Recombinant Human Cytidine Deaminase: Expression, Purification, and Characterization

Vincenzetti, Silvia; Cambi, Alessandra; Neuhard, Jan; Garattini, Enrico; Vita, Alberto

doi:10.1006/prep.1996.0097

Cited by 61 publications

(62 citation statements)

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“…The purity of all proteins was .90%, as demonstrated by the major band at an expected size of 16 kDa (CDAs) (Vincenzetti et al, 1996) and 30.5 kDa (DCKs) (Sabini et al, 2003) on SDS-PAGE (Supplemental Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

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Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase

Baker

Wickremsinhe

et al. 2012

Drug Metab Dispos

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Gemcitabine (dFdC, 29, is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 29,29-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K m and V max for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K m and 6-fold lower V max for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K m (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

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Section: Resultsmentioning

confidence: 99%

“…The molecular mass of the recombinant CDAs was determined by gel filtration using a fast protein liquid chromatography system, and the zinc content of CDA proteins was quantified with inductively coupled plasma optical-emission spectrometry, as described previously (Vincenzetti et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase

Baker

Wickremsinhe

et al. 2012

Drug Metab Dispos

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“…Cell debris was removed by centrifugation at 15,000 ϫ g for 30 min. CDA assays were performed as described previously (23). The recombinant CDA protein was detected by Western blot with Xpress antibody (Invitrogen).…”

Section: ј-Agtgggcattctctggctgtcac-3јmentioning

confidence: 99%

The Role of Cytidine Deaminase and GATA1 Mutations in the Increased Cytosine Arabinoside Sensitivity of Down Syndrome Myeloblasts and Leukemia Cell Lines

Jensen

Stout

et al. 2004

Cancer Research

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“…Human CDA (EC 3.5.4.5) is a 146-amino acid protein (16.1 kDa) and generates Urd or dUrd by hydrolyzing the 4-amino group of Cyd and dCyd (Vincenzetti et al, 1996). In humans, CDA activity has been reported in tumor cells as well as in normal tissues, including the liver, spleen, intestinal mucosa, and lung, with particularly high activity in the liver and spleen (Camiener and Smith, 1965).…”

Section: Introductionmentioning

confidence: 99%

Influence of Cytidine Deaminase on Antitumor Activity of 2′-Deoxycytidine Analogs In Vitro and In Vivo

Yoshida

Endo²,

Obata³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Antitumor 2-deoxycytidine (dCyd) analogs such as gemcitabine (dFdC), cytarabine (Ara-C), and 2-C-cyano-2-deoxy-1-␤-Darabinofuranosylcytosine (CNDAC) are activated by dCyd kinase, whereas cytidine deaminase (CDA) inactivates them by conversion to their uracil forms. To elucidate the relationship between the chemosensitivity to antitumor dCyd nucleosides and CDA expression, we established a stable line of human gastric carcinoma TMK-1 cells constitutively overexpressing CDA (TMK-1/CDA) and examined its chemosensitivity to antitumor dCyd analogs in vitro and in vivo. We observed comparable reactivity for dFdC and Ara-C, and the substrate reactivity of CNDAC to recombinant human CDA was more than 10 times less efficient than those of Ara-C and dFdC. Next, we examined the in vitro chemosensitivity of TMK-1/CDA and observed a marked decrease in the sensitivity of TMK-1/CDA to Ara-C, dFdC, and CNDAC compared with mocktransfected cells. In addition, we transplanted TMK-1/CDA cells into a nude mouse xenograft model and examined their in vivo chemosensitivity to CNDAC. The in vivo antitumor effect of CNDAC on TMK-1/CDA cells was substantially reduced compared with that of mice transplanted with mock-transfected cells. These results indicate that CDA could play an important role in regulating susceptibility to antitumor dCyd analogs in vitro and in vivo. In addition, the expression level of CDA was found to affect the antitumor activity of CNDAC, even though the substrate reactivity of CNDAC to CDA is relatively low.

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Recombinant Human Cytidine Deaminase: Expression, Purification, and Characterization

Cited by 61 publications

References 0 publications

Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase

Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase

The Role of Cytidine Deaminase and GATA1 Mutations in the Increased Cytosine Arabinoside Sensitivity of Down Syndrome Myeloblasts and Leukemia Cell Lines

Influence of Cytidine Deaminase on Antitumor Activity of 2′-Deoxycytidine Analogs In Vitro and In Vivo

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