scholarly journals Neutrophil elastase produces 52-kD and 30-kD glucocorticoid receptor fragments in the cytosol of human leukemia cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 860-868 ◽  
Author(s):  
CW Distelhorst ◽  
KE Janiga ◽  
KJ Howard ◽  
SE Strandjord ◽  
EJ Campbell
Keyword(s):  
Glucocorticoid Receptor ◽  
Neutrophil Elastase ◽  
Glucocorticoid Receptors ◽  
Leukemia Cell ◽  
Specific Inhibitor ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Receptor Fragments ◽  
Limited Digestion

Abstract Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts. We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP). In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds [3H]DFP and correlates with the formation of 52- and 30-kD receptor fragments. This protease is immunoprecipitated by antiserum to neutrophil elastase. Limited digestion of [3H]dexamethasone-21-mesylate- labeled receptors by purified neutrophil elastase produces 52- and 30- kD receptor fragments. Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl- valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase. The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation. Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells. The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions.

scholarly journals Neutrophil elastase produces 52-kD and 30-kD glucocorticoid receptor fragments in the cytosol of human leukemia cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 860-868
Author(s):  
CW Distelhorst ◽  
KE Janiga ◽  
KJ Howard ◽  
SE Strandjord ◽  
EJ Campbell
Keyword(s):  
Glucocorticoid Receptor ◽  
Neutrophil Elastase ◽  
Glucocorticoid Receptors ◽  
Leukemia Cell ◽  
Specific Inhibitor ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Receptor Fragments ◽  
Limited Digestion

Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts. We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP). In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds [3H]DFP and correlates with the formation of 52- and 30-kD receptor fragments. This protease is immunoprecipitated by antiserum to neutrophil elastase. Limited digestion of [3H]dexamethasone-21-mesylate- labeled receptors by purified neutrophil elastase produces 52- and 30- kD receptor fragments. Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl- valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase. The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation. Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells. The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions.

scholarly journals Characterization of glucocorticoid receptors and glucocorticoid receptor mRNA in human leukemia cells: stabilization of the receptor by diisopropylfluorophosphate

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 750-756
Author(s):  
CW Distelhorst ◽  
R Miesfeld
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Lymphoma Cells ◽  
Human Leukemia Cells ◽  
Mouse Lymphoma Cells ◽  
Receptor Fragments ◽  
Mouse Lymphoma

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.

scholarly journals Characterization of glucocorticoid receptors and glucocorticoid receptor mRNA in human leukemia cells: stabilization of the receptor by diisopropylfluorophosphate

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 750-756 ◽  
Author(s):  
CW Distelhorst ◽  
R Miesfeld
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Lymphoma Cells ◽  
Human Leukemia Cells ◽  
Mouse Lymphoma Cells ◽  
Receptor Fragments ◽  
Mouse Lymphoma

Abstract 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.

scholarly journals A single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 679-685
Author(s):  
CW Distelhorst ◽  
BM Benutto ◽  
RC Griffith
Keyword(s):  
Glucocorticoid Receptor ◽  
Cell Lines ◽  
Glucocorticoid Receptors ◽  
Sodium Dodecyl ◽  
Leukemia Cell ◽  
Glucocorticoid Resistance ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Intact Cells ◽  
Human Leukemia Cells

We determined the mol wt of glucocorticoid receptors in human leukemia cells in order to detect glucocorticoid receptor defects that might cause glucocorticoid resistance. Glucocorticoid receptors in intact cells were affinity labeled with [3H]dexamethasone-21-mesylate and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Receptors in normal human peripheral blood mononuclear cells and six human leukemia cell lines had mol wt of 97,000. Malignant cells from ten of 25 patients with leukemia contained electrophoretically abnormal glucocorticoid receptors having mol wt of 55,000 in addition to normal size receptors (Mr = 97,000). The receptor abnormality was not restricted to a particular type of leukemia and was seen in cells from both newly diagnosed patients and patients who had received prior chemotherapy, including prednisone. The abnormal receptor was not generated when cells having only normal size receptors were assayed under conditions that favor proteolysis or when cytosol from cells containing the abnormal receptor form was mixed with cytosol from cells containing only normal size receptors. The mol wt of the abnormal receptors in human leukemia cells was the same as the mol wt of receptors in mutant mouse lymphoma cell lines, S49 143R and S49 55R, which have the nuclear transfer-increased phenotype of glucocorticoid resistance. This work describes for the first time a single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells. Further investigation of glucocorticoid receptor defects in human leukemia cells could lead to an improved understanding of the mechanisms of glucocorticoid resistance in leukemia as well as a method of predicting which patients are likely to be resistant to glucocorticoid therapy.

scholarly journals A single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 679-685 ◽  
Author(s):  
CW Distelhorst ◽  
BM Benutto ◽  
RC Griffith
Keyword(s):  
Glucocorticoid Receptor ◽  
Cell Lines ◽  
Glucocorticoid Receptors ◽  
Sodium Dodecyl ◽  
Leukemia Cell ◽  
Glucocorticoid Resistance ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Intact Cells ◽  
Human Leukemia Cells

Abstract We determined the mol wt of glucocorticoid receptors in human leukemia cells in order to detect glucocorticoid receptor defects that might cause glucocorticoid resistance. Glucocorticoid receptors in intact cells were affinity labeled with [3H]dexamethasone-21-mesylate and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Receptors in normal human peripheral blood mononuclear cells and six human leukemia cell lines had mol wt of 97,000. Malignant cells from ten of 25 patients with leukemia contained electrophoretically abnormal glucocorticoid receptors having mol wt of 55,000 in addition to normal size receptors (Mr = 97,000). The receptor abnormality was not restricted to a particular type of leukemia and was seen in cells from both newly diagnosed patients and patients who had received prior chemotherapy, including prednisone. The abnormal receptor was not generated when cells having only normal size receptors were assayed under conditions that favor proteolysis or when cytosol from cells containing the abnormal receptor form was mixed with cytosol from cells containing only normal size receptors. The mol wt of the abnormal receptors in human leukemia cells was the same as the mol wt of receptors in mutant mouse lymphoma cell lines, S49 143R and S49 55R, which have the nuclear transfer-increased phenotype of glucocorticoid resistance. This work describes for the first time a single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells. Further investigation of glucocorticoid receptor defects in human leukemia cells could lead to an improved understanding of the mechanisms of glucocorticoid resistance in leukemia as well as a method of predicting which patients are likely to be resistant to glucocorticoid therapy.

scholarly journals Characterization of an Amsacrine-resistant Line of Human Leukemia Cells

1989 ◽  
Vol 264 (28) ◽  
pp. 16411-16420
Author(s):  
L A Zwelling ◽  
M Hinds ◽  
D Chan ◽  
J Mayes ◽  
K L Sie ◽  
...  
Keyword(s):  
Leukemia Cells ◽  
Human Leukemia ◽  
Resistant Line ◽  
Human Leukemia Cells

scholarly journals Regulation of myeloblastin messenger RNA expression in myeloid leukemia cells treated with all-trans retinoic acid

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 475-481 ◽  
Author(s):  
C Labbaye ◽  
J Zhang ◽  
JL Casanova ◽  
M Lanotte ◽  
J Teng ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract Retinoic acid is known to induce differentiation of human myeloid leukemia cells in vitro. Recently, all-trans retinoic acid has been used to induce remissions in patients with acute promyelocytic leukemia, probably through differentiation of the leukemia cells. Myeloblastin (mbn) is a protease that has been identified in the human leukemia cell line HL-60. Downregulation of this protease can inhibit proliferation and induce differentiation of HL-60-derived leukemia cells. Here we have investigated the regulation of mbn messenger RNA (mRNA) expression in two human leukemia cell lines, HL-60 and NB4, treated with all-trans retinoic acid. Under this treatment, downregulation of mbn mRNA was observed in both cell lines, but was considerably delayed in NB4 cells that carry the t(15;17) translocation characteristic of acute promyelocytic leukemia. We have found that multiple mechanisms were involved in the control of mbn mRNA expression. These mechanisms were different in HL-60 and NB4 cells. Our results show that in HL-60 cells, all-trans retinoic acid rapidly decreased transcription of mbn. In contrast, in the t(15;17)-positive NB4 cells treated with all-trans retinoic acid, upregulation of mbn mRNA expression was followed by a late downregulation, both achieved via posttranscriptional mechanisms.

scholarly journals Characterization of glucocorticoid receptors in animal lymphoblastic disease: correlation with response to single-agent glucocorticoid treatment

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 380-383 ◽  
Author(s):  
R Bell ◽  
S Cotter ◽  
A Lillquist ◽  
S Sallan ◽  
R McCaffrey
Keyword(s):  
Glucocorticoid Receptors ◽  
Single Agent ◽  
Complete Response ◽  
Single Peak ◽  
Human Leukemia ◽  
Normal Pattern ◽  
Glucocorticoid Treatment ◽  
Normal Tissues ◽  
Low Salt

Abstract The clinical significance of initial DEAE chromatography of glucocorticoid binders in lymphoblastic disease was evaluated in an animal model. Domestic cats and dogs with lymphoblastic disease were treated with prednisone, 2 mg/kg/day, for 14 days, and the outcome of therapy was correlated with DEAE chromatograms of glucocorticoid binders, using 3H-triamcinolone as ligand. Six of 30 animals had a single-peak low-salt binder species, similar to that seen in a subset of human leukemia, and none of these responded. Of the 29 animals with chromatograms identical to normal tissues, 6 had a complete response and another 11 a partial response. This distribution of responders is statistically significant (p = 0.02). Thus, the leukemia-associated single-peak DEAE species appears to be associated with glucocorticoid resistance, as defined by clinical responsiveness. In contrast, the two- peak normal pattern is a necessary, but insufficient, criterion for defining responsive disease.

scholarly journals Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9390
Author(s):  
Monika Jedrzejczyk ◽  
Katarzyna Wisniewska ◽  
Katarzyna Dominika Kania ◽  
Agnieszka Marczak ◽  
Marzena Szwed
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Mononuclear Cells ◽  
Inflammatory Responses ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Morphological Observation ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Normal Peripheral Blood

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin–transferrin (DOX–Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX–Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX–Tf. Studies of expression of TNF-α, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX–Tf conjugate.

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