Kevin John Slater

Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis

In this study the relative levels of ADP and ATP have been measured in cells undergoing apoptosis. Using HL60, CEM7, Jurkat and U937 cell lines and cytotoxic agents known to induce apoptosis, there was a significant correlation (P<0.01 for all models) between the ADP:ATP ratio and the degree of apoptosis measured by TUNEL and estimation of the sub G(0) fraction by propidium iodide staining and flow cytometry. The ratio measured in viable proliferating cells was found to be less than 0.11 compared with ratios between 0.11 and 1.0 seen in cells undergoing apoptosis. The higher the percentage of hypodiploidy the greater the ratio. Necrosis induced by heat shock resulted in ADP:ATP ratios in excess of 15.0. When primary cultures of AML blast cells were used, there was again a significant correlation between the ADP:ATP ratio and the degree of hypodiploidy. Recent evidence suggests that apoptosis is accompanied by opening of the mitochondrial permeability pores, leading to disruption of the mitochondrial transmembrane potential (DeltaPsi(m)). This results in caspase activation due to the release of cytochrome c and apoptogenic factors into the cytosol. In five experiments using CEM7 and dexamethasone the mitochondrial transmembrane potential was assessed using the fluorescent cyanine dye JC-1 and flow cytometry. Functioning mitochondria concentrate the JC-1 to produce red fluorescence. Loss of mitochondrial transmembrane potential results in green fluorescence only. The percentage of cells exhibiting red fluorescence correlated positively with the ATP values and negatively with the ADP:ATP ratio.

Cytotoxicity tests for high-throughput drug discovery.

Despite theoretical obstacles associated with performing cell-based assays in high-density formats (microplates with at least 384 wells), it is becoming clear that the pharmaceutical industry is now routinely running cell-based tests in these formats. This work is revealing the weakness of established cytotoxicity end points, specifically in relation to sensitivity and reproducibility. New assay kits based on bioluminescent detection of ATP and ADP are now providing the answer to these problems.

High-throughput cytotoxicity screening: hit and miss

It has long been established that there is a need for toxicity testing in drug discovery. As the number of lead compounds continues to expand, the problem facing the industry is where to position the toxicity tests in the drug discovery pipeline. To save time and effort, the tests should be performed as early in the process as possible. Historically, the assays available have not lent themselves well to the needs of HTS. This review discusses some of the pitfalls associated with toxicity testing, and covers the advances in technology that enable provision of accurate and reproducible data.

CHANGES IN INTRACELLULAR ADP:ATP RATIOS AS A MARKER OF APOPTOSIS

∆Ψm. We have shown in a number of apoptotic models, that the ADP:ATP ratio can be used reliably as a screen for both early and late apoptosis (2) in human leukaemic cell lines. We have now investigated the effect of the broad spectrum caspase inhibitor, Z-VAD-FMK, on determination of apoptosis by measurement of ADP:ATP ratios. METHOD. HL-60 cells were incubated with increasing concentrations camptothecin (CAM). Time course experiments were also carried out in the presence/absence of 5µM CAM, and apoptosis was determined every 30 minutes for up to 8 hours. The bioluminescent detection of ADP:ATP ratios was determined using the ApoGlow kit (LumiTech (UK) Ltd). Apoptosis was confirmed by Romanowsky staining, and determination of the percentage of hypodiploid cells by propidium iodide (PI) staining and flow cytometry. Collapse of the ∆Ψm was measured using the lipophilic cationic fluorescent probe JC-1. Increasing concentrations of Z-VAD-FMK were added to the cells either prior to addition of the drug, or at increasing time points after CAM to determine at which point the cells could still be ‘rescued’ from apoptosis. RESULTS. There was a concentration dependent increase in ADP:ATP ratio that correlated with an increase in the hypodiploid population and a decrease in % J-aggregates (p<0.0001). The time course experiments with 5µM CAM showed the drop in ATP corresponding to a loss of ∆Ψm, and the increased ratios occurring concomitantly with DNA fragmentation. Cell membrane integrity was maintained for approximately 4 hours after DNA fragmentation was first detected, together with a significant increase in ADP:ATP ratio. After approximately 6.5 hours there was a loss of membrane integrity that correlated with a dramatic increase in ratios. Preliminary washing experiments showed that even when the CAM had been washed from the cells immediately after addition, further incubation for 4 hours still resulted in apoptosis. Addition of 50µM Z-VAD-FMK prior to the addition of CAM showed prevention of apoptosis after 4 hours incubation (see figure). Full protection was no longer observed when the inhibitor was added after 30 minutes incubation with CAM or at concentrations below 40µM. DISCUSSION. Bioluminescent determination of ADP:ATP ratios correlated well with a number of other techniques used for detection of apoptosis. The correlation with loss of the ∆Ψm suggested the change in ratios to be a good indicator of early apoptosis, in the model tested. The use of Z-VAD-FMK reduced ratios back to those of the control cells. As this compound is a broad spectrum inhibitor that appears to act on those caspases associated with

Determination of the Effects of Camptothecin on Cell Cycling and Apoptosis in Human Leukaemic Cell Lines

INTRODUCTION. The measurement of ATP is a recognised method for determination of proliferation or the effect of toxic environments on the cell. More recently, the relationship between ATP and ADP has been shown to be an important factor in determining the apoptotic or necrotic state of the cell (1). The ratio of ADP to ATP shows significant correlation with the degree of apoptosis, as measured by the estimation of the sub-G0 by propidium iodide staining of the nuclei and flow cytometry. Camptothecin is a topoisomerase inhibitor and as such, has effects on the cell cycle prior to inducing apoptosis. This compound has also been used as a chemotherapeutic agent in the treatment of a number of malignancies including leukemias. We have investigated these effects in a number of human leukaemic cell lines, concentrating on U937 and HL-60 cells, together with blast cells isolated from the peripheral blood of patients with acute myeloid leukemia. METHOD. HL-60 cells were incubated for 4 hours, while with the U937 cells a 24-hour incubation was required for induction of apoptosis, with increasing concentrations of camptothecin. The AML blast cells, from 10 patients were incubated with increasing concentrations of camptothecin, Ara-C and etoposide for 4 to 24 hours. The extent of apoptosis was shown by measurement of the ratio of ADP:ATP using the ApoGlow Kit (LumiTech (UK) Ltd). The presence of apoptotic events within the cell population was confirmed using Annexin V, JC-1, and propidium iodide (PI) staining and flow cytometry. The PI staining was also used to determine the relative percentage of cells in different phases of the cell cycle. RESULTS. The HL-60 and U-937 cells showed a concentration dependent effect on apoptosis induction with concentrations of camptothecin from 100 to 500nM. At the highest concentration of camptothecin used (1000nM) there was a significant reduction (n=7, p<0.0001) in the ratios compared with the 500nM sample. Comparison with the PI data revealed emergence of the G0/G1 peak showing that a subpopulation of cells was beginning to cycle again. With HL-60 cells at 1000nM of the drug, there was an increase in the percentage viable cells as shown by exclusion of PI, this correlated with a reduction in the level of ADP relative to ATP with the emergence of a resistant viable population. Of the AML cells investigated there was a marked difference in the response of the patient cells to the different drugs investigated, with respect to induction of apoptosis and drug resistance. DISCUSSION. The U937 cells displayed variations in responses according to the concentration of camptothecin used. The cells arresting in S-phase at low concentrations, could