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.     

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.     

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.     

Staurosporine Sensitizes T Lymphoma Cells to Glucocorticoid-Induced Apoptosis: Role of Nur77 and Bcl-2

Glucocorticoids (GCs) are used for treatment of various hematopoietic malignancies owing to their ability to induce apoptosis. A major obstacle in leukemia therapy is the emergence of GC-resistant cells. Hence, combinatory treatment protocols should be developed that convert GC-resistant leukemia cells into sensitive ones. Here we demonstrate that the broad-acting kinase inhibitor staurosporine (STS) confers GC-sensitivity on GC-resistant T lymphoma cells expressing elevated levels of either Bcl-2 or Bcl-XL, but not on GC-resistant myelogenic leukemia cells expressing Mcl-1 in addition to Bcl-2 and/or Bcl-XL. In T lymphoma cells, STS induces the expression of the pro-apoptotic orphan receptor Nur77 that overcomes the anti-apoptotic effect of Bcl-2, thus enabling GC-induced apoptosis. However, in the myelogenic leukemia cells, STS does not up-regulate Nur77. In these cells, the glucocorticoid receptor (GR) is rapidly downregulated by GC and the anti-apoptotic Mcl-1 protein is upregulated by STS, thereby leading to an even more resistant phenotype. Altogether, our data provide a molecular basis for the differential apoptotic response of T lymphoma versus myelogenic leukemia cells to STS and GC. The former being sensitized to GC-induced apoptosis by STS, whereas in the latter, STS intensifies GC resistance. The cell type specific responses should be taken into consideration when combinatory therapy is used for treating hematopoietic malignancies.     

Glucocorticoids engage different signal transduction pathways to induce apoptosis in thymocytes and mature T cells

Glucocorticoids (GC) induce apoptosis in a variety of cells, but their exact mode of action is controversial. Although initiation relies on the GC receptor (GR) and de novo gene expression, the effector phase differs among cell types. Proteasomal degradation as well as caspase-3, - 8, and -9 activity are essential for GC-induced apoptosis in murine thymocytes, but the same enzymes are dispensable in splenic T cells. Live imaging by confocal microscopy revealed that lysosomal cathepsin B, an unrecognized component of this pathway to date, becomes rapidly activated in thymocytes after GC exposure. This is followed by leakage of cathepsin B into the cytosol, nuclear condensation, and processing of caspase-8 and -3. According to our model, activation of caspase-3 by caspase-9 in thymocytes occurs both directly as well as indirectly via a lysosomal amplification loop. Interestingly, acute T lymphoblastic leukemia cells depend on caspase activity to undergo GC-induced cell death similar to thymocytes. Collectively, the apoptotic program induced by GCs comprises cell type-specific as well as common features.     

The molecular basis of glucocorticoid-induced apoptosis of lymphoblastic leukemia cells

Glucocorticoids (GCs) induce programmed cell death with the morphologic characteristics of apoptosis in cells of the lymphoid lineage at certain stages of differentiation. Although the physiological significance of this phenomenon is not well understood, it has been exploited to a great extent in the therapy of malignant lymphoproliferative disorders. In spite of its clinical importance, the molecular mechanisms underlying GC-induced apoptosis and -clinically even more important resistance to this phenomenon are not known. This review summarizes efforts from our and other laboratories addressing these issues in human lymphoblastic leukemia, with particular emphasis on the CCRF-CEM childhood T-cell leukemia model. These studies have shed some light on the downstream execution phase of GC-induced apoptosis but the critical upstream gene regulatory events have remained a mystery. Very recent DNA chip-based expression profile analyses in our laboratory along with data from the literature have, however, suggested that GC receptor auto-upregulation in sensitive target cells might be the critical event leading to persistent downregulation of central metabolic pathways which is incompatible with cell survival. The validity of this hypothesis and its possible clinical implications are currently being investigated in our laboratory.     

GX15-070 (obatoclax) overcomes glucocorticoid resistance in acute lymphoblastic leukemia through induction of apoptosis and autophagy

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies including acute lymphoblastic leukemia (ALL). The BCL-2 family has an essential role in regulating GC-induced cell death. Here we show that downregulation of antiapoptotic BCL-2 family proteins, especially MCL-1, enhances GC-induced cell death. Thus we target MCL-1 by using GX15-070 (obatoclax) in ALL cells. Treatment with GX15-070 in both dexamethasone (Dex)-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases the Annexin V-positive population, which is indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from the BAK/MCL-1 complex following GX15-070 treatment. Consistently, downregulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, downregulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Downregulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy.     

Glucocorticoid-induced alterations in mitochondrial membrane properties and respiration in childhood acute lymphoblastic leukemia

Mitochondria are signal-integrating organelles involved in cell death induction. Mitochondrial alterations and reduction in energy metabolism have been previously reported in the context of glucocorticoid (GC)-triggered apoptosis, although the mechanism is not yet clarified. We analyzed mitochondrial function in a GC-sensitive precursor B-cell acute lymphoblastic leukemia (ALL) model as well as in GC-sensitive and GC-resistant T-ALL model systems. Respiratory activity was preserved in intact GC-sensitive cells up to 24h under treatment with 100 nM dexamethasone before depression of mitochondrial respiration occurred. Severe repression of mitochondrial respiratory function was observed after permeabilization of the cell membrane and provision of exogenous substrates. Several mitochondrial metabolite and protein transporters and two subunits of the ATP synthase were downregulated in the T-ALL and in the precursor B-ALL model at the gene expression level under dexamethasone treatment. These data could partly be confirmed in ALL lymphoblasts from patients, dependent on the molecular abnormality in the ALL cells. GC-resistant cell lines did not show any of these defects after dexamethasone treatment. In conclusion, in GC-sensitive ALL cells, dexamethasone induces changes in membrane properties that together with the reduced expression of mitochondrial transporters of substrates and proteins may lead to repressed mitochondrial respiratory activity and lower ATP levels that contribute to GC-induced apoptosis.     

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N:-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.     

Protein kinase A, not Epac, suppresses hedgehog activity and regulates glucocorticoid sensitivity in acute lymphoblastic leukemia cells

Cyclic AMP synergizes strongly with glucocorticoids (GC) to induce apoptosis in normal or malignant lymphoid cells. We examined the individual roles that cAMP-dependent protein kinase (PKA) and Epac (exchange protein directly activated by cAMP), two intracellular cAMP receptors, play in this synergistic effect. Our studies demonstrate that PKA is responsible for the observed synergism with GC, whereas Epac exerts a weak antagonistic effect against GC-induced apoptosis. We find that endogenous PKA activity is higher in the GC-sensitive clone than in the GC-resistant clone. In the GC-sensitive clone, higher PKA activity is associated with lower Hedgehog (Hh) activity. Moreover, inhibition of Hh activity by Hh pathway-specific inhibitors leads to cell cycle arrest and apoptosis in CEM (human acute lymphoblastic leukemia, T lineage) cells, and the GC-sensitive clone is more sensitive to Hh inhibition. These results suggest that Hh activity is critical for leukemia cell growth and survival and that the level of Hh activity is in part responsible for the synergism between cAMP and GC.     

Glucocorticoid resistance in chronic lymphocytic leukaemia is associated with a failure of upregulated Bim/Bcl-2 complexes to activate Bax and Bak

Glucocorticoids (GCs) represent an important component of modern treatment regimens for fludarabine-refractory or TP53-defective chronic lymphocytic leukemia (CLL). However, GC therapy is not effective in all patients. The molecular mechanisms responsible for GC-induced apoptosis and resistance were therefore investigated in primary malignant cells obtained from a cohort of 46 patients with CLL. Dexamethasone-induced apoptosis was unaffected by p53 dysfunction and more pronounced in cases with unmutated IGHV genes. Cross-resistance was observed between dexamethasone and other GCs but not fludarabine, indicating non-identical resistance mechanisms. GC treatment resulted in the upregulation of Bim mRNA and protein, but to comparable levels in both GC-resistant and sensitive cells. Pre-incubation with Bim siRNAs reduced GC-induced upregulation of Bim protein and conferred resistance to GC-induced apoptosis in previously GC-sensitive cells. GC-induced upregulation of Bim was associated with the activation of Bax and Bak in GC-sensitive but not -resistant CLL samples. Co-immunoprecipitation experiments showed that Bim does not interact directly with Bax or Bak, but is almost exclusively bound to Bcl-2 regardless of GC treatment. Taken together, these findings suggest that the GC-induced killing of CLL cells results from the indirect activation of Bax and Bak by upregulated Bim/Bcl-2 complexes, and that GC resistance results from the failure of such activation to occur.     

Mitochondrial function, apoptosis and cell cycle delay in the WEHI-3B leukaemia cell line and its variant Ciprofloxacin-resistant WEHI-3B/CPX

OBJECTIVE: The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared. MATERIALS AND METHODS: Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment. RESULTS: We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression. CONCLUSION: The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.