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Rapid In-vitro Testing for Chemotherapy Sensitivity in Leukaemia Patients

Chapter
First Online:
Part of the Advances in Biochemical Engineering/Biotechnology book series (ABE, volume 145)

Abstract

Bioluminescent bacterial biosensors can be used in a rapid in vitro assay to predict sensitivity to commonly used chemotherapy drugs in acute myeloid leukemia (AML). The nucleoside analog cytarabine (ara-C) is the key agent for treating AML; however, up to 30 % of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. To achieve this aim, a self-bioluminescent reporter strain of Escherichia coli has been constructed and evaluated for use as an ara-C biosensor and an in vitro assay has been designed to predict ara-C response in clinical samples. Transposition mutagenesis was used to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine ara-C uptake and phosphorylation by leukemic cells. Intracellular concentrations of 0.025 μmol/L phosphorylated ara-C were detected by significantly increased light output (P < 0.05) from the bacterial biosensor. Results using AML cell lines with known response to ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to ara-C within 8 h. The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced ara-C dose levels. The 8-h assay monitors intracellular ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings.

Graphical Abstract

Keywords

Bioluminescent Biosensor Cytarabine Leukaemia chemotherapy 

Abbreviations

AP

Alkaline phosphatase

AML

Acute myeloid leukemia

ara-C

Cytarabine, cytosine arabinoside

ara-CMP

Cytosine arabinoside monophosphate

ara-CTP

Cytosine arabinoside triphosphate

cdd

Cytidine deaminase

CLA

Cladarabine/cytarabine

DNR

Daunorubicin

dCK

Deoxycytidine kinase

FLA

Fludarabine/cytarabine

hENT1

Human equilibrative nucleoside transporter

IPTG

Isopropyl β-d-1-thiogalactopyranoside

NPM1

Nucleophosmin-1 gene

pyrE

Orotate phospho-ribosyltransferase gene

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© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Institute of Bio-Sensing TechnologyUniversity of the West of EnglandBristolUK

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