1985 — 1990 |
Munck, Allan U |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glucocorticoid-Resistant Leukemic Lymphocytes
These studies have two general goals, both related to the use of glucocorticoids in treatment of leukemias and lymphomas. One goal is to apply and extend our knowledge of glucocorticoid receptors and mechanisms of action to normal and malignant lymphocytes and related cells. For these studies we are applying our recently developed minicolumn procedure to the analysis of nonactivated, activated, and meroreceptor complexes in cytosols from the cells mentioned under various conditions. Of particular current interest to us is the role in stabilizing receptors of calpastatin, a naturally occurring protein inhibitor of a family of calcium-activated proteases called calpains. We have found that the relative stability of receptors in cytosols from cells of patients with chronic lymphocytic leukemia (CLL) compared to those from patients with acute nonlymphocytic leukemia (ANLL) is at least partly due to the presence in the former cells of a stabilizer that closely resembles calpastatin. The other goal is to extend our observations on glucocorticoid effects on lymphocytes and other cells that are mediated by inhibition of, or synergism with, lymphokines such as interleukin 2 (IL-2) and immune or gamma interferon. We have shown that gamma interferon is the major lymphokine that increased Fc-Receptors for IgG on human monocytes, and that glucocorticoids added together with gamma interferon augment this effect as well as antibody-dependent and\-independent tumor cell lysis and expression of class I and class II HLA antigens. We are now investigating the mechanisms of these effects. In relation to IL-2, an autocrine mechanism of neoplastic growth involving IL-2 production and response has been established in MLA 144, an IL-2 producing leukemia cell line derived from a Gibbon ape. (D)
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0.958 |
1985 — 1999 |
Munck, Allan U |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mode of Action of Steroid Hormones
DESCRIPTION (Adapted from the applicant's abstract): This proposal focuses on the relationship between glucocorticoid receptor (GR) function and dynamic transformations of GR by phosphorylation and association with heat shock proteins. Previous work by the principal investigator has identified in detail the sites at which GR is phosphorylated, and has shown cell-cycle dependent changes in GR phosphorylation and heat shock protein association/dissociation. The present proposal will extend these studies, addressing 1) the role of GR phosphorylation in GR function; 2) the role of cell-cycle dependent kinases in this phosphorylation; 3) the possibility that estrogen in progesterone receptors also traverse ATP-dependent cycles.
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0.958 |
1986 — 1987 |
Munck, Allan U |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glucocorticoid-Resistant Leukemic Lymphocutes
The long-term objectives are to elucidate the actions and interactions of glucocorticoids and cytokines such as gamma interferon (IFN-gamma) and interleukin 2 (IL-2) on tumor proliferation and on inactivation of the immune system. We will study these phenomena both in vitro in cell culture systems and in vivo in mice. Proposed experiments are based on numerous recent observations with these substances. They include those made on this project, which demonstrated that glucocorticoids inhibit production of lymphokines, but may inhibit or enhance their actions; and that the lymphokines, in turn, may counteract certain immunosuppressive actions of glucocorticoids, suggesting the possibility of using lymphokines to selectively counteract unwanted side effects of glucocorticoid therapy. The fact that glucocorticoids are already widely used in treatment of leukemias and lymphomas, and that lymphokines, which are now becoming generally available, are being tested experimentally in cancer therapy, emphasizes the importance of understanding interactions and mechanisms of these agents. With respect to tumoricidal activity, the specific aims are directed at two fundamental questions: (i) Do glucocorticoids in vitro or in mice alter tumoricidal activation, and in particular antibody-dependent cellular cytotoxicity (ADCC), of macrophages and other phagocytes by IFN-glamma and lymphotoxin? (ii) Do glucocorticoids alter the tumoricidal activity of interferons and lymphotoxin on glucocorticoid-sensitive and -resistant lymphoid cell lines in culture or in mice? With respect to stimulation of the immune system for bactericidal activity, the questions to be answered are: (i) Do glucocorticoids alter bactericidal activation of cultured human and mouse leukocytes by IFN-gamma plus IL-2? (ii) Can IFN-gamma and/or IL-2 reverse the increased susceptibility to infection in mice caused by glucocorticoids? Finally, using cDNA probes to measure cellular levels of mRNA for IFN-gamma and IL-2, the goals are to answer the following questions: (i) Is reduction by glucocorticoids of lymphokine mRNA levels in human T lymphocytes mediated by induced protein(s)? (ii) Are those levels under negative feedback control by the lymphokines themselves?
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0.958 |
1994 — 1996 |
Munck, Allan U |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cell Cycle and Glucocorticoid Receptor Phosphorylation
Glucocorticoids at high doses are used widely to treat disorders ranging from minor allergies to leukemias and lymphoma. They act on almost all cells. So much effort has gone into maximizing their therapeutic effectiveness and minimizing unwanted side effects. Sensitivity to glucocorticoids varies strikingly through the cell cycle, the hormones being effective only if present during late G1 and S (DNA synthesis) phases. Our long-term goal is to elucidate the basic mechanisms of this variation. In this proposal, the central objective is to test the hypothesis that phosphorylation of glucocorticoid receptors (GRs) changes through the cell cycle and accounts at least in part for the variation in sensitivity. This hypothesis, based on evidence for cell cycle-dependence of GR number, nuclear binding, and phosphorylation, has gained support -- and the means to test it -- from our finding of a rapid, hormone-dependent increase in GR phosphorylation, and our identification by phosphopeptide mapping and sequencing of seven phosphorylated sites in mouse GRs: four lie in consensus sequences for the p34cdc2 kinases that control the cell cycle. The proposal is designed to answer the following specific questions; 1. Do hormone dependent GR hyperphosphorylation and/or GR phosphorylated sites change through the cell cycle? (a) Synchronized WCL2 cells (CHO cells with overexpressed mouse GRs) will be labeled with 32P and treated with hormone to assay hormone-dependent phosphorylation of the GRs; (b) Phosphorylated sites in these GRs will be identified by phosphopeptide mapping, and sequencing of any new peptides. 2. How do glucocorticoid sensitivity and GR number change through the cell cycle in Cho cells? Synchronized CHO cells with overexpressed GRs will be used to: (a) measure hormone effects on activity of a stably transfected gene and thymidine kinase; (b) determine if the increased number of GR binding sites in G1/S is due to increased GR protein per cell. 3. Do GR phosphorylation mutants behave differently from wild-type GRs? CHO cells with overexpressed GRs mutated at normally phosphorylated sites will be studied as in (2a). 4. Are GR phosphorylation and glucocorticoid-induced apoptosis in lymphocytes cell cycle-dependent, and modifiable by chemotherapeutic agents? Synchronized WEHI-7 cells will be used to measure (a) GR phosphorylation as in la; (b) glucocorticoid-induced apoptosis; (c) effects in (a) and (b) of cell cycle-modifying agents. Our results may benefit therapy and enhance understanding of glucocorticoid resistance.
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0.958 |