FOXO3-mediated chemo-protection in high-stage neuroblastoma depends on wild-type TP53 and SESN3.

Forkhead box O class transcription factors are homeostasis regulators that control cell death, longevity and therapy-resistance. In neuroblastoma (NB), nuclear FOXO3 correlates with stage M disease and poor prognosis. To analyze whether FOXO3 contributes to drug-resistance in this childhood cancer, we investigated how different high-stage-derived NB cells respond to the activation of an ectopic FOXO3 allele. We found endogenous FOXO3 mostly localized to the nucleus-upon activation of an ectopic, 4OHT-activated FOXO3(A3)ER fusion protein two of the cell lines underwent apoptosis, whereas in the others FOXO3-activation even increased survival during drug-treatment. In the latter cell type, FOXO3 did not induce the BH3-only protein BCL2L11/BIM due to impaired binding of FOXO3 to the BIM-promoter, but still activated other FOXO3 targets. It was shown before that FOXO3 and TP53 physically interact with each other at two different regions-the TP53-N-terminus binds to the FOXO3-DNA binding domain (DBD) and the FOXO3-C-terminus interacts with the TP53-DBD. Interestingly, cell lines that undergo FOXO3-induced cell death carry homozygous point mutations in the TP53-DBD near the structural hotspot-mutation-site R175H, which abrogated FOXO3-TP53 interaction. In contrast, in FOXO3-death-resistant cells no point mutations in the TP53-DBD were found-in these cells FOXO3-TP53 complexes are formed and FOXO3-binding to the BIM-promoter, but not the induction of the detoxifying protein SESN3, were prevented, which in turn increased chemo-protection in this type of high-stage-derived NB cells. Our combined data suggest that FOXO3 steps in as a death inducer in case of TP53-mutation, whereas functional TP53 alters FOXO3-target-promoter-recognition, which prevents death induction by FOXO3 and instead increases chemo-protection and survival of NB cells. This novel mechanism may explain the low incidence of TP53 mutation in high-stage NB at diagnosis and suggests FOXO3 as a therapeutic target for this childhood malignancy.

C10ORF10/DEPP-mediated ROS accumulation is a critical modulator of FOXO3-induced autophagy.

BACKGROUND: Neuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system. In neuronal tumor cells DNA-damaging chemotherapeutic agents activate the transcription factor FOXO3 which regulates the formation of reactive oxygen species (ROS) and cell death as well as a longevity program associated with therapy resistance. We demonstrated before that C10ORF10/DEPP, a transcriptional target of FOXO3, localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification. In the present study, we investigated the impact of FOXO3 and DEPP on the regulation of autophagy. Autophagy serves to reduce oxidative damage as it triggers a self-degradative process for the removal of aggregated or misfolded proteins and damaged organelles. METHODS: The effect of FOXO3 and DEPP on autophagy induction was analyzed using live cell fluorescence microscopy and immunoblot analyses of SH-EP cells transfected with a plasmid for EYFP-LC3 and with siRNAs specific for LC3, respectively. ROS steady-state levels were measured with reduced MitoTrackerRed CM-H2XROS. Cellular apoptosis was analyzed by flow cytometry and the caspase 3/7 assay. RESULTS: We report for the first time that DEPP induces ROS accumulation and thereby mediates the formation of autophagosomes as inhibition of ROS formation by N-acetyl-cysteine completely blocks autophagy. We further demonstrate that H2O2-treatment triggers autophagy-induction by FOXO3-mediated DEPP expression. Importantly, knockdown of DEPP was sufficient to efficiently inhibit autophagy-induction under different stress conditions such as serum starvation and genotoxic stress, suggesting that DEPP expression is critical for the initiation of autophagy in neuroblastoma. FOXO3-triggered autophagy partially protects neuroblastoma cells from cell death. Consistent with this concept, we demonstrate that inhibition of autophagy by LC3-knockdown significantly increased etoposide- and doxorubicin-induced apoptosis. These results were also confirmed by the use of the autophagy-inhibitor chloroquine that significantly enhanced the chemotherapeutic effect of etoposide and doxorubicin in neuronal tumor cells. CONCLUSION: Targeting FOXO3/DEPP-triggered autophagy is a promising strategy to sensitize neuroblastoma cells to chemotherapy.

Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica.

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3 µg/mL (CI95: 25.9-28.9 µg/mL) corresponding to 9.6 µM (CI95: 9.1-10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR.The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4-6″) and single linked B-type units.This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

BIRC5/Survivin as a target for glycolysis inhibition in high-stage neuroblastoma.

Adverse forms of neuroblastoma (NB), a childhood malignancy that develops from immature neuronal progenitor cells frequently carry a gain of chromosome 17q, which leads to overexpression of the antiapoptotic protein BIRC5/Survivin. We have recently shown that high Survivin expression shuts down mitochondrial complex I activity and shifts NB cells from oxidative phosphorylation to aerobic glycolysis, which further increases resistance to cell death induction. This increased glucose consumption sensitized tumor cells to glycolysis inhibitors. Interestingly, in Survivin-overexpressing cells 2-deoxy-d-glucose (2DG) treatment induces re-fusion of mitochondrial networks after 4 h, which coincides with Survivin repression. 2DG selectively acts on Survivin-expressing NB cells and induces autophagic degradation of Survivin via activation of the E3-ubiquitin ligase Parkin, a downstream target of PINK1. Survivin degradation further releases bound Beclin-1, which enhances autophagy and cell death induction. Knockdown of Parkin, however, reduces the sensitivity of Survivin-expressing NB cells to glycolysis inhibition. The selective activity of 2DG treatment on Survivin-overexpressing tumor cells was also confirmed in a xenograft mouse model, which further supports our hypothesis that glycolysis inhibitors might be useful drugs in the treatment of NB.

BIRC5/Survivin enhances aerobic glycolysis and drug resistance by altered regulation of the mitochondrial fusion/fission machinery.

Gain of chromosome 17q correlates with high-stage disease, an adverse clinical outcome and leads to the overexpression of the antiapoptotic protein BIRC5/Survivin in neuroblastoma (NB). We have shown before that Survivin defines a threshold for the sensitivity of NB cells to DNA-damaging chemotherapeutic agents that require FOXO3 activation for apoptosis induction. To investigate the molecular basis of apoptosis inhibition we analyzed the function of Survivin at mitochondria and uncovered that Survivin induces mitochondrial fragmentation, reduces mitochondrial respiration and represses BCL2L11/Bim. Mitochondrial fission depends on Survivin-induced recruitment of the fission regulator DNM1L/Drp1 to mitochondria. In parallel, Survivin expression inhibits the respiratory complex-I, thereby preventing reactive oxygen species accumulation and, as a consequence, FOXO3-induced apoptosis. The loss of energy production via oxidative phosphorylation is compensated by increased glycolysis in Survivin-overexpressing NB tumor cells. Glycolysis inhibitors neutralize the antiapoptotic effect of Survivin and sensitize high-stage NB to DNA-damaging drugs. This suggests that glycolysis inhibitors target an 'archilles heel' of Survivin-overexpressing NB and may be highly useful as chemosensitizers in the treatment of high-stage NB.

FKHRL1-mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells.

Protein kinase-B (PKB) and its target, the forkhead transcription factor like 1 (FKHRL1)/FoxO3a, have been suggested as regulators of neurotrophin-mediated cell survival in neuronal cells. We analyzed human neuroblastoma cells and found that FKHRL1 was phosphorylated, suggesting its inactivation. To study FKHRL1 function, we infected SH-EP and NB15 cells with a 4OH-tamoxifen-regulated FKHRL1(A3)ER(tm) transgene. Activation of FKHRL1 promoted cytochrome-c release and caspase-dependent apoptosis. FKHRL1 induced TRAIL and the BH3-only proteins Noxa and Bim, implicating both extrinsic and intrinsic death pathways. However, expression of dnFADD did not inhibit FKHRL1-induced cell death, whereas Bcl2 protected against apoptosis. This excluded the death-receptor pathway and suggested that cell death decision is regulated by Bcl2-rheostat. Importantly, RNAi knockdown of Noxa or Bim decreased apoptosis, indicating that Noxa and Bim cooperate to mediate FKHRL1-induced cell death. We conclude that Noxa and Bim establish a connection between FKHRL1 and mitochondria, and that both BH3-only proteins are critically involved in FKHRL1-induced apoptosis in neuroblastoma.

G1 arrest by p16INK4A uncouples growth from cell cycle progression in leukemia cells with deregulated cyclin E and c-Myc expression.

The cell cycle inhibitor p16(INK4A) is frequently inactivated in acute lymphoblastic T-cell leukemia (T-ALL). We analyzed mechanisms and consequences of p16(INK4A) reconstitution in T-ALL cells lacking this tumor suppressor. CCRF-CEM cells with tetracycline-regulated p16(INK4A) expression underwent stable G1-phase cell cycle arrest for 72 h followed by massive apoptosis. p16(INK4A) expression caused pRB hypophosphorylation and repression of certain E2F target genes. Interestingly, cyclin E and c-Myc were not affected, suggesting pRB/E2F-independent expression of these E2F targets. Cyclin E/CDK2, however, was inactive due to stabilization and redistribution of p27(Kip1) from CDK4/CDK6 to CDK2. Analyses of c-Myc target genes suggested that c-Myc was transcriptionally inactive, which correlated with hypophosphorylation of the c-Myc inhibitor p107. Thus, p16(INK4A), although unable to repress the expression of deregulated cyclin E and c-Myc, functionally inactivated these potential oncogenes. p16(INK4A)-arrested cells showed morphologic changes, induction of T-cell-specific surface markers and repression of telomerase activity, suggesting differentiation. Moreover, p16(INK4A) reconstitution was associated with increased cellular volume, normal protein synthesis rates and elevated ATP levels. Taken together, p16(INK4A) reconstitution in p16(INK4A)-deficient T-ALL cells induced cell cycle arrest in the presence of cyclin E and c-Myc expression, uncoupled growth from cell cycle progression and caused a sequential process of growth, differentiation and apoptosis.

Glucocorticoid receptor heterozygosity combined with lack of receptor auto-induction causes glucocorticoid resistance in Jurkat acute lymphoblastic leukemia cells.

Glucocorticoids (GC) induce apoptosis in malignant lymphoblasts, but the mechanism of this process as well as that of the clinically important GC resistance is unknown. We investigated GC resistance in Jurkat T-ALL cells in which ectopic GC receptor (GR) restores GC sensitivity, suggesting deficient GR expression. Jurkat cells expressed one wild-type and one mutated (R477H) GR allele. GR(R477H) ligand-binding-dependent nuclear import, as revealed by live-cell microscopy of YFP-tagged GR, was unaffected. Transactivation and transrepression were markedly impaired; however, GR(R477H) did not act in a dominant-negative manner, that is, did not prevent cell death, when introduced into a GC-sensitive cell line by retroviral gene transfer. Contrary to another GR heterozygous, but GC-sensitive, T-ALL model (CCRF-CEM), Jurkats expressed lower basal GR levels and did not auto-induce their GR, as revealed by 'real-time' RT-PCR and immunoblotting. Absent GR auto-induction could not be restored by transgenic GR and, hence, was not caused by reduced basal GR levels. Thus, inactivation of one GR gene results in haploinsufficiency if associated with lack of GR auto-induction.

Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells.

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27(Kip1). Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27(Kip1) accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.

A conceptual view on glucocorticoid-lnduced apoptosis, cell cycle arrest and glucocorticoid resistance in lymphoblastic leukemia.

Glucocorticoids (GC) control cell cycle progression and induce apoptosis in cells of the lymphoid lineage. Physiologically, these phenomena have been implicated in regulating immune functions and repertoire generation. Clinically, they form the basis of inclusion of GC in essentially all chemotherapy protocols for lymphoid malignancies. In spite of their significance, the molecular mechanisms underlying the anti-leukemic GC effects and the clinically important phenomenon of GC resistance are still unknown. This review summarizes recent findings related to GC-induced apoptosis, cell cycle arrest, and GC resistance with particular emphasis on acute lymphoblastic leukemia (ALL). We hypothesize that under conditions of physiological Bcl-2 expression, GC might induce classical programmed cell death by directly perturbing the Bcl-2 rheostat. In the presence of anti-apoptotic Bcl-2 proteins, cell death might result from accumulating catabolic and/or other detrimental GC effects driven by, and critically dependent on, GC receptor (GR) autoinduction. Although still controversial, there is increasing evidence for release of apoptogenic factors through pores in the outer mitochondrial membrane, rather than deltapsiloss-dependent membrane rupture, with maintenance of mitochondrial function at least in the early phase of the death response. GC-induced cell cycle arrest in ALL cells appears to be independent of apoptosis induction and vice versa, and critically depends on repression of both cyclin-D3 and c-myc followed by increased expression of the cyclin-dependent kinase inhibitor, p27Kip1. Since development of GC-resistant clones requires both cell cycle progression and survival, GC resistance might frequently result from structural or regulatory defects in GR expression, perhaps the most efficient means to target both pathways concurrently.

Resistance to glucocorticoid-induced apoptosis in lymphoblastic leukemia.

Glucocorticoid (GC) resistance is a phenomenon of major significance in a number of clinical situations, including the therapy of lymphoid malignancies. Resistance may concern all, or just selected, GC effects, it may be absolute or just reflect a state of reduced sensitivity and, clinically relevant, be reversible or irreversible. Numerous molecular mechanisms can be envisaged acting either 'upstream' in the GC-triggered signaling pathway, i.e. at the level of the GC receptor (GR), or 'downstream' at the level of the GC-regulated genes responsible for individual GC effects. In lymphoid malignancies, GCs have anti-leukemic effects through the induction of apoptosis and/or cell cycle arrest. In this condition evidence for only a small number of mechanisms for GC resistance has been provided, mostly at the level of the GR. Herein, we review reports and hypotheses regarding 'upstream' and 'downstream' mechanisms for GC resistance in lymphoblastic leukemia and present an in vitro GC resistance model that might allow identification of resistance mechanisms.

The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species.

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.

The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels.

The cyclin-dependent kinase inhibitor p16(INK4A) is frequently inactivated in childhood T-cell acute lymphoblastic leukemia. To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16(INK4A) in the T-cell acute lymphoblastic leukemia line CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16(INK4A) expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G(0)/G(1), documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16(INK4A) expression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a possible explanation for this phenomenon, we found that p16(INK4A)-arrested cells had elevated GC receptor expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16(INK4A) inactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC.

The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels.

The cyclin-dependent kinase inhibitor p16(INK4A) is frequently inactivated in childhood T-cell acute lymphoblastic leukemia. To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16(INK4A) in the T-cell acute lymphoblastic leukemia line CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16(INK4A) expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G(0)/G(1), documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16(INK4A) expression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a possible explanation for this phenomenon, we found that p16(INK4A)-arrested cells had elevated GC receptor expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16(INK4A) inactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC.

Gene expression profiles of proliferating vs. G1/G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis.

Glucocorticoids (GC) have pronounced effects on metabolism, differentiation, proliferation, and cell survival (1). In certain lymphocytes and lymphocyte-related malignancies, GC inhibit proliferation and induce apoptotic cell death, which has led to their extensive use in the therapy of malignant lymphoproliferative disorders (2). Most of these effects result from regulation of gene expression via the GC receptor (GR), a ligand-activated transcription factor (3). Although hundreds of genes are regulated by GC (1), how certain biological GC effects relate to individual gene regulation remains enigmatic. To address this question with respect to GC-induced cell cycle arrest and apoptosis, we applied DNA chip technology (4, 5) to determine gene expression profiles in proliferating and G1/G0-arrested (by conditional expression of the CDK inhibitor p16/INK4a) acute lymphoblastic T cells undergoing GC-induced apoptosis. Of 7074 genes tested, 163 were found to be regulated by dexamethasone in the first 8 h in proliferating cells and 66 genes in G1/G0-arrested cells. An almost nonoverlapping set of genes (i.e., only eight genes) was coordinately regulated in proliferating and arrested cells. Analysis of the regulated genes supports the concept that GC-induced apoptosis results from positive GR autoregulation entailing persistent down-regulation of metabolic pathways critical for survival

Resveratrol causes arrest in the S-phase prior to Fas-independent apoptosis in CEM-C7H2 acute leukemia cells.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), in the concentration range of 20 microM and above, induced arrest in the S-phase and apoptosis in the T cell-derived T-ALL lymphocytic leukemia cell line CEM-C7H2 which is deficient in functional p53 and p16. Expression of transgenic p16/INK4A, which causes arrest in G0/G1, markedly reduced the percentage of apoptotic cells. Antagonist antibodies to Fas or FasL, or constitutive expression of crmA did not diminish the extent of resveratrol-induced apoptosis. Furthermore, a caspase-8-negative, Fas-resistant Jurkat cell line was sensitive to resveratrol-induced apoptosis which could be strongly inhibited in the Jurkat as well as in the CEM cell line by z-VAD-fmk and z-IETD-fmk. The almost complete inhibition by z-IETD-fmk and the lack of inhibition by crmA suggested caspase-6 to be the essential initiator caspase. Western blots revealed the massive conversion of procaspase-6 to its active form, while caspase-3 and caspase-2 were proteolytically activated to a much lesser extent.

Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts.

The histone deacetylase inhibitor and potential anti-cancer drug sodium butyrate is a general inducer of growth arrest, differentiation, and in certain cell types, apoptosis. In human CCRF-CEM, acute T lymphoblastic leukemia cells, butyrate, and other histone deacetylase inhibitors caused G2/M cell cycle arrest as well as apoptotic cell death. Forced G0/G1 arrest by tetracycline-regulated expression of transgenic p16/INK4A protected the cells from butyrate-induced cell death without affecting the extent of histone hyperacetylation, suggesting that the latter may be necessary, but not sufficient, for cell death induction. Nuclear apoptosis, but not G2/M arrest, was delayed but not prevented by the tripeptide broad-range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD) and, to a lesser extent, by the tetrapeptide 'effector caspase' inhibitors benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Glu-Ile-Asp.fluoromethyl-ketone (VEID); however, the viral protein inhibitor of 'inducer caspases', crmA, had no effect. Bcl-2 overexpression partially protected stably transfected CCRF-CEM sublines from butyrate-induced apoptosis, but showed no effect on butyrate-induced growth inhibition, further distinguishing these two butyrate effects. c-myc, constitutively expressed in CCRF-CEM cells, was down-regulated by butyrate, but this was not causative for cell death. On the contrary, tetracycline-induced transgenic c-myc sensitized stably transfected CCRF-CEM derivatives to butyrate-induced cell death.

c-Myc does not prevent glucocorticoid-induced apoptosis of human leukemic lymphoblasts.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. The signal transduction pathways leading to this clinically-relevant form of apoptosis have, however, not been sufficiently elucidated. GC bind to their specific receptor, a ligand-activated transcription factor of the Zn-finger type, that activates or represses transcription of GC-responsive genes. Previous studies in leukemia cells suggested that transcriptional repression of c-myc expression might be the crucial event in GC-induced apoptosis, although in other systems, c-Myc apparently increased the sensitivity to cell-death inducers. To address this controversy, we stably transfected the GC-sensitive human T-ALL cell line CEM-C7H2 with constructs allowing tetracycline-regulated expression of c-Myc. Subsequent analyses of these cell lines showed that overexpression of c-Myc per se had little, if any, effect on cell viability, although it rendered the cells more sensitive to apoptosis induced by low serum, confirming the functionality of the expressed transgene. More importantly, however, when the cells were treated with GC in the presence of exogenous c-Myc, they underwent apoptosis exceeding that in cells treated in the absence of transgenic c-Myc. The data indicate that c-myc downregulation is not critical for induction of cell-death by GC in this system, and support the notion that c-Myc sensitizes cells to apoptosis-inducing agents.

p53-induced apoptosis in the human T-ALL cell line CCRF-CEM.

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.