Introduction

Drug resistance is a major obstacle in chemotherapy and the continuing subject of extensive research. Many cancer patients who are treated with chemotherapeutic drugs will exhibit resistance to the drugs (whether inherent or acquired during the course of treatment), and since many of the drugs are used at their maximum medically allowable dose, even 1.5- to 2-fold resistance presents a serious problem. Thus, a great deal of research is focused on understanding the mechanism of action of the drugs, with the goal of identifying new targets for therapy, as well as enhancing the efficacy of existing drugs. Often such studies can be conveniently performed in experimental model systems, which offer powerful genetics and accessible biochemistry.

Any approach to understanding drug resistance relies on determining cell survival after different treatments, and it requires a rapid assay that can accurately measure cell viability over a wide range of cell concentrations and, at the same time, is sensitive enough to detect even a small number of surviving cells. This has been a significant challenge in performing large-scale genetic screens in a variety of model systems.

Dictyostelium discoideum is being used increasingly as a primary system for drug discovery and for studying the mechanisms that underlie the response to drugs (1,2), including anticancer drugs (3,4). Traditionally, cell viability was measured by plating and counting the plaques resulting from viable cells. The method is labor-intensive and requires considerable experience on the part of the investigator to routinely obtain quality data. Even when using a modification that significantly reduces cost and time (5), this method is not practical for assaying large numbers of samples, as is the case in a high-throughput drug screen. Clearly, a faster robust biochemical assay would benefit such studies.

In the following study, we describe the adaptation of a commercial luciferase-based assay to determine viability in populations of D. discoideum cells. In this assay, survival is determined by assaying for the amount of ATP contained within living metabolically active cells. We describe the optimization and necessary conditions that allowed its use in D. discoideum and show that the assay offers the sensitivity, reproducibility, and ease that are required to perform large-scale screens in this organism. Moreover, it has allowed us to gain preliminary insights into the different mechanisms of cytotoxicity by different drugs.

Materials and Methods

Drug Treatments and Viability Assays

96-Well plates

Aliquots (90 µL) of logarithmically growing D. discoideum cells in HL-5 axenic medium (7 g yeast extract, 14 g proteose peptone, 0.48 g KH₂PO₄, 0.5 g Na₂HPO₄, per liter, pH 6.5) (6) were placed in 96-well opaque white plates (Matrix Technologies, Hudson, NH, USA). Ten microliters of the drug being tested (in the indicated solvent) were added at the indicated concentrations and incubated at 22°C as described for each experiment. Ten microliters of the corresponding solvent were always added to the untreated controls. Following incubation with the drug, 100 µL CellTiter-Glo™ or BacTiter-Glo (Promega, Madison, WI, USA) were added, and the plates were covered and shaken for 30 min at room temperature. Luminescence was measured in a Veritas™ Microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA). Survival was calculated as the percentage of relative luminescence units (RLU) of the treated versus untreated culture. Error bars represent standard error, which was calculated using statistical tools in Microsoft® Excel®. P values were calculated using Student's t-test. Number of replicates is stated in each experiment. All drugs were from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin was prepared as a 1 mg/mL 3.3 mM solution in aqueous Pt buffer (3 mM NaCl/1 mM NaPO₄, pH 6.5). Dimethyl sphingosine (DMS) was prepared as a 1 mM solution in dimethyl sulfoxide (DMSO), and 4-nitroquinone (4NQO) was prepared as a 2 mg/mL solution in DMSO. Both DMS and 4NQO were diluted in aqueous solution prior to adding to the cultures. The final DMSO concentration in cell cultures never exceeded 2%, which is not toxic to D. discoideum.

Shaking cultures

Assays were performed on shaking cultures at a cell density of 2 × 10⁶ cells/mL. Following drug treatments, the cultures were diluted as indicated, and 100-µL samples were assayed as described above.

BacTiter-Glo (www.promega.com/pnotes/88/12162_02/12162_02.pdf) was designed for use with bacteria, and CellTiter-Glo (www.promega.com/pnotes/81/9939_02/9939_02.pdf) was designed to be used with mammalian cells. A detailed description of the basis of the assays and cited literature can be found at the above links. Most of the results presented were obtained with BacTiter-Glo. However, the results with BacTiter-Glo were compared with viability measurements using CellTiter-Glo. Although CellTiter-Glo results in lower RLUs (nearly 8-fold), the dilution curves with both reagents were identical (data not shown) and indicated that either reagent can be used. However, CellTiter-Glo had the advantage that the half-life of the signal is considerably longer than that of BacTiter-Glo (5 h versus 30 min, respectively), making it a more stable and reproducible system for the assays. The rapid decay of the luminescent signal with BacTiter-Glo makes it crucial to read all the samples at precisely the same time after adding the reagent.