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E B Thompson

Publications and source records attributed to E B Thompson.

At least 19 recordsLinked to original sources
PubMed2004

Identification of genes leading to glucocorticoid-induced leukemic cell death.

Glucocorticoidal steroids (GC) are capable of causing apoptotic death of many varieties of lymphoid cells; consequently, GC are used in therapy for many lymphoid malignancies. Gene transcription in the GC-treated cells is required for subsequent apoptosis, but only a few of the actual genes involved have been identified. We employed gene microarray analysis to find the network of genes involved in GC-evoked cell death, using three clones derived from the CEM lymphoid leukemia cell line. Clone C1-15 was resistant to GC-evoked apoptosis, although not necessarily to GC-induced gene transcription; the other two underwent apoptosis in the presence of GC. Clone C7-14 was subcloned from the apoptosis-sensitive parental C7 clone to establish karyotypic uniformity. The second sensitive clone, C1-6, was a spontaneous revertant from parental resistant clone C1. A period of > or = 24 h in the constant presence of receptor-occupying concentrations of synthetic GC dexamethasone (Dex) was necessary for apoptosis to begin. To identify the steps leading to this dramatic event, we identified the changes in gene expression in the 20-h period preceding the onset of overt apoptosis. Cells in the log phase of growth were treated with 10(-6) M Dex, and 2-20 h later, mRNA was prepared and analyzed using the Affymetrix HG_U95Av2 chip, containing probes for about 12,600 genes. Of these, approximately 6,000 were expressed above background. Comparisons of the basal and expressed genes in the three clones led to several conclusions: The Dex-sensitive clones shared the regulation of a limited set of genes. The apoptosis-resistant clone C1-15 showed Dex effects on a largely different set of genes. Promoter analysis of the regulated genes suggested that primary gene targets for GC often lack a classic GC response element.

Animals↗
PubMed2002

Cellular lipid peroxidation end-products induce apoptosis in human lens epithelial cells.

Hydrogen peroxide (H(2)O(2)), an oxidant present in high concentrations in the aqueous humor of the elderly eyes, is known to impart toxicity to the lens---apoptosis being one of the toxic events. Since H(2)O(2) causes lipid peroxidation leading to the formation of reactive end-products, it is important to investigate whether the end-products of lipid peroxidation are involved in the oxidation-induced apoptosis in the lens. 4-Hydroxynonenal (HNE), a major cytotoxic end product of lipid peroxidation, has been shown to mediate oxidative stress-induced cell death in many cell types. It has been shown that HNE is cataractogenic in micromolar concentrations in vitro, however, the underlying mechanism is not yet clearly understood. In the present study we have demonstrated that H(2)O(2) and the lipid derived aldehydes, HNE and 4-hydroxyhexenal (HHE), can induce dose- and time-dependent loss of cell viability and a simultaneous increase in apoptosis involving activation of caspases such as caspase-1, -2, -3, and -8 in the cultured human lens epithelial cells. Interestingly, we observed that Z-VAD, a broad range inhibitor of caspases, conferred protection against H(2)O(2)- and HNE-induced apoptosis, suggesting the involvement of caspases in this apoptotic system. Using the cationic dye JC-1, early apoptotic changes were assessed following 5 h of HNE and H(2)O(2) insult. Though HNE exposure resulted in approximately 50% cells to undergo early apoptotic changes, no such changes were observed in H(2)O(2) treated cells during this period. Furthermore, apoptosis, as determined by quantifying the DNA fragmentation, was apparent at a much earlier time period by HNE as opposed to H(2)O(2). Taken together, the results demonstrate the apoptotic potential of the lipid peroxidation end-products and suggest that H(2)O(2)-induced apoptosis may be mediated by these end-products in the lens epithelium.

Aldehydes↗
PubMed2001

The pathway of leukemic cell death caused by glucocorticoid receptor fragment 465*.

The truncated glucocorticoid receptor mutant gene 465* codes for a protein that is interrupted by a frame-shift mutation in the second zinc finger of the natural DNA binding domain. Thus, 465* represents the natural amino acid sequence 1-465 followed by 21 novel amino acids starting at position 466. The entire ligand binding domain is missing. Prior studies have shown that transient transfection of the glucocorticoid-resistant leukemic T-cell clone ICR-27 with a plasmid expressing 465* rapidly reduces the number of viable cells. This response does not require activation by a steroid, and a hybrid protein consisting of green fluorescent protein fused to 465* is found primarily in the cytoplasm. In the present study, we present evidence that the decrease in cell number is due to a form of cell death that bears many of the classic characteristics of apoptosis. Expression of the 465* protein can be detected a few hours after electroporation and is followed by activation of caspase-3 as well as reduction of the mitochondrial inner transmembrane potential. The caspase-3 inhibitor ZVAD-fmk blocks 465*-dependent cell death when added acutely after electroporation, but fails to do so later. We conclude that the novel 465* gene causes cell death by apoptosis.

Apoptosis↗
PubMed2001

Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells.

Sensitivity to glucocorticoid (GC)-evoked apoptosis in lymphoid cell lines correlates closely with GC-mediated suppression of c-Myc expression. To establish a functional role for c-Myc in GC-mediated apoptosis, we have stably expressed MycER(TM), the human c-Myc protein fused to the modified ligand-binding domain of the murine estrogen receptor alpha, in GC-sensitive CEM-C7-14 cells. In CEM-C7-14 cells, MycER(TM) constitutively imparts c-Myc functions. Cells expressing MycER(TM) (C7-MycER(TM)) exhibited a marked reduction in cell death after 72 h in 100 nM dexamethasone (Dex), with 10-20-fold more viable cells when compared to the parental CEM-C7-14 clone. General GC responsiveness was not compromised, as evidenced by Dex-mediated suppression of endogenous c-Myc and cyclin D3, and induction of c-Jun and the glucocorticoid receptor. MycER(TM) also blunted Dex-mediated upregulation of p27(kipI) and suppression of the Myc target p53. In comparison to parental CEM-C7-14 cells, Dex-evoked DNA strand breaks were negligible and caspase activation was delayed, but the extent of G1 cell cycle arrest was similar in C7-MycER(TM) cells. Myc-ER(TM) did not result in permanent, complete resistance to GC however, and the GC-treated cells eventually died, indicative of redundant or interactive mechanisms in the GC-evoked lytic response of lymphoid cells. Our results emphasize the importance of c-Myc suppression in GC-evoked apoptosis of CEM-C7-14 cells.

Antineoplastic Agents, Hormonal↗
PubMed2001

Involvement of caspases in 4-hydroxy-alkenal-induced apoptosis in human leukemic cells.

4-Hydroxynonenal (HNE), a reactive and cytotoxic end-product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death and in various cell types has been shown to induce apoptosis. We have demonstrated that HNE, at micromolar concentrations, induces dose- and time-dependent apoptosis in a leukemic cell line (CEM-C7). Interestingly, much higher concentrations of HNE (> 15-fold) were required to induce apoptosis in leukocytes obtained from normal individuals. We also demonstrate that HNE causes a decrease in clonogenicity of CEM-C7 cells. Furthermore, our data characterize the caspase cascade involved in HNE-induced apoptosis in CEM-C7 cells. Using specific fluorogenic substrates and irreversible peptide inhibitors, we demonstrate that caspase 2, caspase 3, and caspase 8 are involved in HNE-induced apoptosis, and that caspase 2 is the first initiator caspase that activates the executioner caspase 3, either directly or via activation of caspase 8. Our studies also suggest the involvement of another executioner caspase, which appears to be similar to caspase 8 but not caspases 2 and 3, in its specificity. The demonstration of decreased clonogenicity by HNE in the leukemic cells, and their higher susceptibility to HNE-induced apoptosis as compared to the normal cells, suggests that such compounds may have potential for leukemia chemotherapy.

Aldehydes↗
PubMed2001

The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins.

The activation domains of many transcription factors appear to exist naturally in an unfolded or only partially folded state. This seems to be the case for AF1/tau1, the major transactivation domain of the human glucocorticoid receptor. We show here that in buffers containing the natural osmolyte trimethylamine N-oxide (TMAO), recombinant AF1 folds into more a compact structure, as evidenced by altered fluorescence emission, circular dichroism spectra, and ultracentrifugal analysis. This conformational transition is cooperative, a characteristic of proteins folding to natural structures. The structure resulting from incubation in TMAO causes the peptide to resist proteolysis by trypsin, chymotrypsin, endoproteinase Arg-C and endoproteinase Gluc-C. Ultracentrifugation studies indicate that AF1/tau1 exists as a monomer in aqueous solution and that the presence of TMAO does not lead to oligomerization or aggregation. It has been suggested that recombinant AF1 binds both the ubiquitous coactivator CBP and the TATA box-binding protein, TBP. Interactions with both of these are greatly enhanced in the presence of TMAO. Co-immunoadsorption experiments indicate that in TMAO each of these and the coactivator SRC-1 are found complexed with AF1. These data indicate that TMAO induces a conformation in AF1/tau1 that is important for its interaction with certain co-regulatory proteins.

Humans↗
PubMed2001

Improved response with higher corticosteroid dose in children with acute lymphoblastic leukemia.

PURPOSE: We investigated whether there was a dose-response relationship for the use of corticosteroids in childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Three hundred sixty-nine patients, ages 1 to 18 years with ALL, were randomly assigned to receive one of four different doses of corticosteroid (prednisolone 40 mg/m(2)/d or dexamethasone 6, 18, or 150 mg/m(2)/d) administered as a 3-day, single-drug window before initiation of standard, multidrug induction chemotherapy. Corticosteroid drug response was measured by reduction in bone marrow blast counts and absolute peripheral blast counts after 3 days. Glucocorticoid receptor (GCR) number and the effective concentration of dexamethasone resulting in a 50% reduction of leukemic cell viability in vitro (EC-50) were evaluated at days 0 and 3. RESULTS: Increasing dexamethasone doses resulted in greater marrow blast response (P =.007), with a similar trend in peripheral-blood blast response. High-dose corticosteroid regimens (dexamethasone 18 or 150 mg/m(2)/d) elicited better responses than standard doses of dexamethasone or prednisone (bone marrow, P =.002; peripheral blasts, P =.05). Among patients treated with standard-dose corticosteroids, 38% with resistant (EC-50 > 10(-7)) peripheral blasts had a good response compared with 92% with sensitive (EC-50 < 10(-7)) peripheral blasts (P =.01). In contrast, there was no differential response according to EC-50 group after high-dose corticosteroids. Similarly, an association between response and GCR on peripheral-blood blasts was noted after standard-dose corticosteroid regimens but not after high-dose corticosteroid regimens. CONCLUSION: Response of ALL to glucocorticoid therapy increased with dose. Higher-dose corticosteroid treatment abrogated the effect of relative drug insensitivity and of low GCR on peripheral blasts.

Blood Cell Count↗
PubMed2000

The delayed induction of c-jun in apoptotic human leukemic lymphoblasts is primarily transcriptional.

Because of their ability to induce lymphoid cell apoptosis, glucocorticoids have been used for decades to treat certain human leukemias and lymphomas. Studies presented in this paper complement our previous work demonstrating that sustained induction of the proto-oncogene c-jun plays a crucial role in the glucocorticoid-induced apoptotic pathway in CEM cells, human leukemic lymphoblasts. Results from measurements of c-jun mRNA half-life with RNase protection assays and of transcription by nuclear run-on assays indicate that, in the dexamethasone-sensitive cloned CEM-C7 cells, c-jun is induced at the transcriptional level. Consideration of time-course, however, suggested that this might be a secondary or possibly a delayed primary response. Use of cycloheximide to block protein synthesis strongly induced c-jun mRNA, suggesting that there had been relief from a labile protein repressor of transcription. Comparing the level of induction by cycloheximide with that of dexamethasone indicated that the two did not induce by an identical mechanism. The high induction by cycloheximide obscured simple interpretation of elevated c-jun mRNA levels after concomitant administration of cycloheximide and dexamethasone. This was resolved by nuclear run-on experiments, which showed that the dexamethasone induction of c-jun mRNA in this system does require protein synthesis.

Apoptosis↗
PubMed2000

Hormonal regulation of physiological cell turnover and apoptosis.

Physiological cell turnover plays an important role in maintaining normal tissue function and architecture. This is achieved by the dynamic balance of cellular regeneration and elimination, occurring periodically in tissues such as the uterus and mammary gland, or at constant rates in tissues such as the gastrointestinal tract and adipose tissue. Apoptosis has been identified as the prevalent mode of physiological cell loss in most tissues. Cell turnover is precisely regulated by the interplay of various endocrine and paracrine factors, which modulate tissue and cell-specific responses on proliferation and apoptosis, either directly, or by altering expression and function of key cell proliferative and/or death genes. Although recent studies have provided significant information on specific tissue systems, a clearly defined pathway that mediates cell turnover has not yet emerged for any tissue. Several similarities exist among the various tissues with regard to the intermediates that regulate tissue homeostatis, enabling a better understanding of the general mechanisms involved in the process. Here we review the mechanisms by which hormonal and cytokine factors mediate cell turnover in various tissues, emphasizing common themes and tissue-specific differences.

Adipose Tissue↗
PubMed2000

Structure-apoptotic potency evaluations of novel sterols using human leukemic cells.

Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM-C7-14 human leukemic cells. In addition to testing 15-ketocholestenol (K15), 15-ketocholestenol hydroxyethyl ether (CK15), and 7-ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25-hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC > K15 > CK15 > CK7. Both the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling), which quantifies the amount of DNA nicks caused by a toxic agent, and the MTT assay, which measures cell metabolism and thus reflects cell viability, substantiated the same rank order. An ELISA assay for evaluating release of DNA fragments into the cytosol after treatment gave a similar potency order. The oncogene c-myc mRNA was suppressed by all three oxysterols, with 25OHC and K15 being the most potent suppressors. Hoechst and Annexin V staining documented that these oxysterols kill cells by an apoptotic pathway as evidenced by condensation of nuclear chromatin and plasma membrane inversion, respectively. From these in vitro studies, we believe that 25OHC, K15, and possibly CK15 have the potential to be chemotherapeutic agents.

Apoptosis↗
PubMed2000

Normocortisolemic Cushing's syndrome initially presenting with increased glucocorticoid receptor numbers.

A girl who developed Cushingoid features in peripuberty, but was eucortisolemic, was previously reported to have markedly elevated lymphocyte glucocorticoid receptor sites per cell with normal binding affinity as a potential cause of her phenotype. Her circadian rhythm of cortisol and pituitary-adrenal axis were initially intact, but later proved to be dysregulated. The patient presented at age 10.8 yr with centripetal obesity, moon facies, buffalo hump, and purple striae, but no statural stunting, which is a cardinal sign of Cushing's syndrome. At 11.5 yr she suffered a compression fracture of the L1 vertebra. That prompted treatment with the antiprogestin drug mifepristone (RU486), which was administered at high dose to achieve an antiglucocorticoid effect. From ages 13.75 yr through 15.5 yr, RU486 was administered in various intervals to suppress her Cushingoid features. Once RU486 was introduced, however, a consistent correlation over time between the Cushingoid features and glucocorticoid receptor sites per cell was no longer observed. However, the number of glucocorticoid receptor sites per cell tended to decrease in response to administering RU486. Ultimately, her Cushingoid phenotype proved to be transient.

Adrenocorticotropic Hormone↗
PubMed1999

Interdomain signaling in a two-domain fragment of the human glucocorticoid receptor.

Studies of individual domains or subdomains of the proteins making up the nuclear receptor family have stressed their modular nature. Nevertheless, these receptors function as complete proteins. Studies of specific mutations suggest that in the holoreceptors, intramolecular domain-domain interactions are important for complete function, but there is little knowledge concerning these interactions. The important transcriptional transactivation function in the N-terminal part of the glucocorticoid receptor (GR) appears to have little inherent structure. To study its interactions with the DNA binding domain (DBD) of the GR, we have expressed the complete sequence from the N-terminal through the DBD of the human GR. Circular dichroism analyses of this highly purified, multidomain protein show that it has a considerable helical content. We hypothesized that binding of its DBD to the cognate glucocorticoid response element would confer additional structure upon the N-terminal domain. Circular dichroism and fluorescence emission studies suggest that additional helicity as well as tertiary structure occur in the two-domain protein upon DNA binding. In sum, our data suggest that interdomain interactions consequent to DNA binding imparts structure to the portion of the GR that contains a major transactivation domain.

Circular Dichroism↗
PubMed1999

Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor.

A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive alpha-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1-500 and residues 77-262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the alpha-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1-500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures.

Circular Dichroism↗
PubMed1999

Activation of human O6-methylguanine-DNA methyltransferase gene by glucocorticoid hormone.

O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, removes the mutagenic DNA adduct O6-alkylguanine, which is synthesized both endogenously and after exposure to alkylnitrosamines and alkylating antitumor drugs such as 2-chloroethyl-N-nitrosourea (CNU). The MGMT gene is highly regulated in mammalian cells and its overexpression, observed in many types of tumor cells, is often associated with cellular resistance to CNU. Dexamethasone, a synthetic glucocorticoid hormone, was found to increase MGMT expression in HeLa S3 cells, concomitant with their increased resistance to CNU. Two putative glucocorticoid responsive elements (GREs) were identified in the human MGMT (hMGMT) promoter. Transient expression of the luciferase reporter gene driven by an hMGMT promoter fragment containing these GREs was activated by dexamethasone. DNase I footprinting assays demonstrated the binding of glucocorticoid receptor to these sequences. In vitro transcription experiment showed that these DNA sequences are functional in glucocorticoid receptor signal-mediated activation of transcription. These results suggest glucocorticoid-mediated induction of the MGMT gene contributes to high level expression of MGMT.

DNA Footprinting↗
PubMed1999

Apoptosis induced by oxysterol in CEM cells is associated with negative regulation of c-myc.

Previously we have demonstrated that treatment of the human lymphoblastic leukemic CEM cells with 25-hydroxycholesterol (25OHC) induces apoptosis. In the present study, we show that both c-myc mRNA and c-Myc protein levels are reduced only in oxysterol-sensitive and not in oxysterol-resistant cells after treatment with concentrations of 25OHC that kill the sensitive CEM cells. The repression of c-Myc protein precedes c-myc mRNA reduction, and both events occur before the onset of cell death. Our data suggest that 25OHC-induced suppression of c-myc gene expression in CEM cells results from posttranscriptional regulation. These results demonstrate the regulation by an oxysterol of a gene/gene product important for cell growth and viability and an association between oxysterol-induced apoptosis of CEM cells and the negative regulation of c-myc.

Apoptosis↗
PubMed1999

The structure of the nuclear hormone receptors.

The functions of the group of proteins known as nuclear receptors will be understood fully only when their working three-dimensional structures are known. These ligand-activated transcription factors belong to the steroid-thyroid-retinoid receptor superfamily, which include the receptors for steroids, thyroid hormone, vitamins A- and D-derived hormones, and certain fatty acids. The majority of family members are homologous proteins for which no ligand has been identified (the orphan receptors). Molecular cloning and structure/function analyses have revealed that the members of the superfamily have a common functional domain structure. This includes a variable N-terminal domain, often important for transactivation of transcription; a well conserved DNA-binding domain, crucial for recognition of specific DNA sequences and protein:protein interactions; and at the C-terminal end, a ligand-binding domain, important for hormone binding, protein: protein interactions, and additional transactivation activity. Although the structure of some independently expressed single domains of a few of these receptors have been solved, no holoreceptor structure or structure of any two domains together is yet available. Thus, the three-dimensional structure of the DNA-binding domains of the glucocorticoid, estrogen, retinoic acid-beta, and retinoid X receptors, and of the ligand-binding domains of the thyroid, retinoic acid-gamma, retinoid X, estrogen, progesterone, and peroxisome proliferator activated-gamma receptors have been solved. The secondary structure of the glucocorticoid receptor N-terminal domain, in particular the taul transcription activation region, has also been studied. The structural studies available not only provide a beginning stereochemical knowledge of these receptors, but also a basis for understanding some of the topological details of the interaction of the receptor complexes with coactivators, corepressors, and other components of the transcriptional machinery. In this review, we summarize and discuss the current information on structures of the steroid-thyroid-retinoid receptors.

Amino Acid Sequence↗
PubMed1999

HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system.

Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

Amino Acid Sequence↗