title: Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity.
abstract: While gemcitabine (2'-2'-difluoro-2'-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.
authors.affiliation: School of Molecular Biosciences, College of Veterinary Medicine, P.O. Box 647520, Washington State University, Pullman, WA 99164-7520, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Johnson
authors.forename: Melissa N
authors.initials: MN
authors.lastname: Brown
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Black
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D011993:Recombinant Fusion Proteins; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D000093542:Gemcitabine
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