Elucidating the identity of resistance mechanisms to prednisolone exposure in acute lymphoblastic leukemia cells through transcriptomic analysis: A computational approach

BackgroundIt has been shown previously that glucocorticoids exert a dual mechanism of action, entailing cytotoxic, mitogenic as well as cell proliferative and anti-apoptotic responses, in a dose-dependent manner on CCRF-CEM cells at 72 h. Early gene expression response implies a dose-dependent dual mechanism of action of prednisolone too, something reflected on cell state upon 72 h of treatment.MethodsIn this work, a generic, computational microarray data analysis framework is proposed, in order to examine the hypothesis, whether CCRF-CEM cells exhibit an intrinsic or acquired mechanism of resistance and investigate the molecular imprint of this, upon prednisolone treatment. The experimental design enables the examination of both the dose (0 nM, 10 nM, 22 uM, 700 uM) effect of glucocorticoid exposure and the dynamics (early and late, namely 4 h, 72 h) of the molecular response of the cells at the transcriptomic layer.ResultsIn this work, we demonstrated that CCRF-CEM cells may attain a mixed mechanism of response to glucocorticoids, however, with a clear preference towards an intrinsic mechanism of resistance. Specifically, at 4 h, prednisolone appeared to down-regulate apoptotic genes. Also, low and high prednisolone concentrations up-regulates genes related to metabolism and signal-transduction in both time points, thus favoring cell proliferative actions. In addition, regulation of NF-κB-related genes implies an inherent mechanism of resistance through the established link of NF-κB inflammatory role and GC-induced resistance. The analysis framework applied here highlights prednisolone-activated regulatory mechanisms through identification of early responding sets of genes. On the other hand, study of the prolonged exposure to glucocorticoids (72 h exposure) highlights the effect of homeostatic feedback mechanisms of the treated cells.ConclusionsOverall, it appears that CCRF-CEM cells in this study exhibit a diversified, combined pattern of intrinsic and acquired resistance to prednisolone, with a tendency towards inherent resistant characteristics, through activation of different molecular courses of action.

[1]  W. Tissing,et al.  Genetic Variations in the Glucocorticoid Receptor Gene Are Not Related to Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia , 2005, Clinical Cancer Research.

[2]  R. Kofler,et al.  The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels. , 2001, The Journal of biological chemistry.

[3]  Martin Vingron,et al.  Ontologizer 2.0 - a multifunctional tool for GO term enrichment analysis and data exploration , 2008, Bioinform..

[4]  J. Harmon,et al.  Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells. , 1993, Cancer research.

[5]  Aristotelis A. Chatziioannou,et al.  Evaluating the effect of various background correction methods regarding noise reduction, in two-channel microarray data , 2012, Comput. Biol. Medicine.

[6]  R. Craig.  MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis , 2002, Leukemia.

[7]  Fotini Tzortzatou-Stathopoulou,et al.  Prednisolone exerts late mitogenic and biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene expression. , 2009, Leukemia research.

[8]  Betty H. Johnson,et al.  Regulation of a distinctive set of genes in glucocorticoid-evoked apoptosis in CEM human lymphoid cells. , 2003, Recent progress in hormone research.

[9]  S. Armstrong,et al.  Genomewide identification of prednisolone-responsive genes in acute lymphoblastic leukemia cells. , 2005, Blood.

[10]  E. Thompson,et al.  Characterization of a glucocorticoid-sensitive human lymphoid cell line. , 1977, Cancer research.

[11]  P. Adamson,et al.  Pediatric phase I trial and pharmacokinetic study of dasatinib: a report from the children's oncology group phase I consortium. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[12]  K. Huebner,et al.  Assignment of DAP1 and DAPK--genes that positively mediate programmed cell death triggered by IFN-gamma--to chromosome regions 5p12.2 and 9q34.1, respectively. , 1995, Genomics.

[13]  N. Mulherkar,et al.  MADD, a Splice Variant of IG20, Is Indispensable for MAPK Activation and Protection against Apoptosis upon Tumor Necrosis Factor-α Treatment* , 2009, Journal of Biological Chemistry.

[14]  A. Kimchi,et al.  Death associated proteins (DAPs): from gene identification to the analysis of their apoptotic and tumor suppressive functions , 1998, Oncogene.

[15]  R. Kofler,et al.  MECHANISMS OF STEROID ACTION AND RESISTANCE IN INFLAMMATION Resistance to glucocorticoid-induced apoptosis in lymphoblastic leukemia , 2003 .

[16]  M. Vihinen,et al.  Overexpression of translocation-associated fusion genes of FGFR 1 , MYC , NPM 1 , and DEK , but absence of the translocations in acute myeloid leukemia . A microarray analysis , 2002 .

[17]  G. Churchill,et al.  Experimental design for gene expression microarrays. , 2001, Biostatistics.

[18]  R. Kofler,et al.  p16INK4A Sensitizes Human Leukemia Cells to FAS- and Glucocorticoid-induced Apoptosis via Induction of BBC3/Puma and Repression of MCL1 and BCL2* , 2009, The Journal of Biological Chemistry.

[19]  Douglas G Altman,et al.  Key Issues in Conducting a Meta-Analysis of Gene Expression Microarray Datasets , 2008, PLoS medicine.

[20]  J. Cerhan,et al.  Germline Variation in Apoptosis Pathway Genes and Risk of Non–Hodgkin's Lymphoma , 2010, Cancer Epidemiology, Biomarkers & Prevention.

[21]  B. Aggarwal,et al.  Evodiamine Abolishes Constitutive and Inducible NF-κB Activation by Inhibiting IκBα Kinase Activation, Thereby Suppressing NF-κB-regulated Antiapoptotic and Metastatic Gene Expression, Up-regulating Apoptosis, and Inhibiting Invasion* , 2005, Journal of Biological Chemistry.

[22]  W. Cleveland,et al.  Locally Weighted Regression: An Approach to Regression Analysis by Local Fitting , 1988 .

[23]  Gregory D. Schuler,et al.  Database resources of the National Center for Biotechnology Information: update , 2004, Nucleic acids research.

[24]  David L. Wheeler,et al.  GenBank , 2015, Nucleic Acids Res..

[25]  Minoru Kanehisa,et al.  The KEGG database. , 2002, Novartis Foundation symposium.

[26]  R. Kofler,et al.  The Cell Cycle Inhibitor p16INK4A Sensitizes Lymphoblastic Leukemia Cells to Apoptosis by Physiologic Glucocorticoid Levels* , 2001, The Journal of Biological Chemistry.

[27]  D. Coppola,et al.  IKKϵ Phosphorylation of Estrogen Receptor α Ser-167 and Contribution to Tamoxifen Resistance in Breast Cancer* , 2009, The Journal of Biological Chemistry.

[28]  Cheng Cheng,et al.  The expression of 70 apoptosis genes in relation to lineage, genetic subtype, cellular drug resistance, and outcome in childhood acute lymphoblastic leukemia. , 2006, Blood.

[29]  J. Qian,et al.  Aberrant methylation of the death‐associated protein kinase 1 (DAPK1) CpG island in chronic myeloid leukemia , 2009, European journal of haematology.

[30]  A. Stevens,et al.  Glucocorticoid receptor-mediated apoptosis: mechanisms of resistance in cancer cells. , 2011, The Journal of endocrinology.

[31]  Chih-yuan Chiang,et al.  A Human MAP Kinase Interactome , 2010, Nature Methods.

[32]  S. Dudoit,et al.  STATISTICAL METHODS FOR IDENTIFYING DIFFERENTIALLY EXPRESSED GENES IN REPLICATED cDNA MICROARRAY EXPERIMENTS , 2002 .

[33]  Min Zhang,et al.  Multiplicative background correction for spotted microarrays to improve reproducibility. , 2006, Genetical research.

[34]  T. Hiragun,et al.  Dexamethasone Suppresses Antigen-Induced Activation of Phosphatidylinositol 3-Kinase and Downstream Responses in Mast Cells , 2004, The Journal of Immunology.

[35]  N. Hay,et al.  Akt-phosphorylated Mitogen-activated Kinase-activating Death Domain Protein (MADD) Inhibits TRAIL-induced Apoptosis by Blocking Fas-associated Death Domain (FADD) Association with Death Receptor 4* , 2010, The Journal of Biological Chemistry.

[36]  Ronit Vogt Sionov,et al.  Mechanisms regulating the susceptibility of hematopoietic malignancies to glucocorticoid-induced apoptosis. , 2008, Advances in cancer research.

[37]  R. Crazzolara,et al.  Identification of glucocorticoid-response genes in children with acute lymphoblastic leukemia. , 2006, Blood.

[38]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[39]  H. L. Le Roy,et al.  Proceedings of the Fifth Berkeley Symposium on Mathematical Statistics and Probability; Vol. IV , 1969 .

[40]  Peter J. Rousseeuw,et al.  Robust Regression and Outlier Detection , 2005, Wiley Series in Probability and Statistics.

[41]  Weihong Yan,et al.  Expression-based monitoring of transcription factor activity: the TELiS database , 2005, Bioinform..

[42]  Gregory D. Schuler,et al.  Database resources of the National Center for Biotechnology , 2003, Nucleic Acids Res..

[43]  David Tritchler,et al.  BMC Bioinformatics BioMed Central Methodology article Filtering Genes for Cluster and Network Analysis , 2009 .

[44]  Tongbin Li,et al.  Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis. , 2003, Genomics.

[45]  Roland Eils,et al.  Cross-platform analysis of cancer microarray data improves gene expression based classification of phenotypes , 2005, BMC Bioinformatics.

[46]  Holger Karas,et al.  TRANSFAC: a database on transcription factors and their DNA binding sites , 1996, Nucleic Acids Res..

[47]  Thompson Eb,et al.  Regulation of a distinctive set of genes in glucocorticoid-evoked apoptosis in CEM human lymphoid cells. , 2003 .

[48]  B. Zhivotovsky,et al.  Dexamethasone-induced apoptosis in acute lymphoblastic leukemia involves differential regulation of Bcl-2 family members , 2007, Haematologica.

[49]  Antonio Gnoni,et al.  Dasatinib: an anti-tumour agent via Src inhibition. , 2011, Current drug targets.

[50]  E. Forgy,et al.  Cluster analysis of multivariate data : efficiency versus interpretability of classifications , 1965 .

[51]  Ali S. Hadi,et al.  Finding Groups in Data: An Introduction to Chster Analysis , 1991 .

[52]  Jun Hua,et al.  Extending the loop design for two-channel microarray experiments. , 2006, Genetical research.

[53]  David L. Wheeler,et al.  GenBank: update , 2004, Nucleic Acids Res..

[54]  M. Schrappe,et al.  Initial leukemic gene expression profiles of patients with poor in vivo prednisone response are similar to those of blasts persisting under prednisone treatment in childhood acute lymphoblastic leukemia , 2008, Annals of Hematology.

[55]  M. D. Boer,et al.  Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[56]  Purvesh Khatri,et al.  Ontological analysis of gene expression data: current tools, limitations, and open problems , 2005, Bioinform..

[57]  Wendy R. Fox,et al.  Finding Groups in Data: An Introduction to Cluster Analysis , 1991 .

[58]  Rainer Spang,et al.  Assessment of five microarray experiments on gene expression profiling of breast cancer , 2003 .

[59]  G. Churchill,et al.  Statistical design and the analysis of gene expression microarray data. , 2007, Genetical research.

[60]  Andrew B. Nobel,et al.  Merging two gene-expression studies via cross-platform normalization , 2008, Bioinform..

[61]  David Botstein,et al.  SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data , 2003, Nucleic Acids Res..

[62]  S. P. Lloyd,et al.  Least squares quantization in PCM , 1982, IEEE Trans. Inf. Theory.

[63]  W. Cleveland.  Robust Locally Weighted Regression and Smoothing Scatterplots , 1979 .

[64]  Roslin Russell,et al.  Microarray Technology in Practice , 2008 .

[65]  P. Bork,et al.  G2D: a tool for mining genes associated with disease , 2005, BMC Genetics.

[66]  John Quackenbush.  Microarray data normalization and transformation , 2002, Nature Genetics.

[67]  B. Aggarwal,et al.  Evodiamine abolishes constitutive and inducible NF-kappaB activation by inhibiting IkappaBalpha kinase activation, thereby suppressing NF-kappaB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion. , 2005, The Journal of biological chemistry.

[68]  Gregory D. Schuler,et al.  Database resources of the National Center for Biotechnology Information , 2021, Nucleic Acids Res..

[69]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[70]  H. Varmus,et al.  MYC-induced myeloid leukemogenesis is accelerated by all six members of the antiapoptotic BCL family , 2009, Oncogene.

[71]  Patrik Rydén,et al.  Challenges in microarray class discovery: a comprehensive examination of normalization, gene selection and clustering , 2010, BMC Bioinformatics.

[72]  Robert V. Brill,et al.  Applied Statistics and Probability for Engineers , 2004, Technometrics.

[73]  H. Russcher,et al.  Two polymorphisms in the glucocorticoid receptor gene directly affect glucocorticoid-regulated gene expression. , 2005, The Journal of clinical endocrinology and metabolism.

[74]  E. Thompson,et al.  Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells , 2001, Oncogene.

[75]  Peter J. Rousseeuw,et al.  Robust regression and outlier detection , 1987 .

[76]  Yanying Zhao,et al.  Survivin gene silencing sensitizes prostate cancer cells to selenium growth inhibition , 2010, BMC Cancer.

[77]  E. Kalkhoven,et al.  Distinct Domains of the RelA NF-κB Subunit Are Required for Negative Cross-talk and Direct Interaction with the Glucocorticoid Receptor* , 1997, The Journal of Biological Chemistry.

[78]  Martin Vingron,et al.  Improved detection of overrepresentation of Gene-Ontology annotations with parent-child analysis , 2007, Bioinform..

[79]  Hubert Hackl,et al.  PathwayExplorer: web service for visualizing high-throughput expression data on biological pathways , 2005, Nucleic Acids Res..

[80]  D. Weisenburger,et al.  Downregulation of Death-Associated Protein Kinase 1 (DAPK1) in Chronic Lymphocytic Leukemia , 2007, Cell.

[81]  Bing Zhang,et al.  GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies , 2004, BMC Bioinformatics.

[82]  Chuanfeng Wu,et al.  C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice , 2011, Proceedings of the National Academy of Sciences.

[83]  Gregory D Schuler,et al.  Sequence mapping by electronic PCR , 1997, Genome research.

[84]  Francis D. Gibbons,et al.  Judging the quality of gene expression-based clustering methods using gene annotation. , 2002, Genome research.

[85]  M. Schrappe,et al.  Clinical Outcome of Patients with Childhood Acute Lymphoblastic Leukaemia and an Initial Leukaemic Blood Blast Count of Less than 1000 per Microliter , 2001, Klinische Padiatrie.

[86]  G. Church,et al.  A computational analysis of whole-genome expression data reveals chromosomal domains of gene expression , 2000, Nature Genetics.

[87]  Ronit Vogt Sionov,et al.  Protein kinase networks regulating glucocorticoid-induced apoptosis of hematopoietic cancer cells: fundamental aspects and practical considerations , 2010, Leukemia & lymphoma.

[88]  Jerry Li,et al.  Within the fold: assessing differential expression measures and reproducibility in microarray assays , 2002, Genome Biology.

[89]  H. Stunnenberg,et al.  Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes. , 2011, Genome research.

[90]  George I. Lambrou,et al.  cDNA microarray analysis of a glucocorticoid treated acute lymphoblastic leukemia cell line , 2008, 2008 8th IEEE International Conference on BioInformatics and BioEngineering.

[91]  A. Kimchi,et al.  DAP1, a Novel Substrate of mTOR, Negatively Regulates Autophagy , 2010, Current Biology.

[92]  Michalis Vazirgiannis,et al.  c ○ 2001 Kluwer Academic Publishers. Manufactured in The Netherlands. On Clustering Validation Techniques , 2022 .

[93]  J. Harmon,et al.  Biochemical evidence that glucocorticoid-sensitive cell lines derived from the human leukemic cell line CCRF-CEM express a normal and a mutant glucocorticoid receptor gene. , 1991, Cancer research.