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

Abstract

BACKGROUND The nucleoside analog cytarabine (Ara-C [cytosine arabinoside]) is the key agent for treating acute myeloid leukemia (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. This project aimed to construct and evaluate a self-bioluminescent reporter strain of Escherichia coli for use as an Ara-C biosensor and to design an in vitro assay to predict Ara-C response in clinical samples. METHODS We used transposition mutagenesis 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. RESULTS 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. CONCLUSIONS 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.

Existing Biosensing Platforms for the Nucleotide Markers Detection

“…As an analytical signal generators in this case, the photoproteins, such as obelin [36] or aequorin [37] may be considered singled out from bioluminescent organisms. Due to the high quantum yield of photochemical reactions, the possibility of obtaining the recombinant photoproteins and their stable conjugates with other biomolecules, as well as the availability of current photosensitive technique, such labels are successfully used in the analyzed systems to determine biomolecular markers [37][38][39], including DNA [36] . Bioluminescent detection systems can reliably detect the DNA sequence analyzed in a concentration of 10 -11 M [36].…”

Biosensors in Clinical Practice: Focus on Oncohematology

“…created a pyrimidine-requiring cdd -deficient Escherichia coli mutant that expressed the human dCK gene and was made luminescent by the introduction of the luxCDABE operon, which was used to determine Ara-C uptake and phosphorylation by leukemic cells [73]. …”

“…If fully validated, the biosensor-based assay could be used routinely in the clinical setting to predict the sensitivity of leukemic cells to Ara-C before exposing patients to chemotherapy, and promote the development of tailored treatment strategies [73]. …”

Engineered cells as biosensing systems in biomedical analysis

“…However, it is reported that over 30% of patients do not respond to Ara-C, leading to high morbidity and mortality associated with drug cytotoxicity and side effects. Alloush et al have developed a bioluminescent microbial cell-based biosensor for detection of Ara-C to be used in an in vitro assay to predict response of leukemic cells to Ara-C [56]. In vivo, Ara-C is transported into cells and converted to its active triphosphate form, Ara-CTP.…”

“…4 Cell lysis assay using E. coli HA1 biosensing system for detection of cytarabine. Adapted from Alloush et al [56] …”