REVIEW Molecular Determinants of Glucocorticoid Sensitivity And

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Molecular determinants of glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia WJE Tissing, JPP Meijerink, ML den Boer and R Pieters

University Hospital Rotterdam/Sophia Children’s Hospital, Department of Paediatric Oncology/Hematology, Rotterdam, The Netherlands

Glucocorticoids (GC) are probably the most important drugs in apywith prednisone (including one intrathecal dose of the treatment of ALL. Despite the extensive use of GC for many methotrexate) was shown to be a strong and independent years, little is known about the molecular mechanisms of sensi- 1–3 tivity and resistance. This review summarizes the knowledge prognostic factor. Subgroups with a poor prognosis like on GC cytotoxicity in leukemia. The relevance of polymor- infants, T-ALL patients and patients with a Philadelphia chro- phisms, splice variants and the number and regulation of the mosome-positive ALL, more often show a poor clinical GC receptor are discussed. The role of multidrug resistance response to prednisone.2,4,5 In vitro resistance to glucocortico- proteins, glutathione and glutathione S-transferase is evalu- ids at initial diagnosis is also related to an unfavorable event- ated, as well as the influence of the different heat-shock chap- free survival in childhood ALL.6–9 Leukemic cells from the risk erone (hsp 90 and 70) and co-chaperone proteins (BAG-1 and others) which form a complex together with the GC receptor. groups associated with a poor prognosis (T-ALL, proB-ALL, 10 Finally, the transactivation and transrepression (via NF-␬B and infant ALL) are relatively in vitro resistant to prednisolone. AP-1 binding) of a wide range of genes (like c-myc) which Leukemic cells from adults with ALL, who have an unfavor- initiates the final apoptosis pathway are discussed and sugges- able outcome as compared with children, are also more resist- tions for future directions of research in ALL patients are given. ant to GC in vitro.11,12 Leukemic cells of patients with relapsed Leukemia (2003) 17, 17–25. doi:10.1038/sj.leu.2402733 ALL are 300-fold more in vitro resistant to prednisolone than Keywords: glucocorticoid; glucocorticoid receptor; childhood; acute lymphoblastic leukemia; apoptosis; resistance the cells taken at diagnosis: in paired initial/relapse samples resistance to GC increased in the majorityof patients at the time of relapse compared to initial diagnosis.13 Introduction The glucocorticoid receptor gene and activation by Glucocorticoids (GC) are involved in manybiological pro- glucocorticoids cesses, including metabolism, development, differentiation, immunity, reproduction and neural activity. The diverse The glucocorticoid receptor gene (GR) is located on chromo- actions of GC have led to their use as therapeutic agents in the some 5 (5q31) and consists of nine exons encoding for three treatment of manydiseases. GC can act in an anti-proliferative characteristic domains of the protein (Figure 1).14 The N-ter- manner in specific cell types, which is the reason why GC are minal region contains a transactivation domain (AF-1) that is used in immunosuppressive, anti-inflammatoryand oncolytic involved in transcriptional activation of target genes.15 An therapy. The effect on lymphoid cells is dramatic and includes internal DNA binding domain consisting of two highlycon- the induction of G1 cell cycle arrest and apoptosis. In newly served ‘zinc fingers’ is crucial for the binding to the glucocort- diagnosed acute lymphoblastic leukemia (ALL), prednisone icoid response elements (GRE) sequence. This domain con- and dexamethasone have significant antileukemic effect in the tains a nuclear localization signal (NLS1). The first zinc finger majorityof children. 1 Although GC are the most important (exon 3) encodes for domains necessaryfor binding NF- ␬B drugs used in the treatment of ALL for more than 50 years, and AP-116 and is therefore important for the transrepression the molecular basis of GC sensitivityand resistance remains mode of the receptor. The second zinc finger domain (exon largelyunknown. Understanding of the molecular mech- 4) encodes for receptor dimerization and GRE-mediated trans- anisms related to GC cytotoxicity is crucial for understanding activation.17–20 The C-terminal part of the protein contains the a major part of treatment success or failure in childhood ALL ligand binding domain that also binds heat-shock proteins and is crucial for the exploration of possibilities to modulate (hsp) and is involved in receptor dimerization. This domain GC resistance. This review summarizes the current knowledge contains a second nuclear localization signal (NLS2) and tran- on molecular determinants of glucocorticoid sensitivityand scription activation (AF-2) site.15,19 The two transactivation resistance in ALL. 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 Clinical aspects of glucocorticoid sensitivity initiation complex in the promotors of glucocorticoid-responsive genes.21 GC enter the cell bypassive diffusion and bind Glucocorticoid sensitivityis a major prognostic factor in child- to the GR which is located in the cytoplasm as a homodimer hood ALL. In BFM trials blast count after 7 days of monother- (Figure 2). The GR belongs to the nuclear hormone receptor super- family, and is highly homologous to the mineralocorticoid, Correspondence: WJE Tissing, UniversityHospital Rotterdam/Sophia progesterone and androgen receptor. Binding of GC to the GR Children’s Hospital, Department of Paediatric Oncology/Hematology, Dr Molewaterplein 60, 3015 GJ, Rotterdam, The Netherlands; Fax: triggers the dissociation of proteins bound to the receptor such 22 0031-10-4636801 as hsp and BAG-1. This discloses and activates the nuclear Received 9 April 2002; accepted 1 July2002 localization signal (NLS) domains of the receptor. The GC– Glucocorticoid resistance in ALL WJE Tissing et al 18

Figure 1 Schematic overview of the GR gene. The GR gene, localized on chromosome 5, consists of nine coding exons. The functional parts of the receptor are indicated.

manystudies, as reviewed earlier. 31 Most authors report a significant correlation between low numbers of receptor per cell and a higher rate of induction failure or relapse. However, a high receptor number does not necessarilypredict 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 probablythe ␣- and the ␥-isoform – see below). In contrast to basal expression levels of the receptor, up- regulation of the number of GR upon GC exposure maybe 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.32–37 Contraryto the down-regulation in those non-lymphoidcell lines, up-regulation of the GR upon GC exposure is reported Figure 2 Model of GC-induced apoptosis in leukemic cells. Gluc- in leukemic and lymphoid cell lines. In the leukemic T cell ocorticoids (GC) enter the cell bypassive transport, and bind to the line, CCRF-CEM, an up-regulation of GR mRNA was shown glucocorticoid receptor (GR). The unbound GR forms heterocom- in the first few hours after exposure to GC.38 Ramdas et al39 plexes consisting of heat-shock chaperone molecules hsp90 and 70, showed that the basal level of GR expression is inadequate to co-chaperone molecules hsp40, Hop (p60), p23 and immunophillins mediate glucocorticoid-induced apoptosis in a leukemic FKBp52 and CyP40, required for optimal configuration of the GR to be human T cell line 6TG1.1. An increase in GR number byauto- able to bind GC. As a homodimer the GC–GR complex translocates to the nucleus. There it interacts with either a GRE (consensus sequence regulation is required to induce apoptosis in these cells. 40 (GGTACA NNN TGT TCT) of a target gene (transactivation), or it Recently, Breslin et al studied the hypothesis that activation interacts with other transcription factors such as AP-1 and NF-␬B of different promotors of the GR gene mayregulate the (transrepression). Both processes mayfinallyresult in the induction of expression of GR under the influence of GC in different cell death (apoptosis). NF-␬B is kept in the cytoplasm in a complex tissues. Exon 1A3 mRNA (one of at least five transcripts from ␬ ␣ with I B . Upon dissociation it can translocate to the nucleus. three different promoters) is expressed most abundantlyin hematological cancer cell lines that are sensitive to GC- GR complex then translocates to the nucleus as a homodimer, induced apoptosis. Furthermore, GC exposure causes up- where it can interact with GREs (consensus sequence GGT 23 regulation of exon 1A3-containing GR transcripts in ACA NNN TGT TCT ). These are located in promotor regions (hematological) CEM-C7 cells. of GC-responsive genes, leading to transcriptional activation 24 No data exist proving the hypothesis that leukemic cells that or inactivation of these genes (the latter hardlyever occurs). are sensitive to GC are able to up-regulate their receptor num- The GC–GR complex can also directlyinteract with transcrip- ber more pronouncedlythan resistant leukemic cells. tion factors like activating protein-1 (AP-1) or nuclear factor ␬B(NF-␬B), byforming transrepression complexes. The formation of these complexes represses the transcriptional activity of both the GR, AP-1 and NF-␬B.25 Both transactivation (via Membrane bound receptor GRE-binding) and transrepression (via interaction with AP-1 or NF-␬B) processes can induce apoptosis of GC-sensitive Whereas the GR is generallydescribed as a cytosolicreceptor, cells, but it is yet unknown if one or both are important in Gametchu et al41 described a membrane-bound variant ALL.16,26–30 (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–G2/M phase when the cells are most Molecular mechanisms of sensitivity and resistance in sensitive to the apoptotic effect of GC. The mGR expression leukemic cells varied among leukemic patients, whereas no mGR was found in the membrane of lymphocytes of healthy individuals, the Number of glucocorticoid receptors latter being highlyresistant to GC. 41,42 In a pilot studyno correlation was found between the expression of mGR and in The relationship between the number of GR per cell and the vitro sensitivityto GC in childhood ALL samples (Gametchu, response to GC in patients with ALL has been the subject of personal communication).

Leukemia Glucocorticoid resistance in ALL WJE Tissing et al 19 Polymorphisms/somatic mutations of the The different mutations can cause a decreased sensitivityto glucocorticoid receptor gene GC in various ways. Decreased sensitivity to GC may be related to the location of the mutation (N-terminal, DNA-bind- A number of endocrinological glucocorticoid resistance syn- ing region or ligand-binding region) or to the preferential dromes have been described that are associated with genetic degradation of mutated GR. The GR transcript can be trunc- mutations in the GR.43–47 (A genetic mutation is defined as an ated bya mutation, which introduces a premature stop codon, inheritable germline mutation present in a limited number of resulting in loss of mRNA expression and loss of GR number individuals (most commonlywithin a family)and associated and associated with a decreased GC sensitivity.43,52 Other with a higher risk to develop a malignancy(eg Li Fraumeni mutations mayinterfere with correct splicing of the pre-mRNA syndrome). A polymorphism is defined as an inheritable gen- transcript. Karl et al43 showed that a four-basepair deletion on etic germline variant of a single locus (most frequentlya single the boundaryof exon 6 resulted in the absence of transcripts nucleotide variation) that is present in at least 1% of the popu- of that allele, so GR is onlyencoded byone wild typeallele. lation. The opposite of polymorphisms are somatic mutations The relevance of polymorphisms and genetic or somatic that are associated with a specific type of disease, and thereby mutations in the GR gene for GC sensitivityand prognosis in restricted to the malignant cells only.) These mutations result childhood leukemia is the subject of ongoing studies in our in a lower number of functional receptors and interfere with laboratory53 and of others. If these genetic aberrations are GC binding or transactivational capacity. Besides genetic important for the cellular response to GC, the presence of mutations, several polymorphisms have been described. these polymorphisms/mutations can be used to identify Although most polymorphisms do not effect receptor func- patients at risk for a poor response to GC treatment. In tion,48,49 one polymorphism has been described which is addition, patients with a polymorphism that is associated with associated with increased sensitivityto GC. This N363S poly- increased GC sensitivitylike the N363S polymorphismmay morphism is present in 3–6% of the population.49–51 Table 1 benefit from GC in their treatment. However, these patients summarizes the literature references on polymorphisms and mayalso suffer from severe side-effects, such as Cushing syn- mutations in the GR as found in healthyindividuals, a patient drome, overweight, depression and avascular necrosis of with childhood ALL and patients with a glucocorticoid resist- bone. These patients maystill have an excellent prognosis ance syndrome. when treated with a lower dosage of GC. The role of polymorphisms or somatic mutations in the GR in relation to GC cytotoxicity in childhood leukemia is largely unknown. Hillman et al52 showed that the GC-resistant CCRF- Splice variants of the glucocorticoid receptor CEM cell line contains one GR allele with the L753F mutation. Analysis of the original biopsy material also revealed the same Five different splice variants of the GR gene have been mutation, but in a substantiallylower frequencythan described, formed byalternative splicing, ie the ␣, ␤, ␥,GR- expected, concordant with the hypothesis that this mutation P and GR-A isoform (see Figure 3). was onlylimited to a leukemic subclone. (1) The ␣ isoform is the functional receptor and is encoded for byexon 2 to 9 ␣.54 It is located in the cytoplasm in Table 1 Polymorphisms and mutations in the glucocorticoid the absence of GC, but migrates to the nucleus upon GC receptor gene described in healthyindividuals, a patient with child- binding.55 The onlystudyof the relationship between hood ALL and patients with a glucocorticoid resistance syndrome splice variants and GC sensitivityin leukemic patients was done byLongui et al56 Theydescribed a reduced GR ␣ Identity of genetic alteration GC sensitivity Ref. expression in the leukemic blasts of 13 ALL patients in comparison with EBV transformed lymphocytes of nine Polymorphisms normal controls. The GR␣ expression was lowest in T-ALL Healthy individuals samples and no concomitant decrease in GR␤ expression + Double mutation codon 22 23 Unchanged 57 in the leukemic cells of these patients was observed. The F29L Unchanged 56 ␣ L112F Unchanged 56 authors suggest that decreased GR expression levels may D233N Unchanged 56 explain the lower GC sensitivityin T-ALL patients as com- K293K Unchanged 56 pared to pre B-ALL. However, the proof for the relevance N363S Increased 51 Intron 3: 46 nucleotides upstream Unchanged 57 from exon 4: G–C Intron 4: 16 nucleotides upstream Unchanged 57 from exon 5: G–C D677D Unchanged 56 Codon 766: Asn–Asn Unchanged 57/56

Somatic mutations Patients with GC resistance syndromes: R477H Decreased 55 I559N Decreased 53 ⌬4 deletion 3Ј boundary exon 6 Decreased 51 D641V Decreased 52 G679S Decreased 55 V729I Decreased 54 Patient with ALL: L753F Decreased 60/61 Figure 3 The glucocorticoid receptor gene and ﬁve different splice variants.

Leukemia Glucocorticoid resistance in ALL WJE Tissing et al 20 of the alpha splice variant is indirect as there was no in Phosphorylation of the GR vitro or in vivo assessment of GC sensitivityand patient number is verysmall. Phosphorylation of receptors is a general regulation mech- (2) The ␤ isoform is the result of alternative splicing of exon anism in cells. Phosphorylation of the GR modulates the GR 9␤ instead of 9␣, resulting in 15 unique C-terminal amino function as reviewed byBodwell et al.78 The phosphorylation acids. As a consequence, GR␤ cannot bind GC.54 At rate is maximal in the S-phase and enhances the transactivat- mRNA level, GR␤ expression is 0.2–1% of the total GR ing and transrepressing activities of the GR. Furthermore, the expression.57,58 Strikingly, at protein level, the data are GR is destabilized byphosphorylation,resulting in a shorter controversial and varyfrom levels comparable to mRNA half-life of the protein. The clinical relevance of phosphoryl- levels59,60 to five-fold higher expression levels compared ation of the GR is unknown. with GR␣.61 The reason for these conflicting data might be lack of specificityof the antibodies used in these studies. The ␤ isoform might have a dominant negative effect Multidrug resistance and GC sensitivity over the ␣ isoform. Carlstedt-Duke62 and Vottero and Chrousos63 reviewed the controversial data on this P-glycoprotein (P-gp) is a drug-efflux pump responsible for hypothesis. In GC-resistant asthma patients, Leung et al64 multidrug resistance (MDR) and encoded for bythe mdr-1 gene. In a murine thymoma cell line it was shown that found a relativelyhigher expression of GR ␤ in peripheral increased resistance to dexamethasone was linked to an blood mononuclear cells as compared to GC-sensitive increased expression of the mdr1 gene of P-gp. Verapamil, asthma patients, but Gagliardo et al could not confirm able to restore intracellular drug concentrations byblocking this.65 Transfection studies in cell lines are also inconclus- the drug-efflux pump, was able to increase the intracellular ive. Some transfection studies showed a negative effect of level of dexamethasone.79,80 However, contraryto these cell ␤ ␣ 66,67 60,68,69 GR over GR , whereas others did not. As the line studies, in a report on leukemic cells of 112 children with expression level of the beta isoform is verylow compared ALL no correlation between GC resistance in vitro and the to the alpha isoform, the relevance of the beta expression expression or function of P-gp activitywas found. 81 Further- or resistance in childhood leukemia is unlikely. more, functional P-gp activitywas not related to in vivo ␥ 70 (3) The isoform has onlyrecentlybeen described. In this prednisone response in a group of 90 ALL patients.82 In splice variant exon 4 is alternativelyspliced to exon 3 addition, GC resistance was not associated with an increased therebyincluding three basepairs of the intron region expression of other multi-drug resistance-related proteins such resulting in an additional arginine residue. This isoform is as major vault protein/lung resistance protein (MVP/LRP) and expressed at 3.8–8.7% of total GR mRNA in different multidrug resistance-associated protein (MRP-1).81 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 Glutathione and glutathione S-transferase of the ␥ isoform is reduced to 50% of the wild-type receptor. Gerdes et al71 reported preliminaryresults showing a Increased glutathione (GSH) levels and glutathione S-transfer- possible role for the ␥ isoform in poor prednisone response ase overexpression contribute to resistance to various cytos- in childhood ALL. tatic drugs. Maung et al83 found in their studyof 21 newly (4) The GR-P isoform is encoded byexons 2–7 plus several diagnosed patients a positive correlation between GSH level basepairs from the subsequent intron region.73 As a conse- and prednisolone resistance. However, in a recent report con- quence, this isoform lacks the ligand binding domain and cerning 62 newlydiagnosed children with ALL, this relation- therefore cannot bind GC. The GR-P transcripts account ship was not confirmed.84 In addition, the expression levels for up to 10–20% of total GR mRNA, but has been of GST (␣, ␮ and ␲-class) were not related to in vitro predniso- reported to be up-regulated in a small group of hematolog- lone sensitivityin childhood ALL. 85 Anderer et al86 reported ical malignancies (ALL, non-Hodgkin’s lymphoma and the implication of polymorphisms in the GST genes for GC multiple myeloma, up to 54% of total GR mRNA).57,74–76 sensitivityin childhood ALL. In a case–control studyof 45 in In a studyof de Lange et al,77 transfection of GR-P receptor vivo poor responders and 90 good responders there was a increased the activityof the GR ␣ receptor. In the same reduced risk of prednisone poor response in patients with a studyno relationship was found between sensitivityto GC homozygous deletion of GSTT1 (null genotype), although this = and the ratios of the GR␣,GR␤ and GR-P isoforms in a difference was not statisticallysignificant ( P 0.071). group of multiple myeloma patients. (5) The fifth variant described, the GR-A variant, has an exci- Protein interaction with the GR in the cytoplasm sion of exons 5, 6 and 7, resulting in the in frame juxtapo- sition of exon 8 to 4. No further information is known The unstimulated GR is sequestered within the cytoplasm about the expression levels and function of this variant.73 where it can bind into heterocomplexes consisting of heat- shock chaperone molecules hsp90 and 70, co-chaperone Studying the relevance of the alpha, beta and hGR-P splice molecules hsp40, Hop (p60), p23 and immunophillins variants for GC sensitivityin childhood ALL, preliminarydata FKBP52 and CyP40. This heterocomplex is required for opti- from our laboratorydid not show a relation between mRNA mal configuration of the GR to be able to bind GC.87 In a levels of the three splice variants and in vivo or in vitro sensi- recent study, only Hsp 90 and Hsp 70 were found to be tivityto GC. Further work is needed to define the importance required and were sufficient for GC binding to its receptor.88 of the different splice variants in relation to GC sensitivityin A second function of the heat-shock chaperone molecules is ALL and to delineate the mechanisms of GR mRNA splicing the formation of a heterocomplex with GR, which induces a regulation. conformational change in the ligand-binding domain of the

Leukemia Glucocorticoid resistance in ALL WJE Tissing et al 21 receptor, facilitating nuclear transport of the receptor.22 Kojika al105 reported that c-myc down-regulation maynot be critical et al89 reported the adverse effect of abnormal expression of for induction of cell death byGC since transfection of c- myc hsp90 and low expression of hsp70 on sensitivityto GC in 2 into the human T cell leukemia CEM cell line increased the human leukemic cell lines. sensitivityto GC-induced cell death. One of the reasons for Another co-chaperone molecule BAG-1 (also named these conflicting results maybe that increased c- myc RAP46) has been found to bind to manydifferent proteins expression sensitizes the CEM cell line to apoptosis in a simi- including hsp70. This molecule mayinhibit hsp70 function lar wayas shown byEvan et al,106 ie an enhancement of and GR-heterocomplex formation therebyinterfering with GC apoptosis byenforced c- myc expression following growth-fac- binding to the GR.90,91 It is tempting to speculate that over- tor deprivation which was observed in the absence of GC expression of BAG-1 mayresult in cellular resistance to GC, treatment. and indeed, cell line studies have demonstrated that BAG-1 Another important target gene, known to be up-regulated overexpression resulted in cellular resistance against dexame- in a leukemic cell line but down-regulated in cells not thasone-induced apoptosis.91 undergoing apoptosis after GC exposure is the GR gene itself.29,32–34,36,39 The relationship between the potencyto up- regulate the GR number and sensitivityto GC in ALL is cur- Glucocorticoids and transcription factors rentlythe subject of studyin different laboratories. Using gene chip arrays, many genes have been found to be Besides binding to the GRE, the GC–GR complex is thought to regulated under the influence of GC. A notable cluster of the recruit NF-␬B and AP-1 into a transrepression complex. This repressed genes seemed to be of importance for the processes complex abrogates the transcriptional activation of GR, NF-␬B of transcription, mRNA splicing and protein synthesis. It and AP-1 responsive genes (transrepression). Besides physical remains to be shown whether GC trigger one specific apop- interaction and inactivation of NF-␬B bythe GC–GR com- totic pathwayor that a more general suppression of macro- plex,92 NF-␬B is also inactivated bya second mechanism. molecule synthesis is the cause of apoptosis.107 Since I␬B␣ is a GR-responsive gene,93–95 induction of GR leads to higher levels of I␬B␣ which decreases the level of nuclear NF-␬B. Enhanced expression of I␬B␣ maythereby Apoptosis pathway stimulate GC-induced apoptosis.95,96 Contraryto this hypothesis are the results from Heck et al97 who conclude bydimer- Glucocorticoids induce apoptosis in leukemic cells. Both ization-defective GR mutant studies that I␬B␣ synthesis is transactivation of GRE-containing genes and transrepression neither required nor sufficient for the hormone-mediated of genes via NF-␬B and AP-1 interactions are probablyessen- down-regulation of NF-␬B activity.97 The onlystudyusing leu- tial.16,26–28,30 In rat thymocytes it was shown that glucocort- kemic blasts from children with ALL did not find a difference icoid-induced apoptosis depends on protein synthesis and de in NF-␬B level in a group of 42 good and poor in vivo novo gene expression.108 There are two major apoptosis path- responders to GC.98 The second transcription factor inter- ways: the first starting with a disruption of the mitochondrial acting with GR is AP-1, a heterodimer of Fos and Jun proteins. membrane potential and cytochrome c and SMAC release Since AP-1 target genes are involved in cell proliferation, a after which caspase 9 is activated. This pathwayseems to be reduction of functional AP-1 levels maytherefore facilitate important for GC-induced apoptosis.109,110 The second apoptosis. However, Bailey et al99 did not find a relationship apoptosis pathway, induced by membrane death receptors as between levels of AP-1 and in vitro prednisolone resistance reviewed byAshkenzi and Dixit, 111 is not criticallyinvolved in ALL and chronic lymphoid leukemia blasts, nor between in the apoptotic response to GC.29 Brady et al112 showed that prednisolone-induced changes in AP-1 binding activityand in the apoptosis pathwayinduced byGC is p53 independent. vitro GC resistance. An important protein studied in relation to GC-induced cell It is not known whether GR-GRE binding, GR interactions death is the anti-apoptotic protein Bcl-2. Hartmann et al113 with transcription factors or both are of importance for GC- reported that Bcl-2 protects cells against GC-induced induced apoptosis in ALL. Favoring the importance of GR- apoptosis in the human T-ALL cell line (CCRF-CEM) for up to GRE binding are the results from a studybyReichardt et al27 48 h. However, when cultured for another 24 h, these cells An A458T point mutation was introduced in the DNA-binding undergo massive apoptosis. Bcl-2 did not affect GC-mediated region of mouse GR, therebyabolishing GRE binding. T lym- growth arrest, therebyseparating the anti-proliferative effect phocytes of these mutant mice were refractory to dexame- of GC from the apoptosis-inducing.113 This was confirmed in thasone-induced apoptosis, whereas wild-type T lymphocytes other studies, as GC in the presence of high levels of Bcl-2 were highlysensitive to dexamethasone-induced apoptosis. onlyhas an anti-proliferative effect but not an apoptotic effect, while in the presence of low Bcl-2 expression, GC induced apoptosis.114–116 In different studies in children with ALL, Genes regulated by GC expression of Bcl-2 or Bcl-2 familymembers could not be related to in vitro or in vivo GC resistance.117–119 Contraryto Manygenes have been proposed to be regulated byGC. 29,100 the hypothesis that the anti-apoptotic protein Bcl-2 is related GC induce G0/G1 arrest of proliferating cells. This suggests to resistance to GC-induced apoptosis are the results of a that GC reduce the expression of genes related to cell growth studyof 110 patients treated according to the German BFM and cell viability. An important gene is c-myc, a known target protocols. Good responders to initial prednisone therapyhad gene of NF-␬B. Thompson et al concluded in their studies that higher Bcl-2 expression levels than poor responders. The negative regulation of c-myc expression is a significant step authors speculate that this high Bcl-2 expression might be due in the initial pathwayleading to apoptosis in the CEM cell to up-regulation bycytokines,which mayalso be important line.101–103 Non-obese diabetic (NOD) mice with T lympho- for in vivo tumor cell survival.82 Bcl-2 is a member of a larger cytes that are resistant to GC-inducible apoptosis show no Bcl-2 family, consisting of both pro- as well as anti-apoptotic reduced c-myc levels after GC.104 On the contrary, Lo¨ffler et proteins. It could be that not the absolute expression of Bcl-

Leukemia Glucocorticoid resistance in ALL WJE Tissing et al 22 2, but the ratio of all familymembers are the most important drug resistance and prognosis in newlydiagnosed childhood in determining sensitivityto GC treatment in ALL. However acute lymphoblastic leukemia. Blood 1997; 90: 2723–2729. Salomons et al117 did not find a relationship between the 8 Hongo T, Yajima S, Sakurai M, Horikoshi Y, Hanada R. In vitro drug sensitivitytesting can predict induction failure and early Bax:Bcl-2 ratio and the response to 7 days monotherapy of relapse of childhood acute lymphoblastic leukemia. Blood 1997; prednisone in a group of 76 patients. 89: 2959–2965. The final activation of caspases seems to be a downstream 9 Kaspers GJ, Pieters R, Van Zantwijk CH, Van Wering ER, Van effector event during apoptosis, but the trigger for this pathway Der Does-Van Den Berg A, Veerman AJ. Prednisolone resistance remains unknown. Kofler suggests that the continuous in childhood acute lymphoblastic leukemia: vitro–vivo corre- repression of metabolic pathways by GR up-regulation con- lations and cross-resistance to other drugs. Blood 1998; 92: 259–266. tributes to cell cycle arrest, ultimately leading to apoptosis. 10 Pieters R, den Boer ML, Durian M, Janka G, Schmiegelow K, Particularlycritical in this context might be the down-regu- Kaspers GJ, van Wering ER, Veerman AJ. Relation between age, lation of lactate dehydrogenase, an enzyme controlling gly- immunophenotype and in vitro drug resistance in 395 children colysis in cells.29,38 The suggestion that metabolic events have with acute lymphoblastic leukemia – implications for treatment to take place before the induction of apoptosis maybe in of infants. Leukemia 1998; 12: 1344–1348. accordance with our own unpublished in vitro observations 11 Maung ZT, Reid MM, Matheson E, Taylor PR, Proctor SJ, Hall that induction of apoptosis in ALL cells derived from patients AG. Corticosteroid resistance is increased in lymphoblasts from adults compared with children: preliminaryresults of in vitro is a late event starting onlyafter 24 h from the start of exposure drug sensitivitystudyin adults with acute lymphoblasticleu- to GC. kaemia. Br J Haematol 1995; 91: 93–100. 12 Styczynski J, Pieters R, Huismans DR, Schuurhuis GJ, Wysocki M, Veerman AJ. In vitro drug resistance profiles of adult versus childhood acute lymphoblastic leukaemia. Br J Haematol 2000; Perspectives 110: 813–818. 13 Klumper E, Pieters R, Veerman AJ, Huismans DR, Loonen AH, In vivo and in vitro sensitivityto GC are associated with prog- Hahlen K, Kaspers GJ, van Wering ER, Hartmann R, Henze G. In nosis in childhood ALL. This review has summarized the vitro cellular drug resistance in children with relapsed/refractory knowledge on genes and proteins that regulate GC sensitivity. acute lymphoblastic leukemia. Blood 1995; 86: 3861–3868. 14 Theriault A, Boyd E, Harrap SB, Hollenberg SM, Connor JM. Future studies should focus on polymorphisms, splice variants Regional chromosomal assignment of the human glucocorticoid and regulation of the GR, on transcription factors activated or receptor gene to 5q31. Hum Genet 1989; 83: 289–291. repressed byGC and on downstream molecules that control 15 Hollenberg SM, Evans RM. Multiple and cooperative trans-acti- the apoptotic cascade. Based upon knowledge derived from vation domains of the human glucocorticoid receptor. Cell 1988; these studies, strategies to modulate GC resistance in ALL can 55: 899–906. be developed. 16 Tao Y, Williams-Skipp C, Scheinman RI. 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