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A Bioluminescent Microbial Biosensor for in Vitro Pretreatment Assessment of Cytarabine Efficacy in Leukemia Habib M

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A Bioluminescent Microbial Biosensor for in Vitro Pretreatment Assessment of Cytarabine Efficacy in Leukemia Habib M View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UWE Bristol Research Repository Clinical Chemistry 56:12 Cancer Diagnostics 1862–1870 (2010) A Bioluminescent Microbial Biosensor for In Vitro Pretreatment Assessment of Cytarabine Efficacy in Leukemia Habib M. Alloush,1,2 Elizabeth Anderson1, Ashley D. Martin,3 Mark W. Ruddock,3 Johanna E. Angell,1 Phil J. Hill,4 Priyanka Mehta,5 M. Ann Smith,6 J. Graham Smith,7 and Vyv C. Salisbury1* BACKGROUND: The nucleoside analog cytarabine intracellular Ara-CTP (cytosine arabinoside triphos- (Ara-C [cytosine arabinoside]) is the key agent for phate) levels and, if fully validated, may be suitable for treating acute myeloid leukemia (AML); however, up use in clinical settings. to 30% of patients fail to respond to treatment. Screen- © 2010 American Association for Clinical Chemistry ing of patient blood samples to determine drug re- sponse before commencement of treatment is needed. This project aimed to construct and evaluate a self- Although cytotoxic chemotherapy is widely used for bioluminescent reporter strain of Escherichia coli for treatment of many forms of cancer, failure to respond use as an Ara-C biosensor and to design an in vitro to anticancer drugs and resistance to treatment are ma- assay to predict Ara-C response in clinical samples. jor obstacles to satisfactory clinical outcome. Over 30% of acute myeloid leukemia (AML)8 patients fail to re- METHODS: We used transposition mutagenesis to create a cytidine deaminase (cdd)-deficient mutant of E. coli spond to the nucleoside analog cytarabine (Ara-C [cy- MG1655 that responded to Ara-C. The strain was tosine arabinoside]), the main drug used for AML transformed with the luxCDABE operon and used as a treatment, and a substantial proportion fail to achieve whole-cell biosensor for development an 8-h assay to long-term remission, whereas others respond to re- determine Ara-C uptake and phosphorylation by leu- duced levels of the drug (1). Currently, patients un- kemic cells. dergo no prescreening for any of the courses of cyto- toxic therapy to determine potential response (1). For RESULTS: Intracellular concentrations of 0.025 ␮mol/L nonresponding patients, this lack of screening can re- phosphorylated Ara-C were detected by significantly sult in a delay in commencement of effective treatment increased light output (P Ͻ 0.05) from the bacterial and unnecessary exposure to cytotoxic drugs with as- biosensor. Results using AML cell lines with known sociated morbidity and mortality due to side effects response to Ara-C showed close correlation between and potential drug-induced secondary malignancies the 8-h assay and a 3-day cytotoxicity test for Ara-C cell (2). There is a need for rapid screening of patient sam- killing. In retrospective tests with 24 clinical samples of ples to determine level of drug response before com- bone marrow or peripheral blood, the biosensor-based mencement of treatment that not only will give in- assay predicted leukemic cell response to Ara-C within creased patient benefits, but also reduce healthcare 8h. costs (1). It is anticipated that such a test could be anal- ogous to the existing antibiotic sensitivity tests rou- CONCLUSIONS: The biosensor-based assay may offer a tinely used in clinical settings before antibiotic treat- predictor for evaluating the sensitivity of leukemic cells ment for infection. to Ara-C before patients undergo chemotherapy and The nucleoside analog Ara-C is one of the most allow customized treatment of drug-sensitive patients active single anticancer agents and is the main drug of with reduced Ara-C dose levels. The 8-h assay monitors chemotherapy for AML (3, 4). In vivo, Ara-C is trans- 1 Faculty of Health and Life Sciences, University of the West of England, Bristol, University of the West of England, Bristol BS16 1QY, UK. Fax ϩ44-117-328- UK; 2 Faculty of Sciences, Division of Natural Sciences, Haigazian University, 2904; e-mail [email protected]. Beirut, Lebanon; 3 Randox Laboratories, Antrim, UK; 4 Division of Food Sciences, Received March 12, 2010; accepted September 3, 2010. Nottingham University, Nottingham, UK; 5 Bristol Haematology and Oncology Previously published online at DOI: 10.1373/clinchem.2010.145581 Centre, University Hospitals Bristol NHS Foundation Trust, Bristol, UK; 6 Stem 8 Nonstandard abbreviations: AML, acute myeloid leukemia; Ara-C, cytarabine Cell Transplant Laboratory, Royal Marsden NHS Foundation Trust, Sutton, (cytosine arabinoside); dCK, deoxycytidine kinase; Ara-CMP, cytosine arabino- 7 Surrey, UK; Department of Haematology, Frimley Park Hospital NHS Founda- side monophosphate; Ara-CTP, cytosine arabinoside triphosphate; FMNH2, re- tion Trust, Camberley, Surrey, UK. duced flavin mononucleotide; IPTG, isopropyl ␤-D-1-thiogalactopyranoside; A, * Address correspondence to this author at: Faculty of Health and Life Sciences, absorbance; AP, alkaline phosphatase. 1862 Bioluminescent Biosensor to Test Cytarabine Efficacy ported into the cell via the human equilibrium (luxB)] are linked to those coding for the fatty acid nucleoside transporter (hENT1) and is rapidly phos- reductase complex [fatty acid reductase (luxC); acyl phorylated by deoxycytidine kinase (dCK) to its mono- transferase (luxD); and acyl-protein synthetase (long- phosphate form, cytosine arabinoside monophosphate chain-fatty-acid-luciferin-component ligase) (luxE)], (Ara-CMP), which is further phosphorylated by nucle- which catalyzes the production of luciferin from the oside kinases and finally to the triphosphorylated and fatty acid pool (11). Because the production of FMNH2 active form, cytosine arabinoside triphosphate (Ara- directly depends on a functional electron transport sys- CTP). Drug inactivation can result from Ara-CMP tem, only metabolically active cells emit light. Hence, conversion into Ara-uracil by cytidine deaminase any alteration of cellular metabolism is reflected by a (cdd), or from dephosphorylation of Ara-CMP by cy- change in light emission. Bioluminescent bacterial bio- toplasmic nucleotidase (3). The antiproliferative and sensors have been used to assess the effect of various cytotoxic effects of Ara-CTP are largely due to its ability chemical, biological, and physical signals, providing an to interfere with DNA polymerase and its incorpora- accurate, nondestructive, and real-time assay technol- tion into DNA strands leading to chain termination ogy (9). Bioluminescence phenotype can be conferred and DNA synthesis arrest (4). upon most bacteria by introducing and expressing the Chemoresistance to Ara-C can arise from a number luxCDABE operon, isolated from Photorhabdus lumi- of factors influencing the rate of Ara-CTP formation and nescens, under the control of constitutive promoters. incorporation into DNA (5), including low drug uptake, These whole-cell bacterial biosensors do not require conversion into Ara-uracil by cytidine deaminase, or de- exogenous substrate and give out light as a direct indi- phosphorylation of active metabolite by cytoplasmic nu- cator of the physiological status of the bacteria in real cleotidase (6). Intracellular concentrations of Ara-CTP time (12). The high metabolic rate of bacterial cells, are reported to correlate with response to Ara-C therapy compared with mammalian cells, means that these re- (4). In vitro assessment of Ara-C efficacy has previously porters are ideal for fast, accurate, real-time in situ tox- involved measurement of cell death, assessed by colony icity testing. Light output from these biosensors can be forming units–blast clonogenic assays (7). This method- accurately measured, with no background interfer- ology involves primary cell culture over Ն3 days (7) and ence, by using either luminometers or low-light cam- is not suitable for routine screening. In clinical practice, eras. Lux gene–based reporter technology in bacterial patients are treated with regimens including Ara-C re- biosensors has been successfully used to monitor phar- gardless of their possible nonresponse to the drug and, as macodynamics of antimicrobial agents in real time a result, suffer from debilitating side effects (2, 8). Patients (13, 14). are given induction therapy over a period of weeks, before sensitivity or resistance is determined by leukemic cell Ara-C has no effect on Escherichia coli, since the count (4). bacteria lack dCK that phosphorylates Ara-C into its Recombinant cell-based bioluminescence tech- active form and they actively deaminate Ara-C into nology is an approach that has received increasing Ara-uracil through the activity of cytidine/deoxycyti- attention for biomedical applications (9) and has dine deaminase (cdd). Wang et al. (15) constructed a previously been used for determination of androgen cdd-deficient E. coli strain (SØ5218) that, upon the ex- bioactivity in clinical samples (10). This technology pression of the human deoxycytidine kinase (DCK) has the potential to detect anticancer drug activity in gene, exhibited reduced relative growth in the presence malignant cells from clinical samples, within a period of Ara-C in minimal growth medium. Therefore, this of hours. Light emission from bioluminescent bacteria indicates that expression of human dCK in SØ5218 is due to the activity of bacterial luciferase, which cata- leads to the incorporation of Ara-CTP into bacterial lyzes the oxidation of reduced flavin mononucleotide DNA. However, the growth inhibitory effect of Ara-C was completely abolished when assayed in rich growth (FMNH2) and a long-chain
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