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Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs.

Vande Voorde J, Vervaeke P, Liekens S, Balzarini J - FEBS Open Bio (2015)

Bottom Line: This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor.The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified.CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, blok x - bus 1030, B-3000 Leuven, Belgium.

ABSTRACT
Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency.

No MeSH data available.


Related in: MedlinePlus

Purity evaluation of the CDAHyor-GST fusion protein. Three different concentrations (i.e. 10, 1 and 0.1 μg) of the purified enzyme preparation were analyzed using SDS–PAGE. Proteins were stained using Bio-Safe™ Coomassie G-250 Stain (Bio-Rad Laboratories, CA, USA).
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f0010: Purity evaluation of the CDAHyor-GST fusion protein. Three different concentrations (i.e. 10, 1 and 0.1 μg) of the purified enzyme preparation were analyzed using SDS–PAGE. Proteins were stained using Bio-Safe™ Coomassie G-250 Stain (Bio-Rad Laboratories, CA, USA).

Mentions: A codon-optimized DNA sequence encoding the M. hyorhinis cytidine deaminase (CDAHyor) was synthetically assembled between the EcoRI and NotI restriction sites of a pIDTsmart vector (Integrated DNA technologies, Coralville, IO). The fragment was subsequently subcloned between the EcoRI and NotI sites of the pGEX-5X-1 bacterial expression vector (Amersham Pharmacia Biotech, Uppsala, Sweden) and CDAHyor was expressed in Escherichia coli as a GST-fusion protein (hereafter referred to as CDAHyor) according to a procedure previously described by Liekens et al. [27]. SDS–PAGE revealed that the protein was of expected size (∼38–40 kDa) and purity (⩾95%) (Fig. 2). Since E. coli CDA consists of 294 amino acids, it would be characterized by a molecular weight of around 31 kDa [28]. Therefore, the contaminating protein bands shown in Fig. 2 are not likely related to E. coli-encoded CDA.


Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs.

Vande Voorde J, Vervaeke P, Liekens S, Balzarini J - FEBS Open Bio (2015)

Purity evaluation of the CDAHyor-GST fusion protein. Three different concentrations (i.e. 10, 1 and 0.1 μg) of the purified enzyme preparation were analyzed using SDS–PAGE. Proteins were stained using Bio-Safe™ Coomassie G-250 Stain (Bio-Rad Laboratories, CA, USA).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541722&req=5

f0010: Purity evaluation of the CDAHyor-GST fusion protein. Three different concentrations (i.e. 10, 1 and 0.1 μg) of the purified enzyme preparation were analyzed using SDS–PAGE. Proteins were stained using Bio-Safe™ Coomassie G-250 Stain (Bio-Rad Laboratories, CA, USA).
Mentions: A codon-optimized DNA sequence encoding the M. hyorhinis cytidine deaminase (CDAHyor) was synthetically assembled between the EcoRI and NotI restriction sites of a pIDTsmart vector (Integrated DNA technologies, Coralville, IO). The fragment was subsequently subcloned between the EcoRI and NotI sites of the pGEX-5X-1 bacterial expression vector (Amersham Pharmacia Biotech, Uppsala, Sweden) and CDAHyor was expressed in Escherichia coli as a GST-fusion protein (hereafter referred to as CDAHyor) according to a procedure previously described by Liekens et al. [27]. SDS–PAGE revealed that the protein was of expected size (∼38–40 kDa) and purity (⩾95%) (Fig. 2). Since E. coli CDA consists of 294 amino acids, it would be characterized by a molecular weight of around 31 kDa [28]. Therefore, the contaminating protein bands shown in Fig. 2 are not likely related to E. coli-encoded CDA.

Bottom Line: This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor.The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified.CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, blok x - bus 1030, B-3000 Leuven, Belgium.

ABSTRACT
Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency.

No MeSH data available.


Related in: MedlinePlus