Molecular determinants of glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia
Received 9 April 2002; Accepted 1 July 2002.
Top of pageAbstractGlucocorticoids (GC) are probably the most important drugs in the treatment of ALL. Despite the extensive use of GC for many years, little is known about the molecular mechanisms of sensitivity and resistance. This review summarizes the knowledge on GC cytotoxicity in leukemia. The relevance of polymorphisms, splice variants and the number and regulation of the GC receptor are discussed. The role of multidrug resistance proteins, glutathione and glutathione S transferase is evaluated, as well as the influence of the different heat shock chaperone (hsp 90 and 70) and co chaperone proteins (BAG 1 and others) which form a complex together with the GC receptor. The diverse actions of GC have led to their use as therapeutic agents in the treatment of many diseases. GC can act in an anti proliferative manner in specific cell types, which is the reason why GC are used in immunosuppressive, anti inflammatory and oncolytic therapy. The effect on lymphoid cells is dramatic and includes the induction of G1 cell cycle arrest and apoptosis. In newly diagnosed acute lymphoblastic leukemia (ALL), prednisone and dexamethasone have significant antileukemic effect in the majority of children.1 Although GC are the most important drugs used in the treatment of ALL for more than 50 years, the molecular basis of GC sensitivity and resistance remains largely unknown. Understanding of the molecular mechanisms related to GC cytotoxicity is crucial for understanding a major part of treatment success or failure in childhood ALL and is crucial for the exploration of possibilities to modulate GC resistance. This review summarizes the current knowledge on molecular determinants of glucocorticoid sensitivity and resistance in ALL.
Clinical aspects of glucocorticoid sensitivityGlucocorticoid sensitivity is a major prognostic factor in childhood ALL. In BFM trials blast count after 7 days of monotherapy with prednisone (including one intrathecal dose of methotrexate) was shown to be a strong and independent prognostic factor.1,2,3 Subgroups with a poor prognosis like infants, T ALL patients and patients with a Philadelphia chromosome positive ALL, more often show a poor clinical response to prednisone.2,4,5 In vitro resistance to glucocorticoids at initial diagnosis is also related to an unfavorable event free survival in childhood ALL.6,7,8,9 Leukemic cells from the risk groups associated with a poor prognosis (T ALL, proB ALL, infant ALL) are relatively in vitro resistant to prednisolone.10 Leukemic cells from adults with ALL, who have an unfavorable outcome as compared with children, are also more resistant to GC in vitro.11,12 Leukemic cells of patients with relapsed ALL are 300 fold more in vitro resistant to prednisolone than the cells taken at diagnosis: in paired initial/relapse samples resistance to GC increased in the majority of patients at the time of relapse compared to initial diagnosis.13
The glucocorticoid receptor gene and activation by glucocorticoidsThe glucocorticoid receptor gene (GR) is located on chromosome 5 (5q31) and consists of nine exons encoding for three characteristic domains of the protein (Figure 1).14 The N terminal region contains a transactivation domain (AF 1) that is involved in transcriptional activation of target genes.15 An internal DNA binding domain consisting of two highly conserved ‘zinc fingers’ is crucial for the binding to the glucocorticoid response elements (GRE) sequence. This domain contains a nuclear localization signal (NLS1). The first zinc finger (exon 3) encodes for domains necessary for binding NF B and AP 116 and is therefore important for the transrepression mode of the receptor. The second zinc finger domain (exon 4) encodes for receptor dimerization and GRE mediated transactivation.17,18,19,20 The C terminal part of the protein contains the ligand binding domain that also binds heat shock proteins (hsp) and is involved in receptor dimerization. This domain contains a second nuclear localization signal (NLS2) and transcription activation (AF 2) site.15,19 The two transactivation domains (AF 1 and AF 2) interact with other nuclear proteins such as CBP (CREB binding protein) and P300 that are important for stabilization and activation of the transcription initiation complex in the promotors of glucocorticoid responsive genes.21 GC enter the cell by passive diffusion and bind to the GR which is located in the cytoplasm as a homodimer (Figure 2). The GR gene, localized on chromosome 5, consists of nine coding exons. The functional parts of the receptor are indicated. Glucocorticoids (GC) enter the cell by passive transport, and bind to the glucocorticoid receptor (GR). The unbound GR forms heter Swarovski Outlet ocomplexes consisting of heat shock chaperone molecules hsp90 and 70, co chaperone molecules hsp40, Hop (p60), p23 and immunophillins FKBp52 and CyP40, required for optimal configuration of the GR to be able Swarovski Outlet to bind GC. As a homodimer the GC complex translocates to the nucleus. There it interacts with either a GRE (consensus sequence (GGT ACA NNNTGT TCT) of a target gene (transactivation), or it interacts with other transcription factors such as AP 1 and NF B (transrepression). Both processes may finally result in the induction of cell death (apoptosis). NF B is kept in the cytoplasm in a complex with IB. Upon dissociation it can translocate to the nucleus.
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The GR belongs to the nuclear hormone receptor superfamily, and is highly homologous to the mineralocorticoid, progesterone and androgen receptor. Binding of GC to the GR triggers the dissociation of proteins bound to the receptor such as hsp and BAG 1.22 This discloses and activates the nuclear localization signal (NLS) domains of the receptor. The GC complex then translocates to the nucleus as a homodimer, where it can interact with GREs (consensus sequence GGT ACANNN TGT TCT23). These are located in promotor regions of GC responsive genes, leading to transcriptional activation or inactivation of these genes (the latter hardly ever occurs).24 The GC complex can also directly interact with transcription factors like activating protein 1 (AP 1) or nuclear factor B (NF B), by forming transrepression complexes. However, a high receptor number does not necessarily predict a good response to a GC containing regimen. All studies about GR numbers used dexamethasone binding assays in which only functional receptors are measured (ie probably the and the isoform see below).
In contrast to basal expression levels of the receptor, up regulation of the number of GR upon GC exposure may be more important for GC induced apoptosis. In non lymphoid cell lines and in peripheral blood mononuclear cells of children with auto immune disorders, with no apoptotic response upon GC exposure, a decrease in GR mRNA expression and receptor number was observed after exposure to GC.32,33,34,35,36,37 Contrary to the down regulation in those non lymphoid cell lines, up regulation of the GR upon GC exposure is reported in leukemic and lymphoid cell lines. In the leukemic T cell line, CCRF CEM, an up regulation of GR mRNA was shown in the first few hours after exposure to GC.38 Ramdas et al39 showed that the basal level of GR expression is inadequate to mediate glucocorticoid induced apoptosis in a leukemic human T cell line 6TG1.1. An increase in GR number by autoregulation is required to induce apoptosis in these cells. Recently, Breslin et al40 studied the hypothesis that activation of different promotors of the GR gene may regulate the expression of GR under the influence of GC in different tissues. Exon 1A3 mRNA (one of at least five transcripts from three different promoters) is expressed most abundantly in hematological cancer cell lines that are sensitive to GC induced apoptosis. Furthermore, GC exposure causes up regulation of exon 1A3 containing GR transcripts in (hematological) CEM C7 cells.
No data exist proving the hypothesis that leukemic cells that are sensitive to GC are able to up regulate their receptor number more pronouncedly than resistant leukemic cells.
Membrane bound receptorWhereas the GR is generally described as a cytosolic receptor, Gametchu et al41 described a membrane bound variant (mGR). The expression of the mGR is reported to be cell cycle regulated in the CCRF CEM cell line. The highest expression is found during the late S phase when the cells are most sensitive to the apoptotic effect of GC. The mGR expression varied among leukemic patients, whereas no mGR was found in the membrane of lymphocytes of healthy individuals, the latter being highly resistant to GC.41,42 In a pilot study no correlation was found between the expression of mGR and in vitro sensitivity to GC in childhood ALL samples (Gametchu, personal communication).
Polymorphisms/somatic mutations of the glucocorticoid receptor geneA number of endocrinological glucocorticoid resistance syndromes have been described that are associated with genetic mutations in the GR.43,44,45,46,47 (A genetic mutation is defined as an inheritable germline mutation present in a limited number of individuals (most commonly within a family) and associated with a higher risk to develop a malignancy (eg Li Fraumeni syndrome). A polymorphism is defined as an inheritable genetic germline variant of a single locus (most frequently a single nucleotide variation) that is present in at least 1% of the population. The opposite of polymorphisms are somatic mutations that are associated with a specific type of disease, and thereby restricted to the malignant cells only.) These mutations result in a lower number of functional receptors and interfere with GC binding or transactivational capacity. Besides genetic mutations, several polymorphisms have been described. Although most polymorphisms do n Swarovski Outlet ot effect receptor function,48,49 one polymorphism has been described which is associated with increased sensitivity to GC. This N363S polymorphism is present in 3 of the population.49,50,51 Table 1 summarizes the literature references on polymorphisms and mutations in the GR as found in healthy individuals, a patient with childhood ALL and patients with a glucocorticoid resistance syndrome. Hillman et al52 showed that the GC resistant CCRF CEM cell line contains one GR allele with the L753F mutation. Analysis of the original biopsy material also revealed the same mutation, but in a substantially lower frequency than expected, concordant with the hypothesis that this mutation was only limited to a leukemic subclone.
The different mutations can cause a decreased sensitivity to GC in various ways. Decreased sensitivity to GC may be related to the location of the mutation (N terminal, DNA binding region or ligand binding region) or to the preferential degradation of mutated GR. The GR transcript can be truncated by a mutation, which introduces a premature stop codon, resulting in loss of mRNA expression and loss of GR number and associated with a decreased GC sensitivity.43,52 Other mutations may interfere with correct splicing of the pre mRNA transcript. Karl et al43 showed that a four basepair deletion on the boundary of exon 6 resulted in the absence of transcripts of that allele, so GR is only encoded by one wild type allele. The relevance of polymorphisms and genetic or somatic mutations in the GR gene for GC sensitivity and prognosis in childhood leukemia is the subject of ongoing studies in our laboratory53 and of others. If these genetic aberrations are important for the cellular response to GC, the presence of these polymorphisms/mutations can be used to identify patients at risk for a poor response to GC treatment. In addition, patients with a polymorphism that is associated with increased GC sensitivity like the N363S polymorphism may benefit from GC in their treatment. However, these patients may also suffer from severe side effects, such as Cushing syndrome, overweight, depression and avascular necrosis of bone. These patients may still have an excellent prognosis when treated with a lower dosage of GC.
Splice variants of the glucocorticoid receptorFive different splice variants of the GR gene have been described, formed by alternative splicing, ie the , , , GR P and GR A isoform (see Figure 3).
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The isoform is the functional receptor and is encoded for by exon 2 to 9.54 It is located in the cytoplasm in the absence of GC, but migrates to the nucleus upon GC binding.55 The only study of the relationship between splice variants and GC sensitivity in leukemic patients was done by Longui et al56 They described a reduced GR expression in the leukemic blasts of 13 ALL patients in comparison with EBV transformed lymphocytes of nine normal controls. The GR expression was lowest in T ALL samples and no concomitant decrease in GR expression in the leukemic cells of these patients was observed. As a consequence, GR cannot bind GC.54 At mRNA level, GR expression is 0.2 of the total GR expression.57,58 Strikingly, at protein level, the data are controversial and vary from levels comparable to mRNA levels59,60 to five fold higher expression levels compared with GR.61 The reason for these conflicting data might be lack of specificity of the antibodies used in these studies. The isoform might have a dominant negative effect over the isoform. Carlstedt Duke62 and Vottero and Chrousos63 reviewed the controversial data on this hypothesis. In GC resistant asthma patients, Leung et al64 found a relatively higher expression of GR in peripheral blood mononuclear cells as compared to GC sensitive asthma patients, but Gagliardo et al could not confirm this.65 Transfection studies in cell lines are also inconclusive. This isoform is expressed at 3.8 of total GR mRNA in different human tissues but it is unknown whether this variant is a separate splice variant or part of the , and GR P isoforms.70,71 Ray et al72 reported that the biological activity of the isoform is reduced to 50% of the wild type receptor. The GR P transcripts account for up to 10 of total GR mRNA, but has been reported to be up regulated in a small group of hematological malignancies (ALL, non Hodgkin’s lymphoma and multiple myeloma, up to 54% of total GR mRNA).57,74,75,76 In a study of de Lange et al,77 transfection of GR P receptor increased the activity of the GR receptor. No further information is known about the expression levels and function of this variant.73Studying the relevance of the alpha, beta Swarovski Outlet and hGR P splice variants for GC sensitivity in childhood ALL, preliminary data from our laboratory did not show a relation between mRNA levels of the three splice variants and in vivo or in vitro sensitivity to GC. Further work is needed to define the importance of the different splice variants in relation to GC sensitivity in ALL and to delineate the mechanisms of GR mRNA splicing regulation.
Phosphorylation of the GRPhosphorylation of receptors is a general regulation mechanism in cells. Phosphorylation of the GR modulates the GR function as reviewed by Bodwell et al.78 The phosphorylation rate is maximal in the S phase and enhances the transactivating and transrepressing activities of the GR. Furthermore, the GR is destabilized by phosphorylation, resulting in a shorter half life of the protein. The clinical relevance of phosphorylation of the GR is unknown.
Multidrug resistance and GC sensitivityP glycoprotein (P gp) is a drug efflux pump responsible for multidrug resistance (MDR) and encoded for by the mdr 1 gene. In a murine thymoma cell line it was shown that increased resistance to dexamethasone was linked to an increased expression of the mdr1 gene of P gp. Verapamil, able to restore intracellular drug concentrations by blocking the drug efflux pump, was able to increase the intracellular level of dexamethasone.79,80 However, contrary to these cell line studies, in a report on leukemic cells of 112 children with ALL no correlation between GC resistance in vitro and the expression or function of P gp activity was found.81 Furthermore, functional P gp activity was not related to in vivo prednisone response in a group of 90 ALL patients.82 In addition, GC resistance was not associated with an increased expression of other multi drug resistance related proteins such as major vault protein/lung resistance protein (MVP/LRP) and multidrug resistance associated protein (MRP 1).81