Effects of proteasome inhibition on cisplatin induced DNA damage responses in human cells

iv Chapter Introduction . 1 1.1 Cancer. 2 1.2 The DNA Damage Response. 4 1.3 PIKK mediated protein phosphorylation in the DNA damage response. 5 1.4 The ubiquitin-proteasome pathway. 7 1.4.1 Ubiquitination . 7 1.4.2 The proteasome. 10 1.5 Proteasomal regulation of cell cycle progression. 13 1.6 Cisplatin. 16 1.7 DNA Repair. 19 1.7. 1 Nucleotide Excision Repair (NER) . 19 1.7. 2 The Fanconi Anemia (FA) pathway. 21 1.7. 3 Tolerence to DNA damage by translesion synthesis (TLS) . 24 1.7. 4 DNA double strand break repair. 25 1.7. 4.1 Homologous Recombination (HR) . 28 1.8 Replication Protein A (RPA). 29 1.8. 1 RPA1 . 29 1.8. 2 RPA2 . 30 1.8. 3 RPA3 . 31 1.8. 4 RPA DNA binding and role in DNA Replication. 32 1.8. 5 Phosphorylation of RPA2 . 33 1.8. 6 RPA-protein interactions . 34 1.9 Proteasome inhibition in the DNA damage response . 35 1.10 Research objectives: . 38 Chapter 2 Materials and Methods . 39 2.1 Cell lines . 40 2.2 Cell culture. 40 2.3 Cryopreservation. 41 2.4 Resuscitation. 41 2.5 Cell treatment. 41 2.5. 1 Cis-diammine platinum (II) dichloride (cisplatin) treatment. 41 2.5. 2 MG132 treatment. 42 2.5. 3 Phosphatidylinositide-3 kinase-like kinase (PIKK) inhibitor treatment . 42 2.6 XTT cell viability assay. 42 2.7 Flow cytometry, propidium iodine staining and BrdU incorporation. 43 2.8 Analysis of RPA2-phosphoSer4/Ser8 by flow cytometry. 44 2.9 Preparation of protein lysates. 44 2.10 Preparation of protein lysates under denaturing conditions. 45 2.11 Subcellular fractionation. 45 2.12 Determination of protein concentration. 46 2.13 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDSPAGE) . 47 2.14 Coomassie blue staining. 48 2.15 Silver staining. 48 2.16 Western blotting. 48 2.17 Calculation of approximate protein molecular weight . 51 2.18 Immunofluorescence . 51 2.19 Phosphoprotein purification. 53 2.20 Affi-Gel Blue enrichment of RPA . 54 2.21 Transformation of competent cells. 55 2.22 Plasmid DNA purification. 55 2.23 Transient transfection . 55 2.24 Immunoprecipitation . 56 2.24.1 Immunoprecipitation using magnetic Dynabeads® . 56 2.24.2 Immunoprecipitation using protein A/G agarose beads . 57 2.25 Isopeptidase-T treatment. 57 2.26 λ -phosphatase treatment. 58 2.27 Preparation of samples for Mass Spectrometry . 58 Chapter 3 Results . 60 3. Effect of proteasome inhibition on cisplatin induced DNA damage responses. 61 3.1 Effect of MG132 on protein ubiquitination. 62 3.2 Characterisation of the sensitivity of DNA polymerase η-deficient human fibroblasts (XP30RO). 64 3.3 Effect of MG132 on the cisplatin sensitivity of DNA polymerase ηdeficient human fibroblasts. 65 3.4 Effect of proteasome inhibition on cisplatin-induced cell cycle arrest in pol eta-deficient XP30RO cells. 67 3.5 Effect of proteasome inhibition on cisplatin-induced protein phosphorylation. 75 3.6 Characterisation of RPA2-pS4S8*. 82 3.6. 1 Analysis of RPA4. 82 3.6. 2 Kinetics of RPA2-p-S4S8* formation. 83 3.6. 3 λphosphatase treatment. 85 3.6. 4 PIKK dependence of RPA2-pS4S8* . 87 3.6. 5 Isolation of cellular phosphoproteins. 88 3.6. 6 Affi-Gel Blue RPA enrichment. 90 3.6. 7 Immunoprecipitation of RPA2-pS4S8. 92 3.6. 8 Subcellular localisation of RPA2-pS4S8*. 94 3.6. 9 Cell cycle distribution of novel RPA2-pS4S8* . 95 3.6. 10 Cellular localisation of RPA2-pS4S8 . 97 3.7 Effect of removal of MG132 on cisplatin-induced DNA damage responses. 102 3.7. 1 λ-phosphatase treatment. 106 3.7. 2 PIKK-dependence of modified RPA2. 108 3.7. 3 Affi-gel RPA enrichment.109 3.7. 3 Immunoprecipitation of RPA2 from cell extracts.111 3.7. 4 Characterisation of modified RPA2. 112 3.7. 4.1 Sumoylation . 114 3.7. 4.2 Ubiquitination.115 (i) Immunoprecipitation of RPA2 from native cell extracts.115 (ii) Immunoprecipitation of RPA2 from denatured cell extracts. 118 (iii) Isopeptidase-T treatment.120 (iv) HA-Ubiquitin labelling . 121 (v) Analysis of modified RPA2 by mass spectrometry. 123 Chapter 4 Results . 131 4.1 Effect of pol η expression on cisplatin-induced monoubiquitination of FANCD2. 132 4.2 Cisplatin induced FANCD2 foci in pol ηdeficient and –proficient cells . 138 4.3 DNA damage response protein phosphorylation in FANCD2-deficient human cells.