Sequential gene regulatory events leading to glucocorticoid-evoked apoptosis of CEM human leukemic cells:interactions of MAPK, MYC and glucocorticoid pathways.

Gene expression responses to glucocorticoid (GC) in the hours preceding onset of apoptosis were compared in three clones of human acute lymphoblastic leukemia CEM cells. Between 2 and 20h, all three clones showed increasing numbers of responding genes. Each clone had many unique responses, but the two responsive clones showed a group of responding genes in common, different from the resistant clone. MYC levels and the balance of activities between the three major groups of MAPKs are known important regulators of glucocorticoid-driven apoptosis in several lymphoid cell systems. Common to the two sensitive clones were changed transcript levels from genes that decrease amounts or activity of anti-apoptotic ERK/MAPK1 and JNK2/MAPK9, or of genes that increase activity of pro-apoptotic p38/MAPK14. Down-regulation of MYC and several MYC-regulated genes relevant to MAPKs also occurred in both sensitive clones. Transcriptomine comparisons revealed probable NOTCH-GC crosstalk in these cells.

Ischaemic Priapism and Glucose-6-Phosphate Dehydrogenase Deficiency: A Mechanism of Increased Oxidative Stress?

Ischaemic priapism is a devastating urological condition that has the potential to cause permanent erectile dysfunction. The disorder has been associated with numerous medical conditions and the use of pharmacotherapeutic agents. The aetiology is idiopathic in a number of cases. There are two prior case reports of the association of ischaemic priapism and glucose-6-phosphate dehydrogenase (G6PD) deficiency. We report on a third case of priapism associated with G6PD deficiency and review recently described molecular mechanisms of increased oxidative stress in the pathophysiology of ischaemic priapism. The case report of a 32-year old Afro-Caribbean male with his first episode of major ischaemic priapism is described. Screening for common causes of ischaemic priapism, including sickle cell disease was negative. Glucose-6-phosphate dehydrogenase deficiency was discovered on evaluation for priapism. Penile aspiration was performed and erectile function was good post treatment.Glucose-6-phosphate dehydrogenase deficiency is a cause for ischaemic priapism and should be a part of the screening process in idiopathic causes of the disorder. Increased oxidative stress occurs in G6PD deficiency and may lead to priapism.

Inequalities in outcomes of acute kidney injury in England.

BACKGROUND: Renal replacement is managed by renal specialists and is well documented in national registries. In contrast, nation-wide data on acute kidney injury (AKI) are difficult to capture as it presents in many different ways to all acute hospitals. This paucity impacts on the coordination of appropriate services. AIMS: We have set out to use all the information submitted by all hospitals in England to identify emergency patients in whom AKI was a major contributor to their hospital stay. We then examined workload in relation to specialist provision and outcomes of care. DESIGN AND METHODS: All English hospitals submit a sequential list of International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD 10) codes to describe the diagnosis of each admission. An algorithm was applied to all emergency admissions over a 2-year period to identify AKI. The level of renal specialist care available within each hospital trust was compared with patient outcomes, including 30-day mortality. RESULTS: The incidence of AKI was 1.34% of all emergency admissions. The numbers and types of AKI cases were similar in all trusts, regardless of the service available. Thirty-day mortality was 30.0%. More than half the acute hospitals did not have on-site renal specialists and their AKI mortality rates were significantly higher (P < 0.001). These differences persisted despite adjusting for multiple variables. CONCLUSION: The country has created specialist renal units in 45% of hospital trusts, but AKI presents as emergencies to all hospitals and there is an increased risk of mortality in the 55% of trusts without renal specialists.

Folding of the glucocorticoid receptor N-terminal transactivation function: dynamics and regulation.

The glucocorticoid receptor (GR) mediates biological effects of glucocorticoids at the level of gene regulation, and plays important roles in many aspects of physiology. In recent years, it has become quite evident that GR behaves very dynamically, controlled by its reversible interactions with a variety of coregulatory proteins at various DNA and non-DNA sites. The N-terminal activation function domain (AF1) of the GR exists in an intrinsically disordered (ID) state, which promotes molecular recognition by providing surfaces capable of binding specific target molecules. Several studies suggest that when in action, the GR AF1 gains structure. Thus, it is hypothesized that the GR AF1 domain may be structured in vivo, at least when directly involved in transcriptional activation. Our recent work supports this conclusion. We propose that by allowing AF1 to rapidly and reversibly adopt various configurations through structural arrangements, AF1 can create protein surfaces that are readily available for selective binding to coregulatory proteins, resulting in GR-mediated transcriptional regulation of target genes.

Influence of flanking sequences on signaling between the activation function AF1 and DNA-binding domain of the glucocorticoid receptor.

Despite their importance in gene regulation, the exact mechanisms of glucocorticoid receptor's (GR's) N-terminal activation function region, AF1, which exists in an intrinsically disordered (ID) conformation remains largely unknown. For its interaction with critical coregulatory proteins, AF1 must be malleable and capable of presenting varied interaction surfaces. We hypothesize that various confluences of effects, including intra-molecular signaling between the AF1 and the GR DNA-binding domain (DBD) cause functional structure to form in AF1. In this study, we tested the effect of the amino acid sequences surrounding AF1 on the propensity of AF1 to gain structure when connected to DBD. Removal of amino acids between AF1 and DBD results in the formation of more ordered conformation in AF1. In addition, sequences flanking the AF1 may play an inhibitory role in AF1 activity. These results suggest a mechanism as to why certain GR isoforms with truncated N-terminal domains show altered transcriptional activity.

Effects of different osmolytes on the induced folding of the N-terminal activation domain (AF1) of the glucocorticoid receptor.

In order to understand gene regulation by glucocorticoids, it is pivotal to know how the major transactivation domain AF1 of the glucocorticoid receptor (GR) functions. Located in the N-terminal region of the GR, AF1 is quantitatively important for transcriptional regulation, but only in recent years have we begun to understand how AF1 works. This is in part due to the fact that the recombinant AF1 (rAF1) peptide exists as a random ensemble of conformers. Algorithms that predict structure support the view that AF1 is also not well ordered in the holo-GR, and the properties of the amino acids in AF1 suggest that it is intrinsically disordered. However, it is generally believed that intrinsically disordered sequences of the GR AF1 must achieve one or more ordered conformation(s) to carry out transactivation activity. Based on our previous published work and available literature, we hypothesize that a confluence of effects that operate under physiological conditions cause functionally active conformation(s) to form in AF1. We have shown that when rAF1 is incubated in increasing concentrations of a naturally occurring osmolyte trimethylamine-N-oxide (TMAO), the peptide folds into functionally active conformation(s) that selectively binds several critical coregulatory proteins. Because cells contain various organic osmolytes whose effects may be cumulative, and in light of cell-specific effects of GR AF1 action, we tested whether it can be folded by other natural organic osmolytes representative of three classes: certain amino acids (proline), methylamines (sarcosine), and polyols (sorbitol). The osmolyte-induced folding of rAF1 shows greatly increased affinity for specific binding proteins, including TATA box-binding protein (TBP), CREB-binding protein (CBP), and steroid receptor coactivator-1 (SRC-1). Consistent with theory and published data with other proteins, our results show that different osmolytes have differential effects on rAF1 folding. The cell-specific functions of the GR AF1--and by extension the AF1s of other nuclear hormone receptors--may in part be affected by the presence and concentrations of particular osmolytes within a particular cellular environment.

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.

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.

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.

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.

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.

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.

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.

Glucocorticoid mediated transcriptional repression of c-myc in apoptotic human leukemic CEM cells.

Suppression of c-myc has been implicated as a critical event in some glucocorticoid-evoked apoptotic systems. It is therefore of interest to understand the mechanism of glucocorticoid-regulation of the c-myc gene. In the present study, a detailed analysis of dexamethasone (Dex)-evoked regulation of the human c-myc gene in human leukemic CEM-C7 cells has been performed. Dex suppresses c-myc mRNA and immunoreactive protein expression in clone CEM-C7 and subclone CEM-C7-14 cells. Nuclear run-on assays suggested that the regulation occurred at the level of transcription initiation. The half-life of c-myc mRNA was approximately 30 min and its stability was not affected by Dex treatment. In addition, Dex suppressed luciferase gene expression driven by -2052 to +34 bp c-myc promoter in transfected CEM-C7-14 cells. This result further supports that c-myc gene is suppressed by Dex at the transcriptional level in apoptotic human leukemic cells.

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.

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.

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.

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.

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.

Glucocorticoids, oxysterols, and cAMP with glucocorticoids each cause apoptosis of CEM cells and suppress c-myc.

In clones of the CEM human acute lymphoblastic leukemic cell line, glucocorticoids, oxysterols and activators of the cAMP pathway acting synergistically with glucocorticoids, each can cause apoptotic cell death. Morphologically and kinetically, these deaths resemble one another. The kinetics are striking: in each case, after addition of the lethal compound(s), an interval of approximately 24 h follows, during which cell growth continues unabated. During this "prodromal" period, removal of the apoptotic agent leaves the cells fully viable. We hypothesize that a sequence of biochemical events occurs during the prodrome which eventually results in the triggering of the full apoptotic response as evidenced by the activation of caspases and DNA fragmentation. At some point, the process is irreversible and proceeds relatively rapidly to cell death. Suppression of c-Myc seems a universal early event evoked by each of these lethal compounds or combinations, and we conclude that the negative regulation of this proto-oncogene is an important aspect of the critical pre-apoptotic events in these cells.