and examined whether pharmacological elevation of cAMP in IVM cumulus-oocyte complexes alters ATP levels. Oocytes cultured with isotopic 13C5-AMP dose-dependently produced 13C5-ATP, however total cellular ATP remained constant. Pharmacological elevation of cAMP using forskolin and IBMX prior to IVM decreased oocyte ATP and ATP:ADP ratio, and promoted activity of the energy regulator AMPK. Conversely, cumulus cells exhibited higher ATP and no change in AMPK. Culture of oocytes without their cumulus cells or inhibition of their gap-junctional communication yielded lower oocyte 13C5-ATP, indicating that cumulus cells facilitate ATP production via the adenosine salvage pathway. In conclusion, this study demonstrates that mouse oocytes can generate ATP from AMP via the adenosine salvage pathway, and cAMP elevation alters adenine nucleotide metabolism and may provide AMP for energy production via the adenosine salvage pathway during the energetically demanding process of meiotic maturation.

scholarly journals Hepatic NOD2 promotes hepatocarcinogenesis via a RIP2-mediated proinflammatory response and a novel nuclear autophagy-mediated DNA damage mechanism

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yi Zhou ◽  
Liang Hu ◽  
Wenqing Tang ◽  
Dongping Li ◽  
Lijie Ma ◽  
...  
Keyword(s):  
Dna Damage ◽  
Inflammatory Response ◽  
Genomic Instability ◽  
Muramyl Dipeptide ◽  
Lamin A ◽  
Double Knockout ◽  
Proinflammatory Response ◽  
Gut Dysbiosis

Abstract Background Key hepatic molecules linking gut dysbiosis and hepatocarcinogenesis remain largely unknown. Gut-derived gut microbiota contains pathogen-associated molecular patterns (PAMPs) that may circulate into the liver and, consequently, be recognized by hepatic pattern recognition receptors (PRRs). NOD2, a general intracellular PRR, recognizes muramyl dipeptide (MDP), present in both gram (+) and gram (−) bacteria. Here, we investigated the role of NOD2 as a molecular sensor translating gut dysbiosis signaling into hepatocarcinogenesis. Methods NOD2 expression was measured in clinical hepatocellular carcinoma (HCC) samples using qPCR (80 pairs), western blotting (30 pairs) and immunostaining (141 pairs). The role of NOD2 in hepatocarcinogenesis was examined in the hepatocyte-specific Nod2-knockout (Nod2△hep), Rip2-knockout (Rip2△hep), Lamin A/C-knockout (Lamn△hep) and Rip2/Lamin A/C double-knockout (Rip2/Lamn△hep) mice models of diethylnitrosamine (DEN)/CCl4-induced HCC. Results NOD2 was upregulated and activated in HCC samples, and high NOD2 expression correlated with poor prognosis in HCC patients. Hepatic NOD2 deletion in vivo decreased DEN/CCl4-induced HCC by reducing the inflammatory response, DNA damage and genomic instability. NOD2 activation increased liver inflammation via RIP2-dependent activation of the MAPK, NF-κB and STAT3 pathways. Notably, a novel RIP2-independent mechanism was discovered, whereby NOD2 activation induces the nuclear autophagy pathway. We showed that NOD2 undergoes nuclear transport and directly binds to a component of nuclear laminae, lamin A/C, to promote its protein degradation, leading to impaired DNA damage repair and increased genomic instability. Conclusions We reveal a novel bridge, bacterial sensor NOD2, linking gut-derived microbial metabolites to hepatocarcinogenesis via induction of the inflammatory response and nuclear autophagy. Thus, we propose hepatic NOD2 as a promising therapeutic target against HCC.

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium

1982 ◽  
Vol 152 (3) ◽  
pp. 1111-1116
Author(s):  
G Liu ◽  
J Foster ◽  
P Manlapaz-Ramos ◽  
B M Olivera
Keyword(s):  
Nicotinic Acid ◽  
Salmonella Typhimurium ◽  
Pyridine Nucleotide ◽  
Salvage Pathway ◽  
Nad Biosynthesis ◽  
Nicotinamide Mononucleotide ◽  
Nucleoside Salvage

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.

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