ABSTRACT
In this report, inhibitors of the gamma-secretase enzyme have been exploited to characterize the antiproliferative relationship between target inhibition and cellular responses in Notch-dependent human T cell acute lymphoblastic leukemia (T-ALL) cell lines. Inhibition of gamma-secretase led to decreased Notch signaling, measured by endogenous NOTCH intracellular domain (NICD) formation, and was associated with decreased cell viability. Flow cytometry revealed that decreased cell viability resulted from a G(0)/G(1) cell cycle block, which correlated strongly to the induction of apoptosis. These effects associated with inhibitor treatment were rescued by exogenous expression of NICD and were not mirrored when a markedly less active enantiomer was used, demonstrating the gamma-secretase dependency and specificity of these responses. Together, these data strengthen the rationale for using gamma-secretase inhibitors therapeutically and suggest that programmed cell death may contribute to reduction of tumor burden in the clinic.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Leukemia-Lymphoma, Adult T-Cell/drug therapy , Leukemia-Lymphoma, Adult T-Cell/pathology , Receptors, Notch/antagonists & inhibitors , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cyclic S-Oxides/pharmacology , Flow Cytometry , Humans , Leukemia-Lymphoma, Adult T-Cell/enzymology , Leukemia-Lymphoma, Adult T-Cell/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Thiadiazoles/pharmacologyABSTRACT
Neural stem cells reside in the subventricular zone and the dentate gyrus of the hippocampus in adult mammalian brain. In the hippocampus, a number of factors are reported to modulate the rate of neural progenitor proliferation in the hippocampus, such as exercise, corticosteroids, and many pharmacological agents including several classes of antidepressants. It is currently unclear whether this increased proliferation is physiologically relevant, but it provides a potentially useful biomarker to assess novel antidepressant compounds. Changes in neurogenesis are typically quantified by administration of bromodeoxyuridine (BrdU) in vivo, and subsequent quantification of labelled nuclei. A robust and rapid means of quantifying BrdU labelling in adult hippocampus in vivo would allow higher throughput screening of potential antidepressant compounds. In this study we describe a FACS-based method for quantification of BrdU labelled cells in fixed cell suspensions from BrdU-treated adult mouse hippocampus. A variety of experimental conditions known to modulate proliferation were tested, including administration of corticosterone and the antidepressants imipramine and fluoxetine. The robust changes compared to control groups observed in these models were similar to previously reported studies, thus offering a more rapid and streamlined means to quantify effects of compounds on hippocampal proliferation.
Subject(s)
Flow Cytometry/methods , Hippocampus/cytology , Immunohistochemistry/methods , Neurons/physiology , Organogenesis/physiology , Animals , Bromodeoxyuridine/administration & dosage , Bromodeoxyuridine/metabolism , Cell Count/methods , Dactinomycin/administration & dosage , Dactinomycin/analogs & derivatives , Dose-Response Relationship, Drug , Mice , Reproducibility of ResultsABSTRACT
We have developed an assay system suitable for assessment of compound action on the Edg4 subtype of the widely expressed lysophosphatidic acid (LPA)-responsive Edg receptor family. Edg4 was stably overexpressed in the rat hepatoma cell line Rh 7777, and a Ca(2+)-based FLIPR assay developed for measurement of functional responses. In order to investigate the mechanisms linking Edg4 activation to cytosolic Ca(2+) elevation, we have also studied LPA signalling in a human neuroblastoma cell line that endogenously expresses Edg4. LPA responses displayed similar kinetics and potency in the two cell lines. The Ca(2+) signal generated by activation of LPA-sensitive receptors in these cells is mediated primarily by endoplasmic reticulum. However, there is a substantial inhibition of the LPA response by FCCP, indicating that mitochondria also play a key role in the LPA response. Partial inhibition of the response by cyclosporin A could indicate an active Ca(2+) release role for mitochondria in the LPA response. The inositol 1,4,5-triphosphate receptor antagonist 2-aminoethyl diphenyl borate markedly inhibits, but does not abolish, the Ca(2+) response to LPA, suggesting further complexity to the signalling pathways activated by Edg receptors. In comparing Edg signalling in recombinant and native cells, there is a striking overall similarity in receptor expression pattern, agonist potency, and the effect of modulators on the Ca(2+) response. This indicates that the Edg4-overexpressing Rh7777 cell line is a very useful model system for studying receptor pharmacology and signalling mechanisms, and for investigating the Edg4 receptor's downstream effects.
