Three way comparison of DISC, MTT, and ATP endpoints: Justification for meta-analysis of clinical studies using different cell death endpoints.

Introduction: First described in 1981, the DISC assay is the prototype for studies of cell death endpoints for cell culture drug resistance testing (CCDRT). Later studies introduced semi-automated endpoints for detecting cell death, first showing them to correlate with the DISC assay, as reviewed in the manuscript prepared at the invitation of the journal ONCOLOGY (click here for full text of paper entitled: "Current Status of Cell Culture Drug Resistance Testing").

The DISC assay measures delayed loss of membrane integrity, which has been shown to be a robust surrogate for apoptotic cell death. Other assay endpoints which have been shown (in many published studies) to correlate with the DISC assay include the MTT, ATP, and fluorescein diacetate assays.

We have carried out more than 120,000 head to head comparisons between the DISC and MTT assays in fresh human tumor specimens. Omitting only taxanes and fluoropyrimidines, for which the two assay endpoints often differ (for perfectly understandable biological reasons), the overall correlation coefficient between these two endpoints exceeds 0.82, in specimens containing >60% tumor cells (the DISC assay is specific for tumor cells in a mixed cell population, while the MTT, ATP and fluorescein diacetate assays are not specific for tumor cells and require a relatively pure population of tumor cells in order to be reliable).

Shown here in three X - Y graphs is a recent study1 from our laboratory, in which 20 different drugs were simultaneously tested in 5 fresh specimens of human adenocarcinomas, with three different cell death endpoints: DISC, MTT, and ATP. Shown are comparisons between (1) DISC and MTT endpoints, (2) MTT and ATP endpoints, and (3) DISC and ATP endpoints. These data, along with previously-published data referenced in the review prepared for publication in ONCOLOGY, provide the justification for the meta-analysis of clinical correlations with different cell death assay endpoints.

1Methods in brief: Fresh human tumor biopsies were placed into transport medium and conveyed to our laboratory on ice within 24 hours. Specimens were minced and then digested with collagenase-DNAse. Specimens were enriched for three-dimensional microclusters and these microclusters were cultured for 96 hours in the continuous presence of drugs and appropriate negative and positive controls. At the end of the culture period, % of control cell survival was determined for parallel cultures using the following three endpoints: (a) DISC (Fast green staining of non-viable cells and debris, centrifugation onto Cytospin slides, Hematoxylin/Eosin counterstaining to identify viable cells as being either tumor cells or normal cells); (b) MTT (mitochondrial Krebs cycle activity); and (c) total cellular ATP content (cellular ATP is lost coincident with cell death).