Glucocorticoid-induced apoptosis and glucocorticoid resistance: molecular mechanisms and clinical relevance

The ability of glucocorticoids (GC) to efficiently kill lymphoid cells has led to their inclusion in essentially all chemotherapy protocols for lymphoid malignancies. This review summarizes recent findings related to the molecular basis of GC-induced apoptosis and GC resistance, and discusses their potential clinical implications. Accumulating evidence suggests that GC may induce cell death via different pathways resulting in apoptotic or necrotic morphologies, depending on the availability/responsiveness of the apoptotic machinery. The former might result from regulation of typical apoptosis genes such as members of the Bcl-2 family, the latter from detrimental GC effects on essential cellular functions possibly perpetuated by GC receptor (GR) autoinduction. Although other possibilities exist, GC resistance might frequently result from defective GR expression, perhaps the most efficient means to target multiple antileukemic GC effects. Numerous novel drug combinations are currently being tested to prevent resistance and improve GC efficacy in the therapy of lymphoid malignancies.     

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.     

Glucocorticoid-Induced Apoptosis: Revisited: A Novel Role for Glucocorticoid Receptor Translocation to the Mitochondria

Recent data cast new light on the mechanisms by which glucocorticoids (GCs)elicit apoptosis of thymocytes and leukemia cells. Here we attempt to integrate recentstudies by others and us, which provide a novel insight to this apoptotic process. In thelast few years it was made clear that there is a tight cooperation between genomic andnon-genomic effects exerted by GC receptors (GRs). GC invokes major alterations in thegene expression profile through GR-mediated transactivation and transrepression, whichultimately tip the balance between pro-survival and pro-apoptotic proteins. Althoughessential in shaping the cell’s proteome, these genomic effects are insufficient to elicitapoptotic death and additional signals are required for activating the pro-apoptoticproteins. Several non-genomic effects have been described that occur immediatelyfollowing exposure to GC, which are imperative for the induction of apoptosis. We haverecently observed that GC induces instant GR translocation to the mitochondria in GCsensitive,but not in GC-resistant, T lymphoid cells. This response contrasts the nucleartranslocation of GR occurring in both cell types. We propose that the sustained elevationof GR in the mitochondria following GC exposure is crucial for triggering apoptosis.     

Non-genomic events determining the sensitivity of hemopoietic malignancies to glucocorticoid-induced apoptosis

Glucocorticoid (GC) hormones have been introduced as therapeutic agents in blood cancers six decades ago. The effectiveness of GC treatment stems from its ability to induce apoptotic death of hemopoietic cells. A major impediment in GC therapy is the acquisition of resistance to the drug upon repeated treatment. In addition, some blood cancers are a priori resistant to GC therapy. Usually, resistance to GC correlates with poor prognosis. Albeit the wide use of GC in clinical practice, their mode of action is not fully understood. The cellular response to GC is initiated by its binding to the cytosolic GC receptor (GR) that translocates to the nucleus and modulates gene expression. However, nuclear activities of GR occur in both apoptosis-sensitive and apoptosis-resistant cells. These apparent controversies can be resolved by deciphering non-genomic effects of GCs and the mode by which they modulate the apoptotic response. We suggest that non-genomic consequences of GC stimulation determine the cell fate toward survival or death. Understanding the cellular mechanisms of GC apoptotic sensitivity contributes to the development of new modalities for overcoming GC resistance.     

Repression of the BH3-only molecule PMAIP1/Noxa impairs glucocorticoid sensitivity of acute lymphoblastic leukemia cells

Glucocorticoid (GC)-induced apoptosis plays a major role in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Members of the BCL2 family of pro- and anti-apoptotic proteins are regulated by GC, but to what extent these regulations contribute to GC-induced cell death and resistance development is poorly understood. Using primary lymphoblasts from ALL children during systemic GC monotherapy and related cell lines, we have previously shown that the response of the BCL2 rheostat to GC was dominated by induction of the pro-apoptotic BH3-only molecules BMF and BCL2L11/Bim, but we also observed an unexpected significant repression of the pro-apoptotic BCL2 protein PMAIP1/Noxa. Here, we report that GC represses Noxa mRNA levels and also interferes with its protein stability in a proteasome-dependent manner. Prevention of GC-mediated Noxa repression by conditional expression of transgenic Noxa changed the kinetics of GC-induced apoptosis to resemble cell death induced by BimEL alone. Hence, GC appear to activate functionally relevant pro- as well as anti-apoptotic pathways in ALL cells. Interfering with the anti-apoptotic component of the GC response might contribute to improved therapeutic approaches and circumvention of resistance to this therapy.     

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.     

Identification of an alternative form of caspase-9 in human gastric cancer cell lines: a role of a caspase-9 variant in apoptosis resistance

Overcoming apoptosis resistance to chemotherapy and radiation may lead to a reduction in gastric cancer death. We hypothesize that the apoptotic machinery in gastric cancer cells is dependent upon specific cellular conditions. In the course of our study of the expression of apoptosis-related genes in human gastric cancer cell lines, we have identified a cDNA clone which predicts an alternative form of caspase-9. The caspase-9 variant, which we designated as caspase-9 beta, retained a truncated structure of native caspase-9 without its catalytic domain and was expressed in seven cell lines from human gastric cancer. Among the cell lines examined, MKN-28 cells, which exhibited the most resistance against apoptotic stimuli, expressed the highest level of caspase-9 beta. The induction of apoptosis by staurosporine or actinomycin D was markedly suppressed in caspase-9 beta-transfected HeLa cells. These results are consistent with our hypothesis that the caspase-9 beta may be an endogenous dominant-negative molecule which attenuates apoptotic activity in human gastric cancer cells.     

Silencer of Death Domains Controls Cell Death through Tumour Necrosis Factor-Receptor 1 and Caspase-10 in Acute Lymphoblastic Leukemia

Resistance to apoptosis remains a significant problem in drug resistance and treatment failure in malignant disease. NO-aspirin is a novel drug that has efficacy against a number of solid tumours, and can inhibit Wnt signaling, and although we have shown Wnt signaling to be important for acute lymphoblastic leukemia (ALL) cell proliferation and survival inhibition of Wnt signaling does not appear to be involved in the induction of ALL cell death. Treatment of B lineage ALL cell lines and patient ALL cells with NO-aspirin induced rapid apoptotic cell death mediated via the extrinsic death pathway. Apoptosis was dependent on caspase-10 in association with the formation of the death-inducing signaling complex (DISC) incorporating pro-caspase-10 and tumor necrosis factor receptor 1 (TNF-R1). There was no measurable increase in TNF-R1 or TNF-α in response to NO-aspirin, suggesting that the process was ligand-independent. Consistent with this, expression of silencer of death domain (SODD) was reduced following NO-aspirin exposure and lentiviral mediated shRNA knockdown of SODD suppressed expansion of transduced cells confirming the importance of SODD for ALL cell survival. Considering that SODD and caspase-10 are frequently over-expressed in ALL, interfering with these proteins may provide a new strategy for the treatment of this and potentially other cancers.     

Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.     

IgH-2 cells: a reptilian model for apoptotic studies

Regulation of proper cell number in tissues depends upon a balance between cell proliferation and cell death. The process of apoptosis has thus far been studied in a variety of multicellular organisms from humans to higher plants. In order to broaden our perspective and identify another metazoan system with which to deepen our understanding of the function and evolution of the apoptotic machinery, we have characterized cell death in a reptilian cell line. We show that the death of IgH-2 Iguana (Iguana iguana) heart cells [Clark, H.F., Cohen, M.M., Karzon, D.T., 1970. Characterization of reptilian cell lines established at incubation temperatures of 23 to 36 degrees. Proc. Soc. Exp. Biol. Med. 133, 1039-1047.] is, in response to DNA damaging agents, accompanied by classic morphological changes of apoptosis including detachment from the substrate, cell shrinkage, nuclear pyknosis and externalization of the plasma membrane phospholipid phosphatidylserine. Our biochemical studies show that the death of IgH-2 cells is accompanied by internucleosomal DNA fragmentation and activation of caspases. Our studies with the pan-caspase inhibitor zVAD.fmk implicate caspases in the apoptotic process we observe. This work represents the first detailed molecular and biochemical analysis of apoptosis in cells of an organism of class Reptilia and establishes IgH-2 cells as a suitable model system with which to investigate the phenomenon of caspase dependent apoptosis and the apoptotic machinery in a reptilian model.     

A new, lineage specific, autoup-regulation mechanism for human glucocorticoid receptor gene expression in 697 pre-B-acute lymphoblastic leukemia cells

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.     

A potential role for apoptosis in neurodegeneration and Alzheimer's disease

Previous studies have shown that β-amyloid (Aβ) peptides are neurotoxic. Recent data suggest that neurons undergoing Aβ-induced cell death exhibit characteristics that correspond to the classical features of apoptosis, suggesting that these cells may initiate a program of cell death. This chapter explores the criteria and precautions that must be applied to evaluate mechanisms of cell death in vitro and in vivo, discusses the evidence supporting an apoptotic mechanism of cell death in response to Aβ in cultured neurons, and describes potential correlations for these findings in the Alzheimer's disease brain. In addition, cellular signaling pathways that may be associated with apoptosis in response to Aβ are examined, and support for apoptosis as a mechanism of cell death for other neurodegeneration-inducing stimuli (e.g., oxidative injury) is described. The connection of multiple stimuli that induce neuronal cell death to an apoptotic mechanism suggests that apoptosis could play a central role in neurodegeneration in the brain.