|
|
British Journal of Cancer (2005) 92, 532-538.
doi:10.1038/sj.bjc.6602363 Published online 1 February 2005
Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP
J Cummings1, T H Ward1, E LaCasse2, C Lefebvre2, M St-Jean2, J Durkin2, M Ranson3 and C Dive1
1Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK
2Aegera Oncology Inc., CHEO Research Institute, 401 Smyth Rd, Ottawa, Ontario, Canada K1H 8L1
3Department of Medical Oncology, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK
Correspondence to: Dr J Cummings, E-mail: jcummings@picr.man.ac.uk
Received 16 September 2004; revised 3 December 2004; accepted 3 December 2004; published online 1 February 2005
The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80°C for at least 60 days. M30-ApoptosenseÔ plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80°C, while at 37°C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.
Keywords: XIAP; antisense; quantitative RT-PCR; Western blot analysis; M30-ApoptosenseÔ Elisa
back to top  |
| Article links | |
| ||
| ||
 | Send to a friend | |
 | Download PDF | |
| ||
| ||
| ||
 | Full text | |
| ||
| ||
| ||
 | Preceding article | |
 | Next article | |
 | Table of Contents | |
| ||
| ||
| ||
| ||
| ||
|
|
|
|
| |||
| Print ISSN: 0007-0920 | Online ISSN: 1532-1827 | © 2005 Cancer Research UK | ||
| |||
| Privacy Policy | |||