A robotic MCF-7:WS8 cell proliferation assay to detect agonist and antagonist estrogenic activity.

Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program's Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17ß-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development.

Hedgehog-induced survival of B-cell chronic lymphocytic leukemia cells in a stromal cell microenvironment: a potential new therapeutic target.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell-induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell-induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target.