Characterization of glucocorticoid receptors and glucocorticoid receptor mRNA in human leukemia cells: stabilization of the receptor by diisopropylfluorophosphate.
We have shown that cytosol samples from human leukemia cells frequently contain glucocorticoid receptor fragments that have a mol wt (Mr) of approximately 52,000. In the present study we demonstrate that the Mr approximately 52,000-receptor fragments are derived from intact glucocorticoid receptors (Mr approximately 97,000) by the action of a serine protease. Mr approximately 52,000-receptor fragments were present in cytosol from 24 of 52 leukemia cell samples. Only normal size glucocorticoid receptors were present in cytosol samples if diisopropylfluorophosphate (DFP), a potent inhibitor of serine proteases, was added to the hypotonic buffer used for cytosol preparation. Receptor proteolysis was not inhibited by hydrolyzed DFP, benzamidine, phenylmethylsulfonylfluoride, aprotinin, iodoacetamide, or mercuric chloride. The leukemia cell protease digests the receptor at a different site than chymotrypsin, which digests the intact receptor to produce a Mr approximately 40,000 receptor fragment. Receptor messenger RNA (mRNA) in S49 mouse lymphoma cells and in human leukemia cells was analyzed by Northern hybridization with a cDNA for the normal glucocorticoid receptor. Mutant S49 mouse lymphoma cells that have abnormally small glucocorticoid receptors (Mr approximately 48,000) make a 5.0-kilobase receptor transcript in addition to the normal size 6.5-kilobase receptor transcript. A normal size receptor transcript of 6.5 kilobases was present in all of the human leukemia cells whether or not Mr approximately 52,000-receptor fragments were present. Therefore, abnormalities of glucocorticoid receptor mRNA, which may give rise to the synthesis of foreshortened receptors in certain mutant mouse lymphoma cells, are apparently absent from human leukemia cells.
D000818:Animals; D003600:Cytosol / Q000032:analysis; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007938:Leukemia / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D011480:Protease Inhibitors; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012697:Serine Endopeptidases
D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011965:Receptors, Glucocorticoid; D007531:Isoflurophate; D010450:Endopeptidases; D012697:Serine Endopeptidases