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At least 19 recordsLinked to original sources
PubMed1975

On the interaction of 3,4,5,6-tetrahydrouridine with human liver cytidine deaminase.

In contrast to the rapid inhibition of bacterial cytidine deaminase by 3,4,5,6-tetrahydrouridine, the onset of inhibition of the enzyme from human liver was found to be relatively slow. Inhibition was found to be reversible, and the corrected rate constants for binding (kon = 2.4 x 10(4) M-1 sec-1) and release (koff = 5.6 x 10(-4) sec-1) were in reasonable agreement with a Ki value (2.9 x 10(-8) M) measured separately under steady-state conditions, which was several orders of magnitude lower than estimates previously reported in the literature. Rates of binding and release of this potential transition state analogue were not appreciably affected by the substitution of deuterium oxide for solvent water. The slow onset of inhibition, which was also observed for cytidine deaminase from HeLa cells, suggests that structural reorganization precedes the formation of a stable enzyme-inhibitor complex. 6-Azacytidine, which favors a "high-anti" configuration at the glycosidic bond, was found to be active as a substrate for cytidine deaminase, with a turnover number exceeding that of cytidine. 2,2'-Anhydro-1-beta-D-arabinofuranosylcytosine, which is restricted to the "syn" configuration, was found to be without activity as a substrate or an inhibitor.

Azacitidine↗
PubMed2024

An in vitro cytidine deaminase assay to monitor APOBEC activity on DNA.

APOBEC enzymes promote the deamination of cytosine (C) to uracil (U) in DNA to defend cells against viruses but also serve as a predominant source of mutations in cancer genomes. This protocol describes an assay to monitor APOBEC deaminase activity in vitro on a synthetic DNA oligonucleotide. The method described here focuses specifically on APOBEC3B to illustrate the different steps of the assay. However, the protocol can be applied to monitor the DNA deaminase activity of any other member of the APOBEC family, such as APOBEC3A. This assay involves preparing APOBEC3B-expressing cell extract or purifying APOBEC3B by immunoprecipitation, followed by incubation with a single-stranded DNA containing a TpC motif. The deaminated cytosine is then removed by recombinant Uracil DNA Glycosylase present in the reaction to form an abasic site. The abasic site creates a weakness in the DNA's backbone, causing the DNA to be cleaved under high temperatures and alkaline conditions. Denaturing gel electrophoresis is used to separate cleaved DNA from full-length DNA, enabling the quantification of the percentage of deamination induced by APOBEC3B. This protocol can be used to determine the presence of APOBEC and the regulation of APOBEC activity in specific cell lines, to study substrate preference targeted by different members of the APOBEC family and different APOBEC mutants, or to determine the efficiency and specificity of inhibitor compounds against APOBEC enzymes.

Cytidine Deaminase↗
PubMed2026

Intratumoral Mycobacterium abscessus promotes cytidine deaminase mutagenesis in non-small cell lung cancer.

The intratumoral microbiota is increasingly recognized as an active component of the tumor microenvironment, yet whether it directly drives tumor mutagenesis remains unclear. Here, integrated multi-omics analysis of human non-small cell lung cancer (NSCLC) identifies Mycobacterium abscessus as a microbial determinant of APOBEC3A-associated mutagenesis. Mechanistically, the bacterial effector nucleoside diphosphate kinase (NDK) directly targets the host transcription factor IRF3 and installs a non-canonical 1-phosphohistidine modification at H263, thereby amplifying type I interferon signaling and sustaining APOBEC3A expression. This inter-kingdom phosphotransfer event links intratumoral microbial colonization to an endogenous mutational process that promotes genomic diversification. Genetic inactivation of NDK, or pharmacologic elimination using an engineered NDK-PROTAC, suppresses APOBEC3A activation and attenuates microbe driven mutagenesis. Together, these findings establish a direct microbial effector mechanism that promotes APOBEC3A-associated mutagenesis and provide a therapeutic framework to intercept microbiome driven mutagenesis in NSCLC.

Humans↗
PubMed1977

Blood lymphocytes in chronic lymphocytic leukaemia and Hodgkin's disease: Immunological features and enzymes of nucleoside metabolism.

Purified lymphocytes from venous blood of sixteen healthy adult subjects, eight patients with chronic lymphocytic leukaemia (CLL) and ten with Hodgkin's disease (HD), were examined for adenosine deaminase (ADA), cytidine deaminase (CDA), purine nucleoside phosphorylase (PNPase) and adenosine monophosphate deaminese (AMPA), after thawing and homogenization. The same cells were examined for the capacity to form E rosettes and to respond to phytohaemagglutinin (PHA) stimulation. In CLL a significant reduction (P less than 0-001) of AMPA, PNPase and ADA activities was observed without variation of CDA. In contrast in HD PNPase, AMPA and CDA were increased (P less than 0-01) while ADA was in the normal range. The E-rosette forming cells were significantly reduced in both diseases and the capacity to respond to PHA-stimulation was strongly impaired in CLL. By this experimental approach it seems possible to demonstrate different states of functional activity of the lymphocytic cells in two diseases characterized by reduced T-cell-mediated immunity.

AMP Deaminase↗
PubMed2025

Biochemical assays for AID/APOBECs and the identification of AID/APOBEC inhibitors.

Activation-induced cytidine deaminase (AID) and apolipoprotein B-mRNA editing catalytic polypeptide 3 (APOBEC3 or A3) proteins belong to the AID/APOBEC family of cytidine deaminases. While AID mediates somatic hypermutation and class-switch recombination in adaptive immunity, A3s restrict viruses and retroelements by hypermutation. Mis-regulated expression and off-target activity of AID/A3 can cause genome-wide mutations promoting oncogenesis, immune evasion, and therapeutic resistance due to tumor and viral evolution. In these contexts, inhibition of AID/A3 represents a promising therapeutic approach. Competitive inhibition could be achieved with different strategies: one class would be small molecules that bind in the catalytic pocket (active site) and block access for the substrate cytidine. Another type of larger molecule inhibitor would bind the enzymes' surface more broadly and compete with the binding of the polynucleotide substrates prior to deamination catalysis. Several biochemical assays developed to assess AID/A3 activity can be employed to screen for potential inhibitors. These include in cellulo and in vitro activity-based as well as binding-based assays. In this chapter, we discuss the key considerations for designing robust enzyme assays and provide an overview of assays that we and others have established or modified for specific applications in AID/A3 enzymology, including measurement of inhibition. We provide detailed protocols for the two most widely used in vitro enzyme assays that directly measure the activities of purified AID/A3s on DNA and/or RNA substrates, namely, the gel-based alkaline cleavage assay and multiple variations of PCR/sequencing-based assays.

Cytidine Deaminase↗
PubMed1975

Deoxycytidine kinase: properties of the enzyme from human leukemic granulocytes.

Deoxycytidine kinase, which phosphorylates deoxycytidine (CdR) and its analog, cytosine arabinoside (ara-C), has been purified 71-fold from human leukemic cells. Biochemical properties of the partially purified enzyme included a molecular weight of 68,000, Kms of 7.8 muM for CdR and 25.6 muM for ara-C, and optimal activity with ATP and GTP as phosphate donors. Ara-C phosphorylation was strongly inhibited by CdR (Ki = 0.17 muM) and dCTP (Ki = 7.3 muM) and was weakly inhibited by ara-CTP (Ki = 0.13 mM). Purification by calcium phosphate gel elution and DEAE chromatography effectively separated this enzyme from cytidine deaminase, which deaminates both CdR and ara-C, and from uridine-cytidine kinase, the enzyme which phosphorylates 5-azacytidine. CdR kinase activity was found to decrease and cytidine deaminase to increase with maturation of normal and leukemic granulocytes. Myeloblasts purified by Ficoll sedimentation revealed an average kinase activity of 15.4 U/mg protein in acute myelocytic leukemia and 12.3 U/mg protein in blastic crisis of chronic myelocytic leukemia (CML). The average ratio of CdR kinase to deaminase activity in crude cell extracts varied from 0.197 in AML and 0.089 in blastic crisis to 0.0004 in normal granulocytes, reflecting the changes which take place with cellular maturation. The absolute levels of kinase and deaminase and the ratio of these two enzymes varied considerably among patients with AML, indicating that quantitative differences may be found in the metabolism of CdR and its analogs in leukemic cells.

Azacitidine↗
PubMed1977

Comparative studies of the cytostatic action and metabolism of 5-azacytidine and 5,6-dihydro-5-azacytidine.

5,6-Dihydro-5-azacytidine hydrochloride, a chemically stable, soluble analog of 5-azacytidine, has cytostatic activity against mouse leukemic L1210 cells grown in culture, but concentrations on the order of 10 micronM, 10-fold higher, than the parent drug, are necessary to inhibit cell growth. The addition of either cytidine or uridine protected against growth inhibition by 5-azacytidine and 5,6-dihydro-5-azacytidine, whereas thymidine potentiated the cytostatic action of both drugs. Deoxycytidine also enhanced the action of 5-azacytidine but had no effect with the reduced analog. Cell suspensions of L1210 cells were able to phosphorylate 5-azacytidine and, to a lesser extent, 5,6-dihydro-5-azacytidine. In cell-free extracts in the presence of ATP and Mg2+, both drugs were converted to nucleotides but at less than 5% the rate of cytidine. As a substrate for mouse kidney cytidine deaminase, the apparent Km value for 5,6-dihydro-5-azacytidine (33 micronM) is of the same order of magnitude as that for cytidine (37 micronM) but less than that for 5-azacytidine (2.1 X 10(3) micronM). The Vm for deamination of the reduced analog is one-tenth that for 5-azacytidine. 3,4,5,6-Tetrahydrouridine, a potent inhibitor of cytidine deaminase, is more effective in blocking deamination of 5-azacytidine than 5,6-dihydro-5-azacytidine.

Animals↗
PubMed1978

ARA-C analogs.

Ara-C, a phase-specific antitumor agent, is rapidly deactivated by the enzyme cytidine deaminase. A prolongation of the biological activity of ara-C can be achieved either by the concomitant use of a cytidine deaminase inhibitor or by the development of ara-C derivatives with increased resistance to deamination and a longer half-life in serum. Among such derivatives are cyclocytidine (cyclo-C), anhydro-ara-5-fluorocytidine (AAFC) and the N4-acyl-derivatives. AAFC has been recently shown to be active in human leukemias and in solid tumors of the digestive tract. The tolerance to AAFC is sufficient for clinical use, and AAFC does not produce parotid pains and hypotension, characteristic side effects of cyclo-C. The main toxicity consists of myelodepression, nausea and vomiting. The schedule dependence of AAFC is far less pronounced than for ara-C, so that a weekly application by rapid i.v. injection of 30-40 mg/kg (1,200-1,500 mg/m2) reaches the level of activity with acceptable toxicity. AAFC seems to be as active as ara-C in acute leukemias and is probably active too in malignant lymphomas. In a large phase II trial of the EORTC on selected solid tumor types, AAFC showed a significant activity in GI tract adenocarcinomas with 2 responses/3 evaluable in pancreas, 7/14 in stomach and 2/32 in colorectal tumors (4/30). Hints of activity were also detected in breast cancer (1/17) and anaplastic small cell carcinoma of the lung (1/9). No responses were obtained in 27 patients with epidermoid carcinoma of the lung. These results confirm that ara-C, or newer ara-C analogs, are potentially active in various solid tumor types, and suggest that an extensive further clinical study of such new derivatives is warranted.

Clinical Trials as Topic↗
PubMed1977

[Subcellular distribution of adenosine-, adenosine-5-monophosphate- and cytidine-5-monophosphate desaminase activity in the humna placenta and amnion tissue].

With the method of pH-Stat the adenosinedeaminase-, adenosine-5'-monophosphate deaminase-, cytidine-5'-monophosphate deaminase activities are determined in the nucleus-, mitochondrial-, microsomalfractions and in the cytoplasmatic fractions of the human termplacental and amniotic tissues. The desaminase activities were higher in the amniotic fractions. The possible importance of the adenosinedesaminase and adenosine-5'-monophosphate desaminase for the fetal-placental blood circulation is discussed.

AMP Deaminase↗
PubMed2025

Defining APOBEC-induced mutation signatures and modifying activities in yeast.

APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors. Additionally, the ability to scale yeast experiments to high-throughput screens allows use of this system to also investigate cellular pathways impacting the frequency of APOBEC-induced mutation. Here, we present validated methods utilizing yeast to determine APOBEC mutation signatures, genetic interactors, and chromosomal substrate preferences. These methods can be employed to assess the potential of other human APOBECs and APOBEC orthologs in different species to contribute to cancer genome evolution as well as define the pathways that protect the nuclear genome from inadvertent APOBEC activity during viral restriction.

Humans↗
PubMed2026

Isolation of region-specific factors driving antibody class-switch recombination from the immunoglobulin heavy chain locus.

Activation-Induced Cytidine Deaminase (AID) induces DNA double-strand breaks (DSBs) at the switch (S) regions of the Immunoglobulin heavy chain (IgH) locus, which are essential for class switch recombination (CSR) and somatic hypermutation (SHM), key processes for effective antibody production. While AID activity is critical, its off-target effects, such as DSBs at the Myc locus, can cause chromosomal translocations like IgH-Myc fusions, contributing to B-cell lymphomas. The factors assembled on the IgH locus that help restrict AID-induced DSBs and subsequently CSR, remain unknown. To address this, we developed a method to isolate CSR-specific factors by inserting a 5×-GAL4-UAS sequence at the switch-mu (Sμ) region in CH12 cells. This engineered site enables recruitment of a 3-FLAG-GAL4 DNA-binding protein (3F-GAL4-DBD), allowing specific pulldown of proteins enriched at the Sμ region. Successful recovery of the known CSR regulator BRD2 from the Sμ region, along with enrichment of the DNA repair factors 53BP1 and gH2AX, validated this approach. Identification and characterization of IgH-enriched factors establish a validated methodological framework to facilitate future proteomic discovery of CSR regulators and highlight mechanisms that balance antibody diversification with genomic integrity in B cells.

Immunoglobulin Class Switching↗
PubMed1977

Detection after electrophoresis of enzymes involved in ammonia metabolism using L-glutamate dehydrogenase as a linking enzyme.

The use of L-glutamate dehydrogenase (GLUD) as a reagent in staining mixtures to detect the isozymes of enzymes which catalyze the production of ammonia has been investigated. Methods have been devised for the electrophoresis and detection, using GLUD, of seven enzymes: cytidine deaminase, adenosine deaminase, adenosine monophosphate deaminase, arginase, argininosuccinase, D-amino acid oxidase, and D-aspartate oxidase. GLUD-linked staining methods appear to be sensitive, specific, and of general application.

AMP Deaminase↗
PubMed1977

N4-Behenoyl-1-beta-D-arabinofuranosylcytosine as a potential new antitumor agent.

N4-Acyl-1-beta-D-arabinofuranosylcytosines, which are lipophilic antitumor analogs of 1-beta-D-arabinofuranosylcytosine, were dissolved by the use of a detergent, HCO-60, and the differences in the antitumor activities when the drugs were administered in the forms of solution or suspension were compared. N4-Stearoyl-1-beta-D-arabinofuranosylcytosine (NSC 201290), which was the most active compound when administered as an aqueous suspension, diminished in its activities after it had been dissolved into a clear solution, whereas N4-behenoyl-1-beta-D-arabinofuranosylcytosine (NSC 239336) exhibited activities superior to those of the parent compound 1-beta-D-arabinofuranosylcytosine when administered as a solution. Moreover, the high efficacy of this compound was long lasting in the host animal, regardless of the treatment schedules or the presence of the 1-beta-D-arabinofuranosylcytosine-inactivating enzyme, cytidine deaminase.

Animals↗