Cytidine Deaminase Activity Research Articles

Sensitization to X ray by 5-chloro-2′-deoxycytidine co-administered with tetrahydrouridine in several mammalian cell lines and studies of 2′-chloro derivatives

5-Chloro-2′-deoxycytidine (CIdC) + tetrahydrouridine (H 4U) sensitizes mammalian cells (HEp-2, RIF-1, 5-180) to X ray. This sensitization, as demonstrated previously with HEp-2 cells, is heightened when cells are pre-incubated with inhibitors of pyrimidine synthesis. CHO cells, which intrinsically lack both cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD), are sensitized to X ray by 5-chlorodeoxyuridine (CIdU) but display no significant sensitization with CIdC + H4U. The presence and level of these deaminases appears to correlate with X ray sensitization in cell culture. From experiments in cell culture, it can be inferred that one pathway of conversion, (a) deoxycytidine kinase → dCMPD, or (b) CD → thymidine kinase, may be sufficient for metabolizing C1dC to a radiosensitizer. However, if both pathways are blocked, as in CHO cells, no X ray sensitization results. In addition to HEp-2 cells, which are extremely elevated in both CD and dCMPD activities, we have examined the sensitization of S-180 and RIF-1 cells to X ray by CIdC + H 4U. Both cell lines possess an enzymatic profile consistent with their sensitization to X ray by CIdC + H 4U. Dose enhancement ratios of 1.5 to 1.9 for cells treated with CIdC + H 4U + H 4U and ratios of 2.0–2.7 for cells pre-treated with inhibitors of pyrimidine synthesis prior to CldC + H 4U have been obtained. Based on reports of the marked X ray sensitization of bacteria by 2′-chloro-2′-deoxythymidine, we obtained 2′,5-dichloro-2′-deoxycytidine and 5-bromo-2'-chloro-2-deoxycytdine and found these analogs to be X ray sensitizers of mammalian cells. The strategy that we propose with CIdC + H 4U and the related 2'-chloro derivatives, based on the elevation of CD and dCMPD in human tumors, offers a degree of selectivity that is not necessarily related to differences in cell kinetics; such that malignancies other than brain tumors may be amenable to this therapy.

Cytidine deaminase activity as a measure of acute inflammation in rheumatoid arthritis.

Cytidine deaminase (CD), a cytoplasmic enzyme, is thought to leak out of damaged cells and can be measured in fluids by a simple biochemical assay. This study has shown that serum CD activity is raised in rheumatoid arthritis (RA) compared with osteoarthritis (OA). Synovial fluid (SF) CD activity was always less than the corresponding serum activity (mean SF/serum ratio = 0.6) in OA but up to 22 times greater than the corresponding serum activity in RA (mean SF/serum ratio = 13.1), suggesting CD production in inflammatory joints. Evidence to support the SF neutrophil as a cell of CD origin is provided by the CD gradient running from cells to SF to synovium. The close correlation between SF CD activity and neutrophil count (r = 0.93) indicates that SF CD activity is an accurate measure of acute synovial inflammation. Weak correlation of serum CD activity with erythrocyte sedimentation rate (ESR) (r = 0.44) and C-reactive protein (CRP) (r = 0.49) implies that CD estimations supply different though related information about rheumatoid disease activity. We suggest that CD released from damaged neutrophils diffuses from all inflamed joints into the blood, so that serum CD activity may provide an integrated measure of joint inflammation more specific than traditional measures such as the ESR.

ON THE ROLE OF CYTIDINE DEAMINASE IN CELLULAR METABOLISM: 146

Cytidine deaminase (CDD) catalyzes the deamination of various cytosine nucleosides to their corresponding uracil nucleosides. Due to its ability to inactivate cytidine analogs, it has been a target of considerable interests. The levels of CDD differ greatly among the various organisms studied, in some organisms and cell types CDD appears to be absent. In Enterobacteriaceae cytidine and deoxycytidine are used as carbon source, through the intermediate formation of uridine and deoxyuridine respectively. It is the pentose moiety which is catabolized. Uracil is utilized as pyrimidine source and excess uracil is excreted. When nucleosides are added to growing cells the synthesis of the nucleoside catabolizing enzymes is induced. The role of CDD in mammalian cells has been less well defined. The activity of CDD has been measured in human white blood cells, leukemic cells and in various cell lines. Low enzyme levels have been found in immature cells. An inverse relationship with respect to adenosine deaminase levels is seen, indicating the absence of a coordinate regulation of purine and pyrimidine nucleoside deamination. The determination of CDD levels might prove useful in studies to identify maturating steps of human blood cells.

CYTIDINE DEAMINASE DEFICIENCY AND IMMUNODEFICIENCY MORE THAN A COINCIDENCE ?: 155

A 9 month-old boy, the fourth and the only surviving child of consanguineous parents of tunisian origin, was investigated for severe immunodeficiency associated with autoimmune anemia and thrombocytopenia. Increased serum immunoglobulin levels contrasted with an absence of antibody response after sensitization and blood T lymphocytes increased in number were incapable of proliferating normally in the presence of mitogens, antigens or of OKT3 antibody. Defective secretion of IL2 and of IFNrby patient's leukocytes was observed whatever the inducer used. Proliferative response as well as IFNr secretion were restored by incubation in the presence of exogenous IL2. Enzyme study disclosed a profound deficiency in cytidine deaminase (CDA) in the patient's lymphocytes (10-21-23 pmol.min−1.10−6 cells, normal value 130 ± 30, n = 39). Low values were found in the father (20-21) and the mother (18-55). Cytidine (100 μM) did reverse the inhibitory effect of pyrazofurin on parents' lymphocytes PHA-induced proliferation but not that of patient's lymphocytes. During a 6-months survey lymphocyte CDA activity of the patient, his father and mother rose to 63, 140 and 130 pmol.min−1.10−6 cells, respectively. The fact that CDA deficiency has been transitory in the parents rules out an hereditary enzymatic defect. Nevertheless, our results suggest that CDA activity should be systematically determined in immunodeficient patients.

Cytidine deaminase--a marker for pre-eclampsia?

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) was measured in blood collected for routine antenatal monitoring from 590 unselected pregnant women. The enzyme measurements were made without prior knowledge of the clinical condition of the patients. Increased activity of cytidine deaminase was found in 42.9% of patients with severe pre-eclampsia, 9.1% of patients with mild pre-eclampsia and in 8.6% of normal pregnancies. These results show a poor correlation of elevated cytidine deaminase activity with pre-eclampsia and a significant false positive elevation of the enzyme activity in normal pregnancies.

Cytidine deaminase activity of human normal and malignant lymphoid cells

Cytidine deaminase activity was determined by a radioisotopic assay in extracts of peripheral blood mononuclear cells of normal individuals and of patients with acute lymphoblastic leukaemia. The normal enzyme activity had a broad pH optimum between pH 6.5 and 8.0; apparent K m values for cytidine and deoxycytidine were 3.6 ± 0.6 μmol/ l and 26.5 ± 3.5 μmol/ l, respectively; the activity was resistant to heat inactivation; of the various effectors tested, only uridine, deoxyuridine and tetrahydroxyuridine had inhibitory effects. Cytidine deaminase activity was markedly decreased in lymphoblasts of patients with acute lymphoblastic leukaemia; enzyme activity was related to the percentage of circulating blast cells, and not to the clinical, cytological or immunological characters of the leukaemia.

Establishment of human KB cells resistant to 1-beta-D-arabinofuranosylcytosine, and mechanisms of cellular resistance in isolated clones.

A subline of human KB cells that was resistant to 1-beta-D-arabinofuranosylcytosine (ara-C) was established by continuous exposure of the cells to increasing concentrations of ara-C. Thirteen resistant clones were isolated from the resistant subline (KB/ara-C). KB/ara-C showed 1,300-fold higher resistance than the parent KB cells to ara-C; the most resistant clones, clones 7 and 10, showed 1,330-fold higher resistance. In the absence of ara-C, the resistance of the parent KB/ara-C cells was stable for at least 14 weeks, whereas that of clone 7 was stable for 10 weeks, but was slightly less after 14 weeks. The ara-C kinase and ara-C deaminase activities of the 13 clones and the cellular uptake of ara-C by several clones were measured. In general the clones showed decreased deoxycytidine kinase activity and decreased cellular uptake of ara-C. Most clones had higher cytidine deaminase activity than KB cells, but some had activity similar to that of the KB cells. A clear inverse relationship was found between the ara-C sensitivity of the clones and their kinase activity, but not their deaminase activity or their ara-C uptake. These results clearly demonstrate that a major mechanism of ara-C resistance of these human KB cells was a decrease in the activity of the ara-C activating enzyme deoxycytidine kinase. The parent KB/ara-C cells showed no clear cross-resistance to various antitumor agents other than an ara-C derivative, including metabolic inhibitors, alkylating agents, DNA binders and mitotic spindle poisons.

Dexamethasone-sensitive and -insensitive responses during in vitro differentiation of friend erythroleukemia cells

We have investigated the mechanism(s) by which dexamethasone inhibit DMSO-induced Friend erythroleukemia cell differentiation in vitro. In particular, we examined the effects of dexamethasone on (a) the early events of differentiation such as cell volume alterations and ‘memory response’ and (b) the onset of biochemical events associated with terminal erythroid cell differentiation. By analysing kinetics of commitment of Freind cells to terminal erythroid differentiation on a clonal basis, we have observed that dexamethasone inhibited the completion of the latent period (time elapsed prior to commitment) and impaired “memory” (ability of inducer-treated cells to continue differentiation after a discontinuous exposure to inducer). Treatment of Friend cells with dexamethasone did not prevent the occurence of DMSO-induced alterations in cell volume. However, dexamethasone treatment prevented a series of biochemical events with terminal Friend cell differentiation. These include the decrease in the rate of both cytoplasmic and nuclear RNA synthesis as well as the induction of cytidine deaminase activity and hemoglobin synthesis. These data indicate the dexamethasone-sensitive process(es) operate during the early stages of Friend cell differentiation and that they are responsible for the inhibition of terminal erythroid maturation. These dexamethasone-sensitive processes, however, appear to be different from those regulating cell volume alterations during the earyl steps of DMSO-induced Friend cell differentiation.

Uridine as the only alternative to pyrimidine de novo synthesis in rat T lymphocytes

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10 −5 M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [ 3H]thymidine incorporation. Proliferation is completely restored by 5 × 10 −5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 10 −5 M each, fail to re-establish proliferation but produce an isotropic dilution of [ 3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (<0.001 nmol.min −1.10 −6 cells).

Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole.

The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.

Activity of serum cytidine deaminase during pregnancy

A simple and reliable method for the estimation of cytidine deaminase (cytidine aminohydrolase; EC 3.5.4.5) activity in pregnancy serum is described. This enzyme does not require magnesium for activation and is also more stable than deoxycytidylate deaminase (dCMP deaminase, dCMP aminohydrolase; EC 3.5.4.12). There was excellent correlation between the two enzymes (r = 0.92). Both enzymes showed increased activity in abnormal pregnancy. Both enzymes activities were found to be similar in 1305 maternal serum samples and therefore due to the simplicity of cytidine deaminase estimation it is recommended for the screening of large numbers of antenatal sera for abnormal pregnancies.

Mode of action of 5-fluorocytidine and 5-fluoro-2'-deoxycytidine in L5178Y cells in vitro.

The mode of antiproliferative action of two 5-fluorocytosine nucleosides, 5-fluoro-cytidine (FCR) and 5-fluoro-2'-deoxycytidine (FCdR), was examined using mouse leukemia L5178Y cells in vitro. FCR and FCdR were markedly active against L5178Y cells, though the cells were deficient in cytidine deaminase activity. Both compounds increased the incorporation of 14C-labeled thymidine into the acid-insoluble fraction of L5178Y cells and decreased labeled deoxycytidine incorporation. In reversal studies, the antiproliferative effects of both compounds were almost abolished by simultaneous addition of thymidine or deoxyuridine. Deoxycytidine completely reversed the growth inhibition caused by FCdR, but not that caused by FCR. These results demonstrate that the cytotoxicity of both compounds is due to inhibition of thymidylate synthetase, presumably through formation of 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) after deamination by deoxycytidylate deaminase in the pyrimidine de novo pathway.

ChemInform Abstract: CYCLIC UREA NUCLEOSIDES. CYTIDINE DEAMINASE ACTIVITY AS A FUNCTION OF AGLYCON RING SIZE

AbstractDie cyclischen Harnstoffe (III) werden zu den Verbindungen (IV) silyliert, die ohne Isolierung in Gegenwart eines Quecksilberkatalysators mit dem aus (I) zugänglichen Bromid (II) in die Nucleoside (V) übergeführt werden.

Increase in liver and kidney deoxycytidine kinase activity linked to neoplastic transformation

Deoxycytidine kinase activity in normal rat liver cytosol was low (0.8 nmol/hr/mg protein); it increased 2–26-fold in 12 lines of chemically-induced, transplantable rat hepatomas of different growth rates. The increased kinase activity correlated positively with the hepatoma growth rate. The kinase activity did not change in the regenerating liver and the activity in the differentiating, neonatal rat liver was similar to values in adult liver. Deoxycytidine kinase activity in 2 chemically-induced, transplantable rat kidney tumors was increased to twice the value found in normal renal cortex. Among 15 normal rat tissues examined the highest kinase activities were observed in thymus, bone marrow and spleen. Of the normal and malignant rat tissues tested, only testis had detectable cytidine deaminase activity.

Nucleoside-catabolizing enzyme activities in primary rabbit kidney cells and human skin fibroblasts

Primary rabbit kidney (PRK) and human skin fibroblast (HSF) cell cultures, two cell systems which are regularly employed in our laboratory to explore the antiviral properties of nucleoside analogues, were analyzed for the specific activities of the following nucleoside catabolizing enzymes: cytidine deaminase (EC 3.5.4.5), pyrimidine nucleoside phosphorylases (uridine phosphorylase: EC 2.4.2.3, and 2′-deoxythymidine phosphorylase: EC 2.4.2.4), purine nucleoside phosphorylase (EC 2.4.2.1) and adenosine deaminase (EC 3.5.4.4). No cytidine deaminase activity was detected in either PRK or HSF cells. Likewise, no 2′-deoxythymidine phosphorylase activity could be demonstrated in PRK and HSF cells. PRK cells contained low levels of uridine phosphorylase (3–;5 U/mg protein) and high levels of purine nucleoside phosphorylase (∼ 100 U/mg protein). For both PRK and HSF cells, relatively high adenosine deaminase activities were recorded (at an average 20 and 36 U/mg protein, respectively). Infection of the PRK or HSF cell cultures with either vaccinia virus, herpes simplex virus (type 1 or 2) or vesicular stomatitis virus at high multiplicity of infection (MOI ∼ 1) did not bring about marked changes in any of the enzymatic activities tested. For uridine phosphorylase (from PRK cells) and adenosine deaminase (from both PRK and HSF cells) the substrate specificities were determined with various uridine and adenosine analogues.

53] Cytidine deaminase from leukemic mouse spleen

This chapter describes the purification procedure of cytidine deaminase enzyme from leukemic mouse spleen. Cytidine deaminase is widely distributed among mammalian tissues. This enzyme is responsible for the inactivation of cytosine arabinoside––one of the most useful chemotherapeutic agents available for the treatment of acute myeloblastic leukemia. In the mouse spleen, a marked increase in the specific activity of cytidine deaminase occurs following infection with Friend leukemia virus. Elevated levels of cytidine deaminase are also found in regenerating mouse liver, in human leukemic cells, following the treatment of the patient with cytosine arabinoside, and in HeLa cells cultured in the presence of cytosine arabinoside. The activity of cytidine deaminase is assayed by measuring the amount of labeled uridine formed by the deamination of 2- 14 C-labeled cytidine. High levels of cytidine deaminase are found in human polymorphonuclear leukocytes.

Pyrimidine nucleotide synthesis in rat liver after the administration of cycloheximide.

After the administration of cycloheximide (2 mg/kg) the utilization of [2(-14C)]orotic acid for the synthesis of pyrimidine nucleotides of acid-soluble extracts of the liver is not affected for about 7 h. The specific activities of uridine and cytidine components are increased later on, and this increase is higher in the case of cytidine components. Analogous changes undergoes the specific activity of RNA pyrimidine nucleotides. The increased utilization of labeled orotic acid for the synthesis of cytidine nucleotides can be observed also in the kidney and in the small intestine. The enhanced degree of labeling of cytidine nucleotides in vivo cannot be correlated with the activity of cytidine triphosphate synthetase (EC 6.3.4.2) of liver cytosol estimated in vitro. The amination of UTP is suppressed at later intervals after the application of cycloheximide. The same holds true for the activity of uridine phosphorylase (EC 2.4.2.3),5'-nucleotidase (EC 3.1.3.5) ATPase (EC 3.6.1.3) and of liver cytosol. The activity of uridine kinase (EC 2.7.1.48) is increased when tested both with uridine and cytidine as substrates. Cytidine deaminase activity (EC 3.5.4.5) raises markedly 3--5 h after the administration of drug; later on it decreases again.

Cytosine arabinoside phosphorylation and deamination in acute myeloblastic leukemia cells

Extracts of peripheral leukocytes from patients with AML and from controls were examined for enzymes involved in the metabolism of ara-C in order to investigate a possible relationship between enzymes activities and improvement of treatment with ara-C. The enzymes are deoxycytidine kinase and cytidine deaminase, which activates and inactivates ara-C respectively. Cytidine deaminase activity was found to be lower in AML cells than in normal cells, while deoxycytidine kinase was higher in AML cells. Furthermore, the ratio between kinase activity with ara-C as substrate and activity with deoxycytidine as substrate was higher in AML cells than in normal leukocytes. However, there was no significant difference between those patients with AML, who respond to ara-C treatment, and the patients who did not respond. The enzymatic differences between normal and AML leukocytes with regard to activity and substrate specificity might suggest that ara-C could be combined with advantage with deoxycytidine in the clinic.

Purine metabolism in murine virus-induced erythroleukemic cells during differentiation in vitro.

Purine metabolism was studied in murine virus-induced erythroleukemia cells stimulated to differentiate in vitro in the presence of dimethylsulfoxide. The activities of the enzymes that catalyze the synthesis of the first intermediate of the de novo purine pathway, phosphoribosyl-1-amine, were decreased while the enzymes that catalyze the conversion of purine bases to purine ribonucleotides remained unchanged at the time the cells acquired the specialized function of hemoglobin synthesis. In addition, cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) activity increased with erythropoietic maturation, as it does during murine erythropoiesis in vivo. Stimulation of cellular proliferation of stationary erythroleukemic cells resulted in a marked increase in the activities of purine biosynthetic enzymes. These data provide a convincing example of repression and derepression of the PRA synthesizing enzymes in mammalian cells in vitro, and further evidence that the regulatory mechanisms operative in the normal development of erythrocytes can be activated by exposure of erythroleukemic cells to dimethylsulfoxide.

Distribution and excretion of 3H-cyclocytidine in rhesus monkeys.

The antineoplasmic agent, cyclocytidine (2, 2'-anhydro-i-β-D-arabinofurano-sylcytosine hydrochloride), was examined for distribution, excretion and metabolism in rhesus monkeys. These monkeys have a high activity of cytidine deaminase in their tissues, as well as human beings. When monkeys were injected intravenously with 3H-cyclocytidine, high concentrations of radioactivity were detectable in the kidney, liver, pancreas and adrenal gland. The cumulative content of 3H in the urine reached approximately 37% of the administered dose for 320 min after the injection. A very small amount of 3H was found in the lumen of the digestive tract. It was suggested that the urinary passage might be a predominant route for the excretion of cyclocytidine. When monkeys received an oral dose of the labeled compound, the concentration of 3H was the highest in the kidney and liver of all the organs tested, as seen in the case of intravenous injection. Orally administered 3H-cyclocytidine underwent a metabolic destruction to 3H-arauridine (1-β-D-arabinofuranosyluracil) through 3H-aracytidine (1-β-D-arabinofuranosylcytosine) and finally to 3H-water in the digestive tract. When monkeys were sacrificed 2, 880 min after oral administration with 3H-cyclocytidine, there was an increase in the relative value of this labeled compound to 3H-aracytidine, 3H-arauridine, and 3H-water accompanied by the transport of the intestinal contents containing the 3H-compounds to the anal portion. The cumulative content of urinary 3H and the residual amount of 3H in the digestive tract were approximately 72 and 15% of the administered dose, respectively, for the same period of exposure. This sufficient recovery of radioactivity may reflect the production of 3H-water from the labeled compound by decomposition.