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,1
* Tyrolean Cancer Research Institute, Innsbruck, Austria;
Northern Institute for Cancer Research, Newcastle upon Tyne, UK;
Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria;
Division of Human Genetics, Innsbruck Medical University, Innsbruck, Austria;
|| Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innsbruck, Austria
2Correspondence: Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria. E-mail: stefan.schmidt{at}i-med.ac.at
SPECIFIC AIMS
Despite being a therapeutic problem in the treatment of hematological malignancies, the development of resistance to glucocorticoid (GC) in these conditions has not been investigated systematically. We therefore generated a model of GC-resistance development from both a T cell acute lymphoblastic leukemia (ALL) cell line and a B-ALL cell line and identified the level along the GC-signaling pathway at which GC-resistance occurred. Because DNA repair deficiency is a tumorigenesis facilitating factor our model, including one mismatch repair (MMR) proficient and one MMR-deficient cell line provides insight into GC-resistance development under these two essentially different conditions.
PRINCIPAL FINDINGS
1. Two in vitro models for GC-resistance in ALL
To systematically study the development of GC-resistance in ALL, 41 GC-resistant descendents from CCRF-CEM-C7H2 childhood T-ALL cells and 30 GC-resistant subclones from B-cell precursor ALL cell line, PreB697, were generated by limiting dilution in the presence of GC. For comparison, 13 GC-sensitive CEM-C7H2 descendants were generated by limiting dilution in the absence of GC. Due to lack of clonability, no GC-sensitive PreB697 subclones could be derived, thus the parental PreB697 cell line served as control. The apoptotic response to 0.1 µM GC after 72 h was determined by flow cytometric evaluation of the sub G1 fraction following PI staining. Mean rates of apoptotic cells were 74.7% ± 10.8 in sensitive CEM-C7H2 subclones (vehicle controls: 12.2%±5.7) and 67.1% ± 13.2 (1.7%±0.4) for the PreB parental line compared with 6.2% ± 5.4 (6.8%±5.8) and 16.0% ± 13.7 (4.3%±3.0) in the GC- resistant subclones of the CEM-C7H2 and PreB697, respectively.
2. Mismatch repair status
Previous studies have shown that CCRF-CEM cells are MMR-deficient due to a splice site mutation in the MLH1 gene leading to a loss of MLH1 expression. In an in vitro functional assay the PreB697 parental cell line proved to be fully MMR-proficient, with PreB697 cells showing 83% of repair compared with 7% for CCRF-CEM. In confirmation of this, PreB697 cells were shown to express a full complement of MMR proteins including, MLH1, MSH2, MSH6, MSH3, and PMS2 as judged by Western blotting, contrasting with the absent MLH1 and its heterodimeric partner, PMS2, in the CCRF-CEM parent line. Thus, our models enable assessment of GC-resistance mechanisms in ALL in a MMR-proficient and -deficient background.
3. GC-resistance is associated with reduced induction of GR and its downstream target GILZ
Investigating the molecular mechanism underlying GC-resistance, we determined the expression level of the glucocorticoid receptor (GR) as the most apical component in the GC-induced apoptosis pathway. Expression levels of the GR, its downstream target GILZ and TBP were measured by real-time polymerase chain reaction (PCR) under basal conditions and after 6 and 24 h exposure to 0.1 µM dexamethasone or vehicle control. While basal levels of GR and GILZ expression were similar in GC-resistant and GC-sensitive cell lines, expression of both was markedly increased in GC-sensitive cell lines after 6 h of GC exposure (Fig. 1
A). The increase in GC-sensitive and the absent or marginal increase in resistant ALL cells were further confirmed at the protein level by Western blotting (Fig. 1B
). Thus, in both models deficiency in GR up-regulation on GC-exposure is a hallmark of GC-resistance.
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4. Transgenic GR restores GC sensitivity
To test whether the downstream components of the GC-induced cell death pathway are intact, we transduced CEM-C7H2 subclones R11G5 and R5C3 with a retroviral construct expressing human wild-type (WT) GR
. As shown by Western blotting in Figure 2
, transgenic GR was expressed at levels resembling those seen after 24 h GC treatment in the GC-sensitive CEM-C7H2 cell line and restored GC sensitivity, i.e., the transfected cells underwent typical GC-induced cell death. These data strongly suggest that the downstream components of the GC-induced death pathway are intact in these cells and that apoptosis resistance occurs at the level of the GR.
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5. GC-resistant CCRF-CEM, but not preB697, show high frequency of GR mutations
To address whether deficiency of GR autoinduction might be caused by GR mutations, we screened GR exons encoding the DNA-binding and ligand-binding domains of all subclones for mutations by denaturing high-pressure liquid chromatography (DHPLC) and consecutively sequenced the detected mutations. A total of 3 from 30 resistant PreB697clones had acquired a GR mutation, i.e., clones R4B11 and R3G3 having the same IVSC-2 A > G splice site mutation and R3E3 with an insertion of C in exon 9 (2447 insC) leading to a frameshift. A hetereoduplex pattern in chromatograms of GR exon 5 in all PreB697 subclones (except clones R3C11 and R4C3) was due to the presence of a common intronic polymorphism, IVSD-16 G 
T. Loss of this heteroduplex pattern in clones R3C11 and R4C3 suggested LOH at the GR locus.
All sublines of the CEM-C7H2 bear the parental L753F mutation in exon 9, however, no additional mutations were found in the 11 GC-sensitive subclones screened. In contrast, 11 mutations were detected in 10 of 37 GC-resistant clones. They included 2 nonsense mutations (G506X, Q713X), 6 missense mutations (A484D, P515H L756N, Y663H, L680P, R714W), 2 frameshift mutations, and 1 donor splice site mutation preceding exon 5.
Thus, GC-resistance due to GR mutations occurs in MMR-proficient and -deficient leukemic cell lines but is more common in the latter. However, surprisingly the majority of GC-resistant subclones had no detectable GR gene mutation and almost all of them had a remaining WT allele raising the question as to how the deficient GR autoinduction might be molecularly explained.
6. GC-resistant CCRF-CEM, but not preB697 show microsatellite instability and a high frequency of GR allele loss
Both GC-sensitive as well as GC-resistant CEM-C7H2 subclones show microsatellite instability in fingerprint profiling performed for 16 DNA loci, however, for marker D5S818, located 
20 Mbp centromeric to the GR locus, 37 of 41 resistant subclones showed a characteristically altered pattern compared with the parental CEM-C7H2, indicating 50% reduction of one of the two alleles. The fact that this pattern was found only in 2 of 13 GC-sensitive subclones, suggested that one of the GR alleles was selectively lost during culture in the presence of dexamethasone. In contrast, the MMR proficient PreB697 cells showed microsatellite stability with all subclones displaying the parental allele patterns at all DNA fingerprint loci. PreB697 cells showed two alleles for marker D5S818 and only subclone R3C11 revealed a signal reduction for one allele being indicative for LOH.
This interpretation was subsequently reconfirmed in a selected subset of cells using fluorescence in situ hybridization (FISH).
CONCLUSIONS AND SIGNIFICANCE
GR resistance acts at the level of the GR
Despite the numerous possibilities of GC-resistance development along the GC-induced apoptosis pathway, in the two models we used resistance occurred exclusively at the level of the GR. With regard to the GR locus we found three different allele compositions in the resistant subclones that resulted from mutations and/or allele loss, i.e., a full set of two WT alleles, loss of one with one remaining WT allele, and no functional GR allele. The higher rate of GR mutations found in the GC-resistant descendants of the CCRF-CEM-C7H2 compared with those of the PreB697 can be attributed to its MMR deficiency; however, allele loss affecting almost all resistant CCRF-CEM-C7H2-subclones is typically not associated with MMR deficiency. The majority of resistant CCRF-CEM-C7H2 lines (26/37) were left with a single functional GR allele. Although being an attractive hypothesis, haploinsufficieny on its own can hardly explain the deficient GR up-regulation, since we observed GR up-regulation in a GC-sensitive CCRF-CEM-C7H2 subclone showing evidence for GR allele loss. Hence, haploinsufficiency might be associated with another mechanism, similar to the one that accounts for GR resistance in 25/30 GC-resistant PreB697 subclones which have a full set of functional GR alleles (see schematic).
GR mutations
Altogether this study has identified 13 novel GC-resistance associated mutations of the GR located predominantly in the ligand binding domain. The P515H mutation is the first one associated with GC-resistance and to be found in the hinge region. Fitting with the MMR state of the model systems used, the GR frameshift mutations in the resistant CEM-C7H2 clones were located in coding mononucleotide repeats, while the one found in a PreB697 subclones was not.
Possible relevance for the in vivo situation
Only very few somatic GR mutations have been detected in clinical samples, i.e., the L753F mutation and a 29 bp deletion (
702). Screening 50 samples from relapsed ALL patients treated with a GC-containing regimen revealed only one mutation and, similarly, in a screen of diagnostic samples assessed for GC sensitivity no mutation at all was found. Thus, acquired somatic GR mutations are not a predominant mechanism of GC-resistance in clinical samples.
Whether deficient GR autoinduction plays a role for GC resistance in patients is currently unknown. Two studies, one using a primary childhood ALL-NOD/SCID xenograft model, the other primary childhood ALL cells treated with GC in vitro, argued against this possibility. In the first, all four xenografts (two GC-sensitive and two GC-resistant ones) showed GR down-regulation, in the second, all 12 GC-sensitive and 10 GC-resistant samples revealed GR up-regulation. Given these contradictory results, it remains unresolved how well these models reflect the situation in the patient. In a recent expression profiling study with 13 childhood-ALL patients undergoing GC monotherapy, we found GR autoinduction in 3 of 3 T-ALL and 2 of 10 B-ALL samples. Though showing evidence for GR up-regulation in GC-sensitive patients, no conclusions regarding GC resistance could be derived because no GC-resistant patients were included.
In summary, the present study shows that mutations in the GR gene and, perhaps more importantly, deficiency in GR autoinduction appear to account for GC resistance in two independent ALL models. While mutations in patients are rare, GR autoinduction occurs more frequently, however, whether defects in GR autoinduction might cause GC resistance will require studies on samples from GC-sensitive and -resistant patients treated in vivo.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6214fje
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