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Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines

ABSTRACT: Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines for the article: Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases (PFKFBs) in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives PFKFB1, 3, and 4 were further analyzed. Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly in T-ALL cells. The 3 other family members, in contrast, were not or weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (15A and 15B) did not have any detectable effect on survival or cell cycle progression. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC. Generation of the GC sensitive and resistant clones is described in Parson et al. FASEB J 2005 (Pubmed id 15637111). In brief GC sensitive clones were generated by limiting dilution subcloning from the GC sensitive T-ALL cell line CCRF-CEM-C7H2. To generate GC resistant clones the CCRF-CEM-C7H2 cell line was clutured in the presence of 10E-7 M dexametasone.

ORGANISM(S): Homo sapiens

PROVIDER: GSE22152 | GEO | 2011/02/01

SECONDARY ACCESSION(S): PRJNA127713

REPOSITORIES: GEO

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Project description:Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines for the article: Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases (PFKFBs) in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives PFKFB1, 3, and 4 were further analyzed. Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly in T-ALL cells. The 3 other family members, in contrast, were not or weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (15A and 15B) did not have any detectable effect on survival or cell cycle progression. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC. Generation of the GC sensitive and resistant clones is described in Parson et al. FASEB J 2005 (Pubmed id 15637111). In brief GC sensitive clones were generated by limiting dilution subcloning from the GC sensitive T-ALL cell line CCRF-CEM-C7H2. To generate GC resistant clones the CCRF-CEM-C7H2 cell line was clutured in the presence of 10E-7 M dexametasone. Gene expression profiles of glucocorticoid (GC) resistant and sensitive T-ALL cells during GC treatment and corresponding control samples (cells treated with carrier control). GC induced regulation of PFKFB2 was determined in the various cell lines based on the expression intensities of the corresponding probe sets in GC treated and control samples.

Project description:Background: Glucocorticoids are important pharmaceutical agents in the treatment of acute lymphoblastic leukemia in children. Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis. Prednisolone is a first-line chemotherapeutic agent in the treatment of acute lymphoblastic leukemia. Here, the effects of prednisolone on the resistant CCRF-CEM leukemic cell line were studied. Methods: Prednisolone’s cytotoxic and cell cycle effects were studied with flow cytometry. NF-κB translocation was studied with Western Blotting and differential gene expression was studied with cDNA microarrays. Results: Prednisolone exerted a delayed biphasic effect, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone’s mitogenic and cell death effects were counterbalanced. NF-κB was constitutively present in the nucleus. Early gene microarray analysis revealed 40 differentially expressed genes upon 4 hours of prednisolone’s exposure. Notable differences in gene regulation were observed between the lowest and the highest glucocorticoid doses. Prednisolone activated genes related to apoptosis/tumor suppression, cell cycle progression, metabolism and intra-/ extra-cellular signaling pathways. Conclusions: The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance. Elucidation of the mechanisms of GC action may lead to identification of gene targets responsible for GC resistance. Key tools in this process are high-throughput technologies such as microarray-based gene expression analysis. For this purpose, the parental CCRF-CEM cell line was chosen as the system of study for the effects of prednisolone treatment. This is a T-cell leukemia cell line characterized by a mutation (L753F) on one GR gene allele that impairs ligand binding (Thompson and Johnson 2003). It is known that both the DNA and ligand binding domains of the GR are required in order to repress NF-κB transactivation (Wissink, van Heerde et al. 1997). Interestingly, concerning the question whether this mutation would affect GC resistance, it has been reported previously that both the GC-resistant as well as the GC-sensitive CCRF-CEM subclones express heterogeneous populations of the GR (GRwt/GRL753F) (Palmer and Harmon 1991; Powers, Hillmann et al. 1993). The CCRF-CEM cell line has been reported to be resistant to GCs, presumably due to the accumulation of more resistant variants after long periods of prolonged culture (Norman and Thompson 1977). It is possible that these cells are clonally inhomogeneous, as possibly the cells obtained in vivo by patients. Moreover, the large number of the CCRF-CEM subclone studies in the literature makes it difficult to choose an appropriate resistant cell model. In addition, the utilization of an in vitro system for this study offered reproducibility, an opportunity to closely examine intracellular signals and avoid interference from other in vivo-participating systems. Thus, the cell line used for this study was considered to be useful in studying GC action and resistance in leukemic cells. The aim of this work was to determine the cytotoxic, cell cycle phase distribution and early cancer-specific gene expression effects of prednisolone in CCRF-CEM cells, as an in vitro model of ALL resistance to glucocorticoids. The early gene expression profile allowed identification of genes initiating pivotal, early onset regulatory mechanisms activated by GC and excluded ensuing feedback responses and further downstream signals. Samples tested were in the form of a loop-design i.e. control vs. 10nM prednisolone (designated 0vs1) , 10nM prednisolone vs.700uM prednisolone (designated 1vs3) and control vs. 700uM prednisolone (designated 0vs3), the sum of the logarithms of the first two should equal the third. In other words if Rj,i is the ratio of the ith gene in the jth experiment then R0vs1,i+R1vs3,i=R0vs3,i. We tested this using a statistical test. We have applied an intensity-dependent z-score where the sum of the ratios was compared to the ratio of the third experiment. If the difference was significant genes, in a standard deviation threshold of ±1.5 in relative units, were rejected from further analysis (Kerr and Churchill 2001; Kerr and Churchill 2001; Altman and Hua 2006; Kerr and Churchill 2007).

Project description: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-7M dexamethasone and 7.0e-7M 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. Additional microarray data set with RNA from CCRF-CEM-C7H2 cells treated with 1.0e-7M dexamethasone, 4.0 and 8.0 e-6M Solu-dacortin (prednisolone 21-(sodium succinate), a water soluble prednisolone) and 0.1% ethanol.

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Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines

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