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CORRELATION OF VISUALIZED GLUCOCORTICOID RECEPTOR AND APOPTOSIS IN INDIVIDUAL CLONES OF NB2 CELLS Devang Patel A thesis submitted in partial fu lfillment of the requirements for the degree of Master in Sc ience at Virginia Commonwealth University. V irginia Commonwealth University, 1 999 Director: Raphael J. Witorsch, Ph.D. , Professor, Department of Physiology The Nb2 lymphoma cell l ine is susceptible to glucocorticoid (GC)-induced apoptosis in the absence of mitogen and this effect is inhibited by the mitogen prolactin (PRL). This process is believed to be mediated by the glucocorticoid receptor (GR) and various downstream signals. The purpose of this study was to use the l imiting dilution method to isolate subsets of Nb2 clones which expressed either h ighly enriched amounts of GR or an absence of GR and to relate the amount of GR to the magnitude of GC induced cytolysis. The presence of GR in these clones was determined by immunocytochemistry (ICC) using an affinity purified polyclonal rabbit antibody directed towards the GR. The presence of GR was then correlated to cytolysis in these cel ls produced by incubation with dexamethasone (Dex), a synthetic GC, in the presence and absence of PRL. x Cytolysis was measured by two different methods, Trypan Blue exclusion counts, to determine the percentage of dead and the TUNEL (TdT-dependent dUTP-biotin Nick End Label ing) assay used to label apoptotic cells. The l imiting dilution produced 28 clones with varying amounts of GR and all were responsive to Dex and this response was inhibited by PRL. A significant correlation (r 2 =0.4 1 9 and p<O.OOO I ) was observed between the percentage of cells stained for GR and the % dead cel ls after Dex treatment. Comparison of mean values for percent dead and percent TUNEL labeled for DMSO, Dex±PRL, and PRL alone for clones and wild types (assayed along with clones) revealed that these two endpoints of apoptosis were in close agreement with one another. The mean values of the percentage of cel ls undergoing apoptosis agreed closely with the percentage of cells labeled for GR. Furthermore the mean values for all treatments of the clones were very similar to those obtained for the wild type cells. It is concluded that the cytolytic response to dexamethasone is representative of the apoptotic response and that the GR observed by ICC is a quantitative predictor of the apoptotic response to Gc.

Correlation of Visualized Glucocorticoid Receptor and Apoptosis In Individual Clones of Nb2 Cells