Analysis of Glucocorticoid Actions on Rat Thymocyte Deoxyribonucleic Acid by Fluorescence-Activated Flow Cytometry*

Abstract

Glucocorticoids induce thymocyte cell death via mechanisms that occur in concert with internucleosomal DNA cleavage. To further study this process at the single cell level, glucocorticoid effects on rat thymocyte DNA were analyzed using fluorescence-activated flow cytometry. Thymocytes were isolated from adrenalectomized rats, treated in vitro with steroid hormones, stained with 0.1 mM acridine orange, and analyzed by flow cytometry. After 5 h of treatment with 10(-7) M dexamethasone a population of thymocytes with reduced acridine orange binding was observed. Thymocyte viability after glucocorticoid treatment was greater than 95%, indicating that the observed changes in dye binding occurred before cell death. Induction of this subpopulation of thymocytes was both time and steroid concentration dependent, being detectable within 2 h of in vitro steroid treatment. This response to glucocorticoids was blocked by the glucocorticoid receptor antagonist RU 486, indicating a steroid receptor-mediated process. Furthermore, both actinomycin D and cycloheximide blocked the appearance of this glucocorticoid effect, thus demonstrating a dependence upon RNA and protein biosynthesis, respectively. Incubation of thymocytes with inhibitors of DNA repair (aminobenzamide or methylnicotinamide) also diminished acridine orange binding to thymocytes. The effects of these DNA repair inhibitors and glucocorticoids were additive, suggesting independent mechanisms of action. Dexamethasone treatment (10(-7) M) of cells that fail to die in response to glucocorticoids (HeLa S3) had no effect on dye binding. These findings are consistent with the concept that glucocorticoids induce a lysis gene product that alters the thymocyte genome and thus leads to cell death.

Comparative evaluation of several DNA binding dyes in the detection of apoptosis‐associated chromatin degradation by flow cytometry

“…These results strongly suggest that a particular binding mechanism is not a requirement for the resolution of the A, region in thymocyte apoptosis. Although intercalating dyes have been utilized most extensively as probes of chromatin structure (7,ll) and have been previously used to distinguish apoptosis (1,6,20,27), the changes in chromatin conformation associated with apoptosis can apparently result in reduced overall fluorescence with nuclear dyes possessing a variety of primary binding mechanisms.…”

“…Apoptotic (A,) Region The formation of a distinct, quantifiable cell cycle region below G,/G, i n mouse thymocytes after incubation with glucocorticoids or prior exposure to ionizing radiation has been previously shown to represent cells undergoing apoptosis-associated DNA degradation (1,6,20,27). The region was designated the A, region, and a percentage of events in this region with respect to the entire cell cycle could be easily calculated.…”

Clarithromycin Delays Progression of Bronchial Epithelial Cells from G 1 Phase to S Phase and Delays Cell Growth via Extracellular Signal-Regulated Protein Kinase Suppression

“…The DNA histogram was analyzed with Modfit (Modfit Verity Software House, Topsham, ME), and the percentage of cells in each fraction of the cell cycle (G 1 , S, and G 2 /M phases) was calculated as described previously (10). Cells located in the sub-G 1 area were considered apoptotic cells (3).…”

The interleukin 1β‐converting enzyme inhibitor CrmA prevents Apo1/Fas‐ but not glucocorticoid‐induced poly(ADP‐ribose) polymerase cleavage and apoptosis in lymphoblastic leukemia cells

“…The mechanisms underlying GC-induced apoptosis are poorly understood and may differ in various cell types and species. Thus, in rodent thymocytes, GC apoptosis is macromolecule neosynthesis-dependent [40,41], employs a Ca 2+ -activated endonuclease [42], and occurs in the absence of functional ICE, as shown in corresponding knock-out mice [4]. In human leukemic cells, in contrast, GC-induced cell death may occur via transrepression rather than transactivation [43], and DNA fragmentation is mediated by a Ca 2+ -independent endonuclease [44].…”