Quantitation of bcl-2 protein in bladder cancer tissue by enzyme immunoassay: Comparison with Western blot and immunohistochemistry

Abstract

Apoptosis (programmed cell death) and the genes regulating this process (e.g., bcl-2), have recently become a focus of interest in the study of cancer development and progression. We adapted and evaluated a new enzyme immunoassay method (EIA) for quanitifying bcl-2 in cell lysates. The range of detection of the assay was 5-400 kilounits/L with inter- and intraassay CVs of 5.5-9.2% and 5.0-8.8%, respectively. The recovery of added bcl-2 protein to cell lysates was 96-104%. The concordance rates with Western blotting and immunohistochemistry were 97.5% and 93.7%, respectively. Bcl-2 concentrations were measured in the cell lysate of bladder tumors. The amount of bcl-2 in 158 bladder cancer (mean rank, 71.3 kilounits/g protein; range, 8.4324 kilounits/g protein), was significantly higher than in nondiseased bladder tissues distant to the tumors (mean rank, 31.5 kilounits/g protein; range, 5- 54.9 kilounits/g protein), P = 0.00001. Bcl-2 expression was correlated to tumor proliferative capacity, which was measured by DNA flow cytometry as the percentage of cells in the synthetic phase of the cell cycle. The enzyme immunoassay provides a rapid, quantitative, and reliable technique for measurement of bcl-2 in tumor tissue. The detection of substantial amounts of bcl-2 in invasive tumors (compared with nondiseased tissues) suggests that the assay should be a useful tool for investigating the prognostic value of bcl-2 in bladder tumors and for selecting patients for future anti-bcl-2 therapy.