Molecular mechanisms conferring resistance/sensitivity to glucocorticoid-induced apoptosis during cytotoxic stress

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Molecular mechanisms conferring resistance/sensitivity to glucocorticoid-induced apoptosis during cytotoxic stress

James Lynch

PhD

2009-10

University of Manchester, School of Pharmacy and Pharmaceutical Sciences

202

Chapter 1: Introduction 161.1 Gene transcription 161.1.1 Pre-initiation complex 161.1.2 Chromatin structure 181.1.3 Activators and repressors of transcription 201.2 Apoptosis 221.2.1 Extrinsic pathway of apoptosis 241.2.2 Intrinsic pathway of apoptosis 241.2.3 Bcl-2 family of proteins 251.2.3.1 Pro-apoptotic Bcl-2 family members 261.2.3.2 Anti-apoptotic Bcl-2 family members 271.2.3.3 NOXA 271.2.3.4 Mcl-1 291.2.3.5 Bcl-2 family members and cancer 301.3 Glucocorticoids 331.3.1 HPA axis 331.3.2 Glucocorticoids and disease 331.3.3 Superfamily of nuclear receptors 361.3.4 Structure of GR 361.3.4.1 AF-1/AF-2 381.3.4.2 DNA binding domain 381.3.4.3 Ligand binding domain 391.3.5 Mechanism of action 401.3.6 Post-translational modifications of GR 431.3.6.1 Phosphorylation 441.3.6.2 SUMOylation 441.3.7 Co-regulators 461.3.7.1 p160 family 461.3.7.2 p300 461.3.7.3 P/CAF 471.3.7.4 Co-repressors 471.3.8 Glucocorticoid-induced apoptosis 481.4 The MAPK family 511.4.1 JNK 531.4.2 Identification and structure of JNK 531.4.3 Mechanism of action 531.4.4 JNK and GR 541.5 Hypoxia 551.5.1 Identification and structure of HIF-1α 551.5.2 Mechanism of action 571.5.3 HIF-1α PTMs and transactivation 591.5.4 Hypoxia and cancer 591.5.4.1 Hypoxia and leukaemia 611.5.5 Regulation of apoptosis by hypoxia 611.5.6 Crosstalk of HIF-1 and GR 631.5.7 Activation of JNK by hypoxia 641.6 Project aims 65Chapter 2: Materials and Methods 6712.1 Cell culture 672.1.1 Cell lines and culture conditions 672.1.2 Passage and maintenance of cells 672.1.3 Cell counting 682.2 Cytotoxic stress conditions 682.3 Plasmid constructs and transfections 682.3.1 Plasmid constructs 682.3.2 Polyfect transfection 682.3.3 Calcium phosphate transfection 692.4 Immunoblotting 692.4.1 Lysis for immunoblotting 692.4.2 Bradford Assay 702.4.3 Lysis for immunoprecipitation 702.4.4 SDS-PAGE 702.4.5 Western blotting and detection of protein 712.5 RNA extraction and qRT-PCR 722.5.1 RNA extraction 722.5.2 qRT-PCR 722.6 Chromatin Immunoprecipitation (ChIP) Assay 732.6.1 Crosslinking 702.6.2 Immunoprecipitation 702.6.3 DNA isolation 712.7 Cloning and luciferase assays 762.7.1 Cloning of promoter fragments 762.7.1.1 Polymerase chain reaction (PCR) 762.7.1.2 Restriction digestion and ligation 762.7.2 Site-directed mutagenesis of promoter fragments 772.7.3 Luciferase assay 772.8 Flow cytometry 782.9 MTT Assay 78Chapter 3: The Bcl-2 family members NOXA and Mcl-1 are regulated by GR 793.1 Dexamethasone differentially regulates members of the Bcl-2 family 793.2 NOXA and Mcl-1 protein levels are regulated by glucocorticoids 843.3 Identification and validation that NOXA and Mcl-1 contain functional GREs 863.4 Differential recruitment of GR and SRC1 to the NOXA and Mcl-1 GREs 903.5 Glucocorticoids regulate NOXA and Mcl-1 in HeLa cells 953.6 Mcl-1 stability is compromised in CEM C7-14, correlating with its apoptotic ability 993.7 The proteasome inhibitor MG-132 enhances glucocorticoid-induced apoptosis 104Chapter 4: Cytotoxic stress regulates glucocorticoid-induced apoptosis through post-translational modifications of the GR 1074.1 GR is phosphorylated by JNK at serine 246 1074.2 Serine 226 phosphorylation regulates GR activity in a gene-specific manner 1124.3 Regulation of NOXA and Mcl-1 by the JNK-dependent phosphorylation of GR 115Chapter 5: Hypoxia regulates glucocorticoid-induced apoptosis through post-translational modifications of the GR 1225.1 Activation of JNK and GR phosphorylation during hypoxic exposure 1225.2 Glucocorticoids and hypoxia differentially regulate both NOXA and Mcl-1 1275.3 Regulation of GR and HIF-1α transcriptional activity at the NOXA and Mcl-1 promoters 1365.4 Hypoxia regulates the ability of glucocorticoids to proliferate and induce apoptosis 142Chapter 6: Discussion 1476.1 NOXA and Mcl-1 regulate glucocorticoid-induced apoptosis 1476.1.1 Cell-type specific regulation of NOXA and Mcl-1 at the mRNA/protein level 1476.1.2 NOXA and Mcl-1 contain putative GREs 1496.1.3 GR and SRC1 differentially occupy the NOXA/Mcl-1 GREs 1506.1.4 Glucocorticoids cause destabilisation of Mcl-1 in CEM C7-14 cells 1536.1.5 NOXA enhances glucocorticoid-induced apoptosis 1536.2 Phosphorylation of GR by JNK regulates GR target genes 1576.2.1 JNK phosphorylates GR at serine 246 1576.2.2 JNK-mediated inhibition of GR target genes 1586.2.3 GR phosphorylation regulates the expression of NOXA and Mcl-1 1606.3 Hypoxia and glucocorticoids differentially regulate NOXA and Mcl-1 expression through multiple mechanisms 1626.3.1 Hypoxia induces JNK activation and downstream GR phosphorylation 1626.3.2 NOXA and Mcl-1 are differentially regulated by glucocorticoids and hypoxia 1636.3.3 Cytotoxic stress regulates GR and HIF-1α transcriptional activity 1656.3.4 Regulation of apoptosis by hypoxia 1686.4 Conclusions 1716.4.1 Role of NOXA and Mcl-1 in glucocorticoid-induced apoptosis 1716.4.2 Phosphorylation regulates GR-mediated transcription 1726.4.3 Crosstalk between glucocorticoids and hypoxia 1726.5 Future work 173Chapter 7: References 174Chapter 8: Appendix 192

During stress conditions, glucocorticoids are secreted and exert most of their physiological responses by binding to and modulating the transcriptional activity of the glucocorticoid receptor (GR). Once activated, GR can regulate numerous cellular processes including inflammation, development, growth, metabolism and apoptosis. Although glucocorticoids have been used in the treatment of leukaemia for over 50 years, with the molecular mechanisms by which steroids exert their pro-apoptotic effect, the pathways responsible for the development of resistance to glucocorticoid treatment, as well as their role in the programmed cell death in other tissue types have not been precisely defined. Research has demonstrated that glucocorticoid-induced apoptosis requires a transcriptionally active form of GR and is executed by the induction of the intrinsic pathway of apoptosis. In addition, GR is regulated by diverse types of cytotoxic stress; including UV irradiation and hypoxia, which alter the receptor’s transcriptional activity through multiple mechanisms. These include post-translational modifications, subcellular localisation and interaction of the receptor with co-regulator proteins.The aims of this study are to identify novel members of the Bcl-2 family that are regulated at the transcriptional level by GR in both leukaemia and other tissue types where glucocorticoids promote cell survival. In addition, the molecular crosstalk between signalling pathways activated by cytotoxic stress conditions and the mechanisms by which they differentially modulate the apoptotic response will be investigated.Results obtained in this study have identified putative glucocorticoid response elements in the promoters of the BH3-only pro-apoptotic gene NOXA and the anti-apoptotic gene Mcl-1 and confirmed that both NOXA and Mcl-1 are direct GR transcriptional targets. The glucocorticoid-mediated expression of NOXA and Mcl-1 alters their protein-protein interaction pattern, leading to the subsequent destabilisation of Mcl-1 in cell lines that undergo glucocorticoid-induced apoptosis. Investigation into the effects that other cytotoxic stress pathways have on GR function have revealed that serine 226 phosphorylation of GR by JNK occurs in a rapid and transient manner. Phosphorylation has inhibitory effects on the transcription of GR targets in a gene-specific manner, including the differential regulation of NOXA gene expression. During hypoxia, glucocorticoids differentially regulate the GR and HIF-1 target genes, NOXA and Mcl-1, altering the apoptotic response. This study has provided additional insight into the molecular mechanisms that govern glucocorticoid-induced programmed cell death and revealed mechanisms by which glucocorticoids and cytotoxic stress pathways crosstalk, regulating apoptosis.

• Transcription
• Acute Lymphocytic Leukaemia
• Glucocorticoid Receptor
• Apoptosis
• Hypoxia
• Phosphorylation

• Demonacos, Constantinos

• Faculty of Medical and Human Sciences
• School of Pharmacy and Pharmaceutical Sciences

uk-ac-man-scw:151882

Demonacos, Constantinos

16th January, 2012, 08:53:45

Demonacos, Constantinos

16th January, 2012, 08:53:45