Detection of intracellular ara-C/ara-CTP following treatment with the new anti-leukemic drug elacytarabine using a bioluminescent bacterial biosensor

Robinson, Gareth M.; Anderson, Elizabeth; Salisbury, Vyv; Mehta, P.; Sandvold, M.L.; Reynolds, Darren M.

Abstract

Cytarabine (ara-C) is an analogue of deoxycytidine with an established role in treating AML. Cytarabine depends on the human equilibrative nucleoside transporter-1 (hENT-1) for cellular entry (1). Loss of hENT-1 function in vitro has been shown to result in ara-C resistance (2), and reduced hENT-1 expression and activity has been associated with adverse therapeutic outcomes in patients treated with cytarabine (3). The lipophilic ara-C-5’ elaidic acid ester of cytarabine (elacytarabine) has shown nucleoside transport independent uptake and long retention of the active nucleotide ara-CTP in a leukemic cell line (4) and may have the ability to overcome resistance due to insufficient transport in the clinical setting. (1). The whole cell bioluminescent biosensor, Escherichia coli HA1 was engineered to produce a measurable bioluminescent output in response to the nucleoside analogue ara-C (5). This was achieved through reduced expression of cytidine deaminase and the insertion of the human deoxycytidine kinase gene and the full lux operon from the Photorhabdus luminescens. The bacterial biosensor is able to monitor both ara-C and ara-CTP.
The aim of this study was to evaluate the feasibility of using the Escherichia coli HA1 biosensor to measure intracellular ara-C and ara-CTP following ex vivo exposure of human cells to elacytarabine and cytarabine.
CCRF-CEM cells or blast cells from patients were treated with ara-C or elacytarabine for up to 4 hours in the presence or absence of the hENT inhibitor dipyridamole (DPM). Cells were washed, lysed and the lysate incubated with E. coli HA1 and monitored on a plate reader luminometer. A cell viability assay was also performed to confirm biosensor results.
E. coli HA1 produced a measurable response to lysate from CCRF-CEM cells treated with either ara-C or elacytarabine. In the case of ara-C, this response was reduced when cells were incubated with DPM. No such reduction was observed for elacytarabine. When used to treat blast cells derived from leukemic patients, elacytarabine treatment produced a stronger signal from E. coli HA1 than an equivalent concentration of ara-C. Furthermore, the efficacy of elacytarabine was not susceptible to hENT inhibition in patient samples, whereas the efficacy of ara-C was. The findings of this project suggest that the E. coli HA1 biosensor is compatible with elacytarabine and that elacytarabine is able to bypass the effect of hENT inhibition on CCRF-CEM cells and patient derived blast cells

• Presentation Conference Type: Poster
• Conference Name: American Association for Cancer Research Annual Meeting
• Start Date: Apr 6, 2013
• End Date: Apr 10, 2013
• Publication Date: Jan 1, 2013
• Peer Reviewed: Peer Reviewed
• Keywords: new anti-leukemic drug elacytarabine, bioluminescent bacterial biosensor
• Public URL: https://uwe-repository.worktribe.com/output/940154
• Additional Information: Title of Conference or Conference Proceedings : American Association for Cancer Research Annual Meeting