School of Molecular Biosciences, College of Veterinary Medicine, P.O. Box 647520, Washington State University, Pullman, WA 99164-7520, USA.
Biochem Biophys Res Commun. 2011 Dec 9;416(1-2):199-204. doi: 10.1016/j.bbrc.2011.11.025. Epub 2011 Nov 10.
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.
虽然吉西他滨（2′-2′-二氟-2′-脱氧胞苷，dFdC）作为单一药物具有广泛的抗肿瘤活性，但可变的反应率和较差的细胞内代谢常常限制其临床疗效。为了提高 dFdC 的细胞毒性并帮助正常化反应率，我们创建了一种双功能融合酶，它将脱氧胞苷激酶（dCK）和尿苷/胞苷一磷酸激酶（UCMK）的酶活性结合在单个多肽中。我们的目标是评估所创建的融合是否可以诱导 dFdC 的有益、功能性变化，加速 dFdC 转化为其活性代谢物，从而放大细胞对 dFdC 的敏感性。虽然动力学分析表明 UCMK/dCK 融合酶具有两种天然活性，但融合使细胞对 dFdC 的细胞毒性作用敏感，其水平与单独表达 dCK 相同。这些结果表明，增加野生型 UCMK 的表达并不能显著增强 dFdC 介导的细胞毒性，可能需要进行旨在工程化具有提高的吉西他滨单磷酸盐活性的 UCMK 变体的研究。
