Development of a Novel Assay Using Bioluminescent Reporter Bacteria To Determine the Sensitivity of AML Cells to Cytosine Arabinoside.

The nucleoside analogue cytosine arabinoside(Ara-C) has been a cornerstone of remission induction therapy for patients with AML for three decades. Even today, approximately 70% of adults fail to achieve durable complete remission with regimens including Ara-C. This project involves the development of a simple, rapid, economical test to determine Ara-C sensitivity of patient cells to enable clinicians to consider the efficacy of this drug prior to patient treatment.

Bacteria expressing lux genes have been successfully used to study real time pharmacodynamics of antimicrobial agents. We have extended this elegant reporter gene technology to determine cellular penetration of Ara-C, into AML cells. AraC has no effect on E. coli since it does not express deoxycytidine kinase (dCK) and has the ability to deaminate AraC into AraU through the activity of cytidine deaminase (cdd). To render E. coli sensitive to AraC, a cdd-deficient strain was used as the host to express the human dCK gene carried on pTrc99-A plasmid under the control of the lac promoter. The expression of dCK, induced by the addition of IPTG, made E. coli sensitive to AraC. E. coli was then made self-bioluminescent by introducing the pAL2 plasmid carrying the lux operon (luxABCDE)

AML cell lines were incubated with therapeutic levels of Ara-C (100 μM),washed, and re-suspended in fresh media containing bioluminescent modified E. coli and 0.1% saponin, to lyse the AML cells. Light levels and optical density were monitored using a microtitre plate reader (Fluorstar Optima, BMG Labtec).

Cell lines minus drug were used so as to generate control lysates and supernantants.

It was possible to detect significant increases in light levels from the lysate and supernatant of an AML cell-line known to be Ara-C sensitive (KG-1a). The lysate from a cell-line (THP-1) known to deaminate Ara-C through the over production of cytidine deaminase showed no increase in light production in the presence of Ara-C. In addition no difference in light levels was observed from the supernatant of these resistant cells. The control supernatant from both cell lines showed no change in light output. These findings indicate that this assay could be used to evaluate the uptake and metabolism of Ara-C by leukaemia cells. With refinement, the assay could be used to specify at which point in Ara-C metabolism AML cells acquire resistance. Furthermore, this technology could be modified to examine the sensitivity of AML cells to other established chemotherapeutic agents such as anthracyclines thus giving a genuinely helpful chemosensitivity profile to clinicians involved in patient care and customising treatment for individual patients.