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Biomedical subjects

E B Thompson

Publications and source records attributed to E B Thompson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Resistance of human leukemic CEM-C1 cells is overcome by synergism between glucocorticoid and protein kinase A pathways: correlation with c-Myc suppression.

Glucocorticoids (GCs) induce apoptosis in lymphoid cells that contain functional GC receptors (GRs). However, GC resistance often is seen in cells with demonstrable GRs; one such line is CEM-C1. We have tested the hypothesis that positive interactions between GC and cyclic AMP (cAMP) regulate GC actions in CEM clones. Treatment of both GC-resistant CEM-C1 [resistant to 1 microM dexamethasone (Dex)] and the sensitive sister clone, CEM-C7 (approximately 65% cell death with 20 nM Dex, approximately 99% death with 1 microM Dex), with a < or = 20 microM concentration of the protein kinase A activator, forskolin, had no significant effect on cell viability. Cotreatment with Dex and forskolin resulted in a strong synergistic death response, with only approximately 10% CEM-C1 cells surviving treatment with 1 microM Dex and 20 microM forskolin. This death was blocked by the GR antagonist RU 38486. However, the extent of apoptosis did not correlate with the amount of GR protein or binding activity in either C7 or C1 cells. As reported previously, Dex-evoked cell death was associated with suppression of c-Myc in C7 cells. In CEM-C1 cells, Dex alone did not affect c-Myc; however, Dex plus forskolin suppressed c-Myc levels. To evaluate mechanisms of Dex-forskolin synergism, fresh subclones of CEM-C7 (clone 14) and CEM-C1 (clone 15) were isolated, to ensure purity of phenotype. In these, forskolin (with or without Dex) caused a similar increase in cAMP (approximately 300-fold) and phospho-cAMP-responsive element binding protein (approximately 4-5-fold) levels, whereas total cAMP-responsive element binding protein expression was not affected. GR transcription function, as tested from a GR-responsive 330-bp mouse mammary tumor virus promoter-luciferase reporter construct, was induced 8- and 4-fold by 1 microM Dex treatment of CEM-C7-14 and CEM-C1-15 cells, respectively. Forskolin (10 microM) significantly potentiated Dex response in CEM-C1-15 cells (13.5-fold) but had only a modest effect (1.5-fold) in CEM-C7-14 cells. These studies suggest that sensitization of CEM-C1 cells by cross-talk between GR and protein kinase A pathways may occur via cooperative effects on GR-mediated gene transcription.

Apoptosis↗
PubMed1998

Estrogen-induced microsatellite DNA alterations are associated with Syrian hamster kidney tumorigenesis.

Exposure to estrogens is associated with an increase in cancers, including malignancies of the breast and uterus in humans, and of the kidney in hamsters. DNA damage induced by metabolic activation of estrogen has been postulated to result in gene mutations critical for the development of estrogen-induced kidney tumors in hamsters. As part of our examination of the genetic consequences of estrogen-induced DNA damage, we searched for estrogen-induced alterations in microsatellite DNA, a frequent site of mutation in tumors. Genomic DNA isolated from kidney of hamsters treated with estradiol, from estrogen-induced kidney tumors and from untreated age-matched controls, was examined by Southern blot analysis with three multi-locus oligonucleotide probes: (GACA)4, (CAC)6 and (CAG)6. Alterations in DNA fragments containing GACA and CAC tandem repeats were detected in kidney DNA of hamsters treated with hormone for 3 and 4 months, whereas no such effects were seen in control animals. In estrogen-induced tumors, microsatellite alterations were observed in fragments that contain these same two repeat sequences and also CAG repeat sequences. The induction of microsatellite alterations by estradiol in kidney DNA preceding estrogen-induced renal malignancy may play a role in hormone-induced tumorigenesis.

Animals↗
PubMed1998

The many roles of c-Myc in apoptosis.

The proto-oncogene c-myc encodes a transcription factor c-Myc, which is of great importance in controlling cell growth and vitality. The quantity of c-Myc is carefully controlled by many mechanisms, and its actions to induce and repress genes are modulated by interactions with other regulatory proteins. Understanding the kinetic and quantitative relationships that determine how and what genes c-Myc regulates is essential to understanding how Myc is involved in apoptosis. Reduction of c-myc expression and its inappropriate expression can be associated with cellular apoptosis. This review outlines the nature and regulation of the c-myc gene and of c-Myc and presents the systems and conditions in which Myc-related apoptotic events occur. Hypotheses of the mechanisms by which expression and repression of c-myc lead to apoptosis are discussed.

Animals↗
PubMed1997

Protein-protein interactions are implied in glucocorticoid receptor mutant 465*-mediated cell death.

Previously we have shown that ICR-27, a clone of glucocorticoid-resistant human leukemic T cells, showed rapid cell loss upon transient transfection with plasmids expressing certain fragments of the human glucocorticoid receptor lacking the ligand binding domain. An extreme example was the frameshift GR mutant 465*, mutated after amino acid 465. This generated a novel 21-amino acid "tail," beginning within the second zinc finger of the human glucocorticoid receptor DNA binding domain, a region required for ICR-27 cell death caused by hologlucocorticoid receptor plus hormone. The cell loss mediated by 465* was faster but quantitatively equivalent to that caused by hologlucocorticoid receptor plus hormone. We are therefore investigating the mechanism of action of 465*. We overexpressed 465* with or without a glutathione S-transferase tag fused to its N terminus and tested its ability to affect glucocorticoid response element (GRE)-driven reactions in vitro. Partially purified 465* showed little binding to a consensus GRE, caused virtually no stimulation of transcription from a GRE, and failed to inhibit GR-driven transcription. However, GST-465* "trapped" several proteins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro. In co-transfection assays of CV-1 cells, 465* expression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) transcriptional activation from a promoter containing multiple AP-1 sites. Further studies proved the repressive activity of 465* was c-Jun-specific and not due to squelching artifacts. The data suggest that interaction of 465* with other proteins, such as c-Jun, might be responsible for its cell killing function.

Animals↗
PubMed1997

Characteristics of 25-hydroxycholesterol-induced apoptosis in the human leukemic cell line CEM.

Cholesterol and related compounds can give rise to oxygenated sterol molecules (oxysterols) which are potent regulators of lymphoid cell growth. Oxysterols added exogenously cause cell death of several lines of cultured cells, and on the basis of limited criteria, it has been suggested that this death is apoptosis. In the present study, we show definitive evidence that 25-hydroxycholesterol (25OHC) kills cells of the clone CEM-C7 by apoptosis and establish the temporal sequence of related cellular and biochemical events. Cell shrinkage was evident as early as 12 h, while cell death was not evident until after 24 h. It mounted rapidly after that, and by 72 h, virtually all cells were dead. Electron microscopic analysis shows that by 24 h after treatment and before the onset of cell death, early ultrastructural features typical of apoptosis were present. DNA breaks were detected by TUNEL assay prior to the onset of cell death. Two types of specific DNA pieces often associated with apoptosis were found as increasing numbers of cells died. DNA fragments of 300 and 50 kbp were not appreciable until 42 h, and internucleosomal cleavage was observed by 48 h after oxysterol addition. None of these effects were seen in an oxysterol-resistant CEM subclone, establishing the specificity for apoptosis of the biochemical and morphological events. z-VAD.FMK, a peptide inhibitor of ICE-related proteases delayed but did not prevent the apoptosis of CEM-C7 cells induced by 25OHC. The addition of mevalonate partially protected CEM-C7 cells from apoptosis but did not restore cell growth.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Relationship between biochemical, virological, and histological response during interferon treatment of chronic hepatitis C.

The present study was conducted to evaluate the relationship between biochemical, virological, and histological response during the course of interferon therapy. Ninety consecutive patients with well-documented chronic hepatitis C virus (HCV) were treated with 5 MU of interferon alfa-2b three times weekly for 6 months. Liver biopsy was performed, and serum HCV RNA titer was measured before and at the completion of interferon treatment. Normalization of serum alanine transaminase (ALT) concentration (biochemical response) was observed in 50% of patients. In these patients, Knodell score declined significantly from 9.6 +/- 0.5 to 5.0 +/- 0.5 (P < .01), and 75% became HCV RNA negative. The remaining patients (50%) were biochemical nonresponders; mean Knodell score declined from 9.6 +/- 0.5 to 7.7 +/- 0.5 (P < .01), and 11% became HCV RNA negative. For both biochemical responders and nonresponders, the decline in Knodell score was confined to the components of hepatic inflammation (piecemeal necrosis + lobular + portal inflammation); no change in fibrosis was observed. Hepatic inflammation declined by 5 points or more in 69% of biochemical responders and 48% of biochemical nonresponders, and by at least 50% from pretreatment values in 74% and 38% of biochemical responders and biochemical nonresponders, respectively. For all patients (both biochemical responders and nonresponders) who remained viremic at the conclusion of interferon therapy, the reduction in hepatic inflammation was a linear function of the decline in HCV RNA titer. We conclude that more than one third of patients who had no biochemical response after 6 months of interferon therapy achieved a similar improvement in hepatic histology as was observed in patients with biochemical response. This improvement in hepatic histology appeared to correlate with a reduction in HCV RNA titer, especially in patients who remained viremic.

Adult↗
PubMed1997

Optimal ligand binding by the recombinant human glucocorticoid receptor and assembly of the receptor complex with heat shock protein 90 correlate with high intracellular ATP levels in Spodoptera frugiperda cells.

The full-length human glucocorticoid receptor (hGR), overexpressed in Spodoptera frugiperda (Sf9) cells, associates with heat shock protein 90 (hsp90) and hsp70 and binds dexamethasone with high affinity. Baculovirus infection of Sf9 cells grown in TNM-FH medium results in the rapid depletion of glucose from the medium within 24 h. Noting a discrepancy between hGR protein levels and ligand binding capacity in such cultures, we hypothesized that the depletion of glucose from the medium could result in intracellular ATP depletion and consequently affect the ligand binding capacity of the recombinant hGR. Supplementation of the Sf9 culture medium with additional glucose resulted in a three-fold increase in intracellular ATP levels, and a three-fold increase in 3H-dexamethasone binding capacity, without altering the protein levels of hGR, hsp90 or hsp70. However, more hsp90 co-immunoprecipitated with hGR from cells grown in glucose supplemented medium. Our data support the hypothesis that high-affinity ligand binding by hGR requires the ATP-dependent formation of the hGR:hsp90 heterocomplex. Besides having practical consequences for the production of recombinant GR and other related proteins, our findings could ultimately have relevance in diseases such as diabetes mellitus.

Adenosine Triphosphate↗
PubMed1997

Glucocorticoid/oxysterol-induced DNA lysis in human leukemic cells.

Both glucocorticoids and oxysterols, steroids with quite different known transduction pathways, cause the death of lymphoid cells. Dual TUNEL/propidium iodide assays on sensitive human leukemic CEM-C7 clones treated with either steroid were clearly positive by 48 h, consistent with apoptosis. Both steroids evoked two distinctive types of DNA lysis: cleavage into large fragments of several different sizes and the classic "ladders", multiples of approximately 200 base pairs. Conventional gel electrophoresis showed that a small proportion of total DNA had undergone laddering 36-48 h after treatment with glucocorticoid or 24 h after oxysterol exposure. On field inversion gel electrophoresis of cellular DNA both steroids caused an increase in an array of large DNA fragments <50 kb in size. A 50 kb fragment appeared 36 h after treatment with either steroid, but only oxysterol treatment caused a significant increase in a 300 kb fragment. Oxysterol treatment did not result in DNA fragmentation in the resistant M10R5 subclone, which retained sensitivity to glucocorticoids. We conclude that glucocorticoids and oxysterols kill these cells with similar, but not identical, patterns of DNA lysis which occur just before or concomitant with the onset of cell death.

Cell Division↗
PubMed1997

Elevated expression of Bcl-2 and Bcl-x by intestinal intraepithelial lymphocytes: resistance to apoptosis by glucocorticoids and irradiation.

Administration of glucocorticoids or exposure to ionizing radiation in vivo results in a rapid cell death of thymocytes. We report that murine small intestinal intraepithelial lymphocytes (IEL) are resistant to both steroid- and radiation-induced deletion. This is due to resistance to apoptosis, as evidenced by the absence of detectable apoptotic IEL nuclei in situ after in vivo glucocorticoid treatment. IEL express normal levels of glucocorticoid receptors and these receptors bind [3H]dexamethasone to equivalent levels as other lymphocyte populations. Thus, their survival is due to post-receptor signaling mechanisms. Many IEL express high levels of Bcl-2 and that of these Bcl-2high IEL are largely TCR gamma delta +. Those IEL that do express high levels of Bcl-2 are CD8 alpha + beta - CD4-. In addition, IEL express Bcl-x, another protein shown to be involved in the protection of cells from apoptotic signals. IEL represent the first lymphocyte population in vivo shown to have high levels of expression of both molecules, that otherwise occur only in activated lymphocytes in vitro. These data suggest that the Bcl-2+Bcl-x+ IEL are activated cells and not an effete population of cells necessarily destined to die. Also, the high levels of Bcl-2 and Bcl-x in this in vivo activated population supports the in vitro correlate of protection from activation-induced cell death.

Animals↗
PubMed1996

Lymphoid cell resistance to glucocorticoids in HIV infection.

In humans infected with the HIV-1 virus there may be a disproportionate severity of signs and symptoms of illness compared to the fraction of CD4+ infected T-lymphoid cells. In part, this may be due to altered intercellular signalling systems and intracellular signal transduction. Glucocorticoids are well known for their effects on the vitality and function of lymphoid cells. Patients with HIV infections often show elevated circulating levels of cortisol, suggesting some misfunction in the regulatory systems that maintain the levels of this critical hormone. At the cellular level, it is known that both acute HIV infection and glucocorticoids can cause apoptotic cell death in thymic lymphocytes. However, chronically HIV-infected cells appear to be resistant to glucocorticoid-evoked cell death. Glucocorticoid receptor-ligand binding studies on patients' cells have shown reduced affinity between the receptor binding sites and test steroids. In vitro, chronically HIV-infected cells of the lymphoid CEM line displayed resistance to glucocorticoid-induced apoptosis. These cells showed reduced numbers of binding sites with little alteration in apparent affinity between ligand and receptor. Thus it appears that there may often be malfunction of the normal glucocorticoid response in HIV-infected cells probably due to altered interactions between the glucocorticoid receptor and its hormone. Such alterations may have clinical consequences, including the possibility of a relatively longer life span of infected CD4+ T-lymphocytes, as well as systemic effects of chronically elevated cortisol level.

CD4-Positive T-Lymphocytes↗
PubMed1996

Use of hepatic lidocaine metabolism to monitor patients with chronic liver disease.

Lidocaine is converted to its primary metabolic product monoethylglycinexylodide (MEGX) via cytochrome P-4503A4 within the liver. A steady-state concentration of MEGX appears in serum within 15 min following the intravenous administration of lidocaine. The present article reviews some of the data suggesting that this MEGX value can be utilized to assess hepatic function. MEGX production declines stepwise with the severity of chronic hepatitis. In patients with cirrhosis, MEGX declines further with worsening Child class. Nearly all persons with MEGX of < 20 ng/ml had cirrhosis confirmed upon histologic evaluation. Severe life-threatening complications of cirrhosis were observed only in patients with MEGX production below 20 ng/ml. One-year survival for patients with an MEGX value of < 10 ng/ml was only 50%. In contrast, 1-year survival for patients with MEGX of > 10 ng/ml was approximately 80%. These data suggest that MEGX could be utilized as an accurate test of hepatic function and to predict morbidity and mortality related to complications of chronic liver disease. However, this test does have several limitations. There is wide interpatient variability between MEGX and hepatic histology, which severely impairs the ability of this test to accurately predict hepatic histology. In addition, MEGX is affected by gender and several medications. However, since MEGX does decline stepwise with advancing histology in any given patient, the available data suggest that serial monitoring of MEGX could be utilized to track hepatic metabolic capacity in patients with chronic hepatitis and cirrhosis.

Anesthetics, Local↗
PubMed1996

Role of c-jun induction in the glucocorticoid-evoked apoptotic pathway in human leukemic lymphoblasts.

Accumulated evidence suggests tI AP-1 family in CEM cell clones exposed to the glucocorticoid dexamethasone (Dex) has been investigated. Dex is known to cause apoptosis of lymphoid cells in general and of sensitive human lymphoid CEM cell clones in particular. This study finds that Dex induces c-jun mRNA and cJun protein in cells of the sensitive clone CEM-C7 and of the lysis-sensitive OEM hybrid clone H10. CEM-O7 cells were screened for several other Jun/Fos family proteins. Both cFos and JunD were expressed but were unaffected by the steroid, and JunB was not detected. In the continual presence of Dex the induction of cJun began about 6 h after addition of Dex, reached a maximum by 24 h, and plateaued for 72 h, while cell death did not begin until 24-48 h. In clone OEM-Cl cells, which contain glucocorticoid receptor (GR) but are resistant to lysis by Dex, the basal, and even the fully induced, cJun levels are below the basal levels in OEM-07 and H10 cells. To test the hypothesis that cJun plays an important role in steroid-evoked apoptosis, stable transfectants expressing Dex-regulable antisense c-jun RNA were established. Mass cultures of these cells showed reduced sensitivity to Dex, and in three of three clones tested, complete resistance to Dex was obtained. This occurred even though endogenous genes (GR, c-jun) normally responsive to Dex were still inducible, indicating that the GR and basic glucocorticoid response apparatus were intact. It is concluded that Dex induces cJun levels in sensitive OEM cells before cell death and that this induction plays a role in the apoptotic process.

Apoptosis↗
PubMed1996

Transfected glucocorticoid receptor and certain GR fragments evoke cell death in malignant lymphoid, not myeloid cell lines.

Previously we have shown that glucocorticoid sensitivity could be restored to a clone of glucocorticoid-resistant leukemic T cells by transfecting them with an expression vector for the glucocorticoid receptor. Furthermore, transfection with plasmids expressing fragments of the receptor containing the DNA-binding domain resulted in constitutive loss of cells. In this paper, we report the results of transfecting both types of constructs into lines of glucocorticoid-resistant human leukemic cells of T cell, B cell, and myeloid origin. In all the lymphoid lines tested, transfection of the holoreceptor gene resulted in appearance of steroid-dependent cell death. In the same lines, transfection of glucocorticoid receptor fragments expressing amino acids 1-465* (465 residues of the normal sequence plus a novel 21 amino acid C-terminus) or expressing only 398-465* caused cell death without the addition of steroids. The amount of cell loss following transfection of these constitutively lethal fragments was in the same range as that following transfection of the holo glucocorticoid receptor plus administration of glucocorticoid. However, the cell loss due to the constitutively active fragments occurred more rapidly. Neither of the myeloid lines tested were sensitive to any of the transfected constructs, with or without added steroid.

Apoptosis↗
PubMed1995

Steroid hormones. Membrane transporters of steroid hormones.

The discovery of a plasma membrane ABC protein that exports steroids in yeast highlights the possibility that similar membrane sorting systems in mammalian cells may modulate the access of steroids to their receptors.

ATP-Binding Cassette Transporters↗
PubMed1995

Glucocorticoid antagonist RU 486 reverses agonist-induced apoptosis and c-myc repression in human leukemic CEM-C7 cells.

A synthetic glucocorticoid dexamethasone (DEX) inhibits proliferation and induces apoptosis of clone CEM-C7 cells, but not in clone CEM-C1 cells, even though they contain glucocorticoid receptors (GR). We previously showed that suppression of c-myc is a critical step in glucocorticoid-induced cell lysis of C7 cells. It is not reduced in C1 cells. In this study we review the basis for this conclusion and present evidence that the glucocorticoid antagonist RU 486 rescues DEX-treated C7 cells from cell death. An increase in DEX-repressed c-myc mRNA levels precedes the recovery of cell growth. A threshold level of Myc expression appears to be required to maintain growth and viability of C7 cells. Although C1 cells are highly resistant to lysis by glucocorticoids, addition of forskolin, an inducer of protein kinase A, synergizes to evoke complete apoptosis. This synergistic effect is prevented by RU 486, indicating direct involvement of the GR.

Apoptosis↗