The synthetic glucocorticoids prednisolone and dexamethasone regulate the same genes in acute lymphoblastic leukemia cells.

BACKGROUND: Glucocorticoids (GCs) cause apoptosis in malignant cells of lymphoid lineage by transcriptionally regulating a plethora of genes. As a result, GCs are included in almost all treatment protocols for lymphoid malignancies, particularly childhood acute lymphoblastic leukemia (chALL). The most commonly used synthetic GCs in the clinical setting are prednisolone and dexamethasone. While the latter has a higher activity and more effectively reduces the tumor load in patients, it is also accompanied by more serious adverse effects than the former. Whether this difference might be explained by regulation of different genes by the two GCs has never been addressed. RESULTS: Using a recently developed GC bioassay based on a GC-responsive reporter construct in human Jurkat T-ALL cells, we found ~7-fold higher biological activity with dexamethasone than prednisolone. Similarly, 1.0e-7 M dexamethasone and 7.0e-7 M prednisolone triggered similar cell death rates in CCRF-CEM-C7H2 T-chALL cells after 72 hours of treatment. Using microarray-based whole genome expression profiling and a variety of statistical and other approaches, we compared the transcriptional response of chALL cells to 6 hour exposure to both synthetic GCs at the above concentrations. Our experiments did not detect any gene whose regulation by dexamethasone differed significantly from that by prednisolone. CONCLUSIONS: Our findings suggest that the reported differences in treatment efficacy and cytotoxicity of dexamethasone and prednisolone are not caused by inherent differences of the 2 drugs to regulate the expression of certain genes, but rather result either from applying them in biologically in-equivalent concentrations and/or from differences in their pharmacokinetics and - dynamics resulting in different bioactivities in tumor cells and normal tissues.

"Bam," a novel glucocorticoid-induced BH3-only transcript from the BCL2L11/Bim locus, does not appear to be translated.

Glucocorticoids (GCs) are steroid hormones that induce cell death and cell cycle arrest in lymphoid tissues. By virtue of this property, GCs are widely exploited in the therapy of acute lymphoblastic leukemia (ALL) in children. We reported a novel BH3-only transcript, "Bam," from the BCL2L11 locus, which was first described in patients with multiple myeloma. The Bam gene consists of two exons, and became of particular interest to us when we found that it was regulated in the majority of children with ALL and many in vitro systems in which GCs induce cell death. Being a BH3-only transcript, Bam retains a BH3 domain identical to that of Bim, although Bam has a unique C-terminus that is totally different from that of its relative Bim. The present work analyzes whether Bam is translated or not. Since we could not detect Bam in the endogenous situation, we evaluated its 5' untranslated region (UTR). This revealed that there are three out-of-frame initiation codons preceding the Bam open reading frame (ORF). Experiments with constructs without out-of-frame initiation codons and constructs harboring such codons in their 5' UTR revealed that Bam translation is handicapped by their presence. Moreover, there was no Kozak translational initiation sequence surrounding any of the AUGs. Taken together, results of the present study strongly suggest that this transcript is translated at a very low rate, if at all.

Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes.

Our laboratory has developed a series of Gateway(®) compatible lentiviral expression systems for constitutive and conditional gene knock-down and over-expression. For tetracycline-regulated transgenic expression, we constructed a lentiviral "DEST" plasmid (pHR-TetCMV-Dest-IRES-GFP5) containing a tetracycline-responsive minimal CMV promoter, followed by an attP site-flanked DEST cassette (for efficient cloning of cDNAs by "Gateway(®)" recombination cloning) and green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES).This lentiviral bicistronic plasmid allows immediate FACS identification and characterization of successfully transfected cell lines. Although this system worked well with several cDNAs, we experienced serious problems with SLA, Bam and BMF. Particularly, we cloned the cDNA for human SLA (Src-like adapter), a candidate gene in GC-induced apoptosis, into this plasmid. The resulting construct (pHR-TetCMV-SLA-IRES-GFP5) was transfected into HEK 293-T packaging cells to produce viral particles for transduction of CEM-C7H2-2C8 cells. Although the construct produced many green fluorescent colonies at the HEK 293-T and the CEM-C7H2-2C8 level, we could not detect any SLA protein with α-SLA antibody from corresponding cell lysates. In contrast, the antibody readily detected SLA in whole cell lysate of HEK 293-T cells transfected with a GST-flagged SLA construct lacking IRES-GFP. To directly address the potential role of the IRES-GFP sequence, we cloned the SLA coding region into pHR-TetCMV-Dest, a vector that differs from pHR-TetCMV-Dest-IRES-GFP5 just by the absence of the IRES-GFP cassette. The resulting pHR-TetCMV-SLA construct was used for transfection of HEK 293-T cells. Corresponding lysates were assayed with α-SLA antibody and found positive. These data, in concert with previous findings, suggest that the IRES-GFP cassette may interfere with translation of certain smaller size cDNAs (like SLA) or generate fusion proteins and entail defective virus production in an unpredictable manner.

Promyelocytic leukemia zinc finger protein (PLZF) enhances glucocorticoid-induced apoptosis in leukemic cell line NALM6.

Glucocorticoids (GC) actuate apoptosis as well as cell cycle arrest in lymphocytes, and included as core element in the lymphoid malignancy treatment. Despite clinical significance of GC and considerable efforts to understand it, the molecular basis of GC regulated cell death and the resistance phenomenon remains, however, poorly understood. Using Affymetrix-based whole genome expression profiling our group has previously identified a number of prominent glucocorticoid-response genes (Blood 107: 2061, 2006). Promyelocytic leukemia zinc finger (PLZF) was one of the best candidate genes. This study was proposed to investigate the possible role of PLZF in GC regulated cell death in leukemic model cell line NALM6. To this end, we generated NALM6 cell line (bulk) transduced with a retroviral expression vectors, pHR-SFFV-PLZF-IRES-Puro (U426) and pHR-SFFV-Venus-IRES-Puro (U417), as control, for constitutive gene-expression. HEK293T cells were transfected transiently to generate viral particles. These cell lines were characterized by Western blotting and used to assay the effect of constitutive PLZF expression. In conclusion, we report that bona fide transcription repressor PLZF, which turned out as prominent GC-regulated gene both in vivo and in vitro situations was found to enhance the GC-induced cell death (basal) in leukemic model cell line NALM6 after 48 and 72h time points.

Expression and glucocorticoid-regulation of "Bam", a novel BH3-only transcript in acute lymphoblastic leukemia.

Apoptosis is a morphologically defined form of cell death that plays a major role in cell physiology, pathology and cancer therapy. The Bcl-2 family of pro- and anti-apoptotic molecules is a key regulator of this phenomenon, with the sub-family of BH3-only molecules serving as activators and/or facilitators. Apoptosis induced by glucocorticoids (GC) is a central component in the therapy of acute lymphoblastic leukemia (ALL), and defining its molecular basis and that of GC resistance is crucial for therapeutic improvements. We recently identified a novel transcript from the BCL2L11/Bim locus, termed "Bam", by affymetrix based whole genome expression profiling performed on 27 children (13 were already published) with ALL and many additional biological systems. Most children that induced Bam also induced Bim as well, in some cases Bam induction was more pronounced than that of Bim and in one patient only the Bam, but not Bim was induced. In C7H2, PreB697, Jurkat-GR(wt) and GC-sensitive (S-lines), this transcript was induced by GC in a translation-independent manner, suggesting that direct transcriptional induction of this BH3-only molecule by GC might cause apoptosis in at least ALL children and other biological systems.

Establishing a sensitive and specific assay for determination of glucocorticoid bioactivity.

Glucocorticoids are hormones that play a major role in energy homeostasis and stress response of the body. As drugs they are most frequently used for immunosuppressive and anti-inflammatory purposes. Glucocorticoids are exploited successfully in the treatment of a wide variety of diseases; however, some patients develop side-effects, while others fail to respond to this form of therapy. Alterations in pharmacodynamic and pharmacokinetic actions might contribute to individual differences in glucocorticoid sensitivity. Antibody-based methods such as RIA (Radioimmunoassay) and ELISA (Enzyme-linked immunosorbent assay) are routinely used to determine glucocorticoid serum levels. However, as these techniques measure the total amount of a specific glucocorticoid and do not discriminate between protein-bound and freely available (i.e. biologically active) glucocorticoids, the results do not necessarily reflect the active levels of glucocorticoid, i.e. the "glucocorticoid milieu" in a patient. Being able to determine glucocorticoid bioactivity in serum or other body fluids could help identifying glucocorticoid-sensitive or -resistant patients and help finding explanations for different responses in individual patients. For this reason, we established a glucocorticoid bioactivity assay that is based on the measurement of glucocorticoid-dependent reporter gene activity. Making use of a human T-cell leukemia line, equipped with the glucocorticoid receptor and the fluorescence protein Venus as the assay's reporter (Jurkat(GR)-MMTV-VNP), glucocorticoid bioactivity can be determined from small amounts of serum or other biologic fluids. The developed glucocorticoid bioassay is both sensitive and reproducible, without any relevant cross-reactivity with steroid hormones other than glucocorticoids and can be practically applied in daily laboratory routine.

Glucocorticoid resistance in two key models of acute lymphoblastic leukemia occurs at the level of the glucocorticoid receptor.

Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.

Cancer cell line identification by short tandem repeat profiling: power and limitations.

Cancer cell lines are used worldwide in biological research, and data interpretation depends on unambiguous attribution of the respective cell line to its original source. Short-tandem-repeat (STR) profiling (DNA fingerprinting) is the method of choice for this purpose; however, the genetic stability of cell lines under various experimental conditions is not well defined. We tested the effect of long-term culture, subcloning, and generation of drug-resistant subclones on fingerprinting profiles in four widely used leukemia cell lines. The DNA fingerprinting profile remained unaltered in two of them (U937 and K562) throughout 12 months in culture, and the vast majority of subclones derived therefrom by limiting dilution after long-term culture revealed the same profile, indicating a high degree of stability and clonotypic homogeneity. In contrast, two other cell lines (CCRF-CEM and Jurkat) showed marked alterations in DNA fingerprinting profiles during long-term culture. Limiting dilution subcloning revealed extensive clonotypic heterogeneity with subclones differing in up to eight STR loci from the parental culture. Similar heterogeneity was observed in subclones generated by selection culture for drug resistance where DNA fingerprinting proved useful in identifying possible resistance mechanisms. Thus, common tissue culture procedures may dramatically affect the fingerprinting profile of certain cell lines and thus render definition of their origin difficult.