demecolcine and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

The human leukemic cell line (CCRF-CEM) and a subline enriched for the plasma membrane-resident glucocorticoid receptor (mGR) were studied for the influence of the cell cycle on the expression and function of mGR. Three synchronization procedures (double thymidine, colcemid, and combined thymidine-colcemid blocks) were used. Fluorescent microscopy and flow cytometry simultaneously assessed antibody-tagged mGR and DNA. In addition, mGR was quantitated and characterized by immunoprecipitation and immunoblotting. Apoptosis was assayed by DNA fragmentation (TUNEL assay) and by cell survival (trypan blue exclusion). All synchronization procedures demonstrated that progression from DNA replication (S) to the second growth phase and mitosis (G2/M) leads to cells having the highest levels of mGR expression and being highly glucocorticoid sensitive in the apoptosis assays: 32 and 80% sensitivity of wild type and mGR-enriched cells, respectively, compared with 12 and 30% sensitivity in asynchronous cells. Therefore, mGR expression appears to be cell cycle regulated, with its highest expression at late S-G2/M, when the cells are most sensitive to the lymphocytolytic effects of glucocorticoids.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Membrane; Demecolcine; DNA, Neoplasm; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kinetics; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Glucocorticoid; S Phase; Thymidine; Tumor Cells, Cultured