Sharon Patricia Mary Crouch

The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity

Adenosine triphosphate (ATP) bioluminescence was used to determine whether there was a linear relationship between cultured cell number and measured luminescence using the luciferin-luciferase reaction. In all the cells tested including peripheral blood mononuclear cells (MNC), MOLT-4, HL-60, TF-1, NFS-60 and L-929 cell lines there was a significant correlation as determined by Spearmans rank correlation coefficient (p > 0.00001). These observations were then used to determine whether ATP bioluminescence could be used as a suitable substitute for tritiated thymidine uptake as a measure of cell proliferation. The cell lines MOLT-4, HL-60, TF-1 and NFS-60 showed a strong correlation between thymidine uptake and ATP bioluminescence (p > 0.00001 for all cell types). Additionally the ATP method could detect the cytokine dependent proliferation on TF-1 and NFS-60 cells by granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) respectively. The tumour necrosis factor alpha (TNF)-induced cytotoxic effect on L-929 cells could also be accurately detected using this method. It would therefore appear to be possible to use ATP bioluminescence in the detection of cytokine activity in a number of different bioassays.

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.

The priming effects of the products of stimulated mononuclear cells on the response of neutrophils to C5a des arg

Certain recombinant human cytokines have been shown to enhance polymorphonuclear leucocyte (PMN) responses to subsequent stimulation. Mononuclear cells (MNC) from normal healthy individuals were stimulated for 5 h with 1 μg/ml bacterial lipopolysaccharide (LPS) in order to induce production and secretion of inflammatory cytokines into the surrounding medium. These mononuclear cell conditioned media (MNCM) were then used to prime PMN isolated from healthy volunteers. Preincubating the PMN with MNCM for 15 min at 4°C followed by washing and warming to 37°C caused a 344% increase (n= 26) in the rate of superoxide anion production in response to zymosan‐activated serum (ZAS), a source of C5a des arg. This effect could not be reproduced with recombinant human forms of interleukin 1 beta (Il‐1 beta) or granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), although, with the latter, there was some effect when the preincubation stage was carried out for 60 min at 37°C. Only recombinant human tumour necrosis factor‐alpha (rh‐TNF‐alpha) gave a similar PMN priming effect to that seen with MNCM. This effect could not be reversed by washing away either the MNCM or rh‐TNF‐alpha. The priming effect could be markedly reduced (74.8%, n= 6) by employing the use of polyclonal antibody to TNF‐alpha in the preincubation step; assaying for TNF‐alpha in these MNCMs showed that the degree of priming corresponded to the amount of TNF‐alpha present. Rh‐TNF‐alpha alone appeared to have very little direct stimulatory effect on respiratory burst activity.

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

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing

&NA; TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro‐inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti‐inflammatories based on their ability to down‐regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through‐put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS‐associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro‐inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10‐fold concentrations on cell lines expressing both wild‐type (WT) TNFR1 and TRAPS‐associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse‐phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y‐TNFR1 transfectants towards the level seen in the WT‐TNFR1 transfectants. Seven high‐ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y‐TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro‐inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose‐dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF‐&kgr;B and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing. Graphical abstract Figure. No caption available.