Homogenate Based Assays for Apoptosis May Cause a Misleading Result
D He, J Slisz, J Johnson, C Miller, B Firestone, L Zawel, J Li, M Caprio, R Mosher. Novartis Institutes for Biomedical Research, Inc., Camdridge
Background: Detection of apoptosis can be used as a pharmacodynamic marker to evaluate the anti-tumor effect. These studies can be performed on tumor homogenates or histological analysis. The purpose of this study was to evaluate both methods that might impact the readout of our studies.
Design: Colo 205 and A2058-derived xenograft tumors were treated with compounds known to induce apoptosis. Xenograft collected both formalin fixed for IHC/histology and frozen for homogenate preparation. Cleaved Caspase 3 IHC was scored using the Aperio Image analysis system. The anti-human CAIX antibody was performed to define hypoxic regions. In parallel, Caspase 3 enzymatic activity was measured in homogenates using the both TruPoint Caspase-3 Assay and Caspase-Glo 3/7 Assay.
Results: Histologic examination revealed the fast growing A2058 tumors to be very heterogeneous in both the vehicle and treated animals. CC3 activation was observed around necrotic areas in both vehicle and treated tumor. Necrotic areas co-localized with CAIX antibody expression suggest that hypoxia may cause activation of CC3. Manual exclusion of necrotic areas of IHC slides allowed differences between treated and non-treated groups to be observed. Following normalization to the vehicle group the CC3 induction attributed to drug treatment was approximately14 fold. Interestingly, the homogenate assay failed to discern this difference. In contrast to the A2058 tumors, slow growing Colo 205 xenograft tumors were well defined with few necrotic areas. Caspase 3 profiling was performed in Colo205 tumors following treatment with the DR5 antibody. The CC3 IHC and CC3 enzymatic homogeneous assay gave concordant results in this non-necrotic xenograft.
Conclusions: It is necessary to understand tumor growth behavior and tissue composition prior to choosing a homogenate or slide based assay.
Category: Special Category for 2009 - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 249, Tuesday Morning