ROLE OF THE SURVIVAL PROTEINS HSP27 AND SURVIVIN IN A SMALL MOLECULE SENSITIZATION TO TRAIL-MEDIATED APOPTOSIS GRÉGORY MELLIER (B.Sc., M.Sc., Claude Bernard University – Lyon 1, Lyon, France) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY YONG LOO LIN SCHOOL OF MEDICINE DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGMENTS “If we knew what it was we were doing, it would not be called research, would it?” Albert Einstein First and foremost, I would like to thank my supervisor, Prof Shazib Pervaiz, for giving me the opportunity to join his lab and embark on this PhD Thank you for your constant support, both in and out of the lab, throughout these last years Of course, huge thanks to all my labmates, both past and present, for making the lab such a lively place To my friend and flatmate, Dr Andrea Lisa Holme for her encouragements during the writing of this thesis and her help during the past few years To Dr Alan Prem Kumar and the rest of the gang for their friendship And last but not least, I would like to thank my family for their never ending support despite the distance Dad, thank you for believing in me i To my mother ii TABLE OF CONTENTS Acknowledgments i Table of contents .iii Summary ix List of tables xi List of Figures xii List of Abbreviations xiv Introduction 1 General apoptotic mechanisms 1.1 Apoptosis and necrosis 1.1.1 Necrosis 1.1.2 Apoptosis 1.1.2.1 Characteristics of apoptosis 1.1.2.2 Apoptosis is an evolutionary conserved mechanism 1.1.2.3 Physiological role of apoptosis 1.2 Mechanisms of apoptosis induction 1.2.1 Molecular players in apoptosis 1.2.1.1 Caspase family 1.2.1.2 Inhibitors of apoptosis 1.2.1.2.1 Survivin 11 1.2.1.3 Bcl-2 family 13 1.2.2 Mitochondrial, or intrinsic, pathway 14 1.2.3 Death receptors, or extrinsic, pathway 17 1.2.3.1 Ligands and receptors of the TNF-! superfamily 17 1.2.3.2 Signalization pathway 18 1.2.3.3 Activation of the mitochondrial pathway by the death receptors 19 TRAIL 21 2.1 TRAIL 21 2.2 TRAIL receptors 21 2.3 TRAIL signal transduction 22 iii 2.4 TRAIL as a therapeutic modality 24 2.5 TRAIL resistance in cancer 26 2.5.1 Resistance in the death receptor pathway 28 2.5.1.1 Resistance involving the death receptors 28 2.5.1.2 DISC components: c-FLIP and caspase-8 29 2.5.2 Resistance in the mitochondrial pathway 29 2.5.2.1 Upstream of the mitochondria: the Bcl-2 family 30 2.5.2.2 Downstream of the mitochondria: the IAP family 30 2.6 Strategies to restore cancer cell sensitivity to TRAIL 31 2.6.1 Modulation of the extrinsic pathway 31 2.6.1.1 Up-regulation of death receptors 32 2.6.1.2 Oligomerization of DRs 32 2.6.1.3 Down-regulation of c-FLIP 33 2.6.2 Modulation of the intrinsic pathway 33 2.6.2.1 Down-regulation of IAPs 34 2.6.2.2 Down-regulation of the anti-apoptotic Bcl-2 proteins 34 2.6.3 Simultaneous modulation of extrinsic and intrinsic pathways 35 2.7 Pre-clinical and clinical evaluation of TRAIL 36 2.7.1 Recombinant human TRAIL 36 2.7.2 Agonistic antibodies 37 2.7.3 Combinational therapies 37 Heat Shock Proteins and Hsp27 39 3.1 High molecular weight Hsps 39 3.1.1 Hsp90 40 3.1.2 Hsp70 40 3.1.3 Hsp60/10 41 3.2 Small Heat Shock Proteins 41 3.2.1 Cellular characteristics 43 3.2.1.1 Cellular localization 43 3.2.1.2 Expression/Regulation 43 3.2.1.2.1 Expression following a cellular stress 44 3.2.1.2.2 Pathological expression 45 3.2.1.2.3 Physiological expression 46 3.2.2 Biochemical characteristics 46 iv 3.2.2.1 Structure 47 3.2.2.1.1 Primary/Secondary 47 3.2.2.1.2 Tertiary/Quaternary 49 3.2.2.2 Phosphorylation 50 3.2.2.2.1 Phosphorylation signaling 51 3.2.2.2.2 Effect of phosphorylation on the quaternary structure 51 3.2.3 Cellular functions 52 3.2.3.1 Protection against heat shock and oxidative stress 54 3.2.3.2 Cytoskeleton protection 57 3.2.3.3 Anti-apoptotic functions 57 3.2.3.4 Chaperone activity 60 3.2.3.4.1 Mode of action 60 3.2.3.4.2 Importance of the large oligomers for the chaperone activity 61 3.2.3.4.3 Dynamics of oligomers 63 3.2.3.4.4 Additional role of Hsp27 chaperone activity 63 LY303511 65 Material and Methods 68 Cell line 68 Reagents and chemicals 68 Antibodies 69 Plasmids and siRNAs 70 Transient transfection of plasmids 70 Transient silencing of protein expression 71 Cell viability assay 71 Colony forming assay 72 Laser Scanning Cytometry (LSC) 72 Cell cycle, PI uptake 72 Mitochondrial membrane potential measurement and mitochondrial aggregation 73 Caspase activity 73 PP2A activity assay 74 Western blot analysis 74 Cellular fractionation 75 v Soluble / Insoluble fractions 75 Mitochodrial fractionation 76 Nuclear fractionation 76 Non-radioactive Electro-Mobility Shift Assay (EMSA) 77 Chromatin Immuno-Precipitation (ChIP) 77 Reverse-Transcriptase Polymerase Chain Reaction 79 Total RNA extraction 79 RT-PCR 79 Hsp27 oligomerization analysis 80 In vivo chaperone activity assay 81 Densitometry 82 Statistical analysis 82 Aims of the study 83 Results 84 LY30 restores HeLa cells sensitivity to TRAIL-induced cell death 84 LY30 and TRAIL combined treatment decreases HeLa cells ability to form colonies 85 LY30, alone or in combination with TRAIL, induces an early mitochondrial membrane potential depolarization ("#m) and mitochondrial aggregation 88 LY30 and TRAIL treatment engages the mitochondrial apoptotic pathway 91 LY30 and TRAIL combined treatment induces, and is dependent on, caspase activation 93 LY30 and LY30+TRAIL treatment decrease Hsp27 protein level in cleared RIPA cell lysates 97 LY30-mediated decrease in Hsp27 protein level is not due to transcriptional regulation nor proteasomal degradation 99 LY30, alone or in combination with TRAIL, does not affect Hsp27 expression but instead induces a long lasting translocation of Hsp27 to a nuclei-enriched fraction 101 Hsp27 specifically translocates to the nucleus upon exposure to LY30 106 Hsp27 phosphorylation increases upon exposure to LY30 108 Hsp27 increased phosphorylation is dependent on LY30-mediated p38 protein kinase activation 111 vi Hsp27 increased phosphorylation is dependent on LY30-mediated Protein Phosphatase 2A (PP2A) inhibition 113 LY30 disrupts the equilibrium between small size and large size Hsp27 oligomers 117 LY30 affects Hsp27 molecular chaperone activity in vivo 119 Modulation of Hsp27 expression can modulate HeLa cells viability upon treatment with TRAIL, alone or in combination with LY30 121 Silencing of Hsp27 reduces long term cell viability/colony formation ability of HeLa cells when combined with TRAIL treatment 124 Modulation of Survivin expression by LY30 at the transcriptional level 127 Modulation of Survivin expression by LY30 at the post-transcriptional level 129 Survivin expression silencing decreases HeLa cells viability and sensitizes them to LY30 and TRAIL, alone or in combination 131 Silencing of survivin expression has a drastic effect on medium- and long-term cell survival alone or in combination with TRAIL 133 LY30 treatment induces the expression of Egr-1, a repressor of Survivin expression 135 Egr-1 binds to the survivin gene promoter upon LY30 treatment 137 Egr-1 silencing protects HeLa cells from cell death by partially reducing LY30mediated down-regulation of Survivin 139 Double knock-down of Hsp27 and survivin sensitizes HeLa cells to TRAIL 141 Discussion 143 LY30 enhances TRAIL-induced cell death 143 LY30 regulates Hsp27 functions 145 LY30 does not affect Hsp27 expression but its localization 145 LY30 mediates Hsp27 phosphorylation through activation of p38 MAPK and inhibition of PP2A 148 LY30-mediated Hsp27 phosphorylation affects its oligomerization and chaperone activity 150 Discussion of the potential effect of LY30 on Hsp27’s other protective functions 152 Hsp27 is a key component in resistance to TRAIL 153 LY30 negatively regulates survivin expression 154 vii LY30 promotes survivin proteasomal degradation and inhibits its transcription 155 Importance of survivin protection in HeLa cells 156 Possible consequences of survivin down-regulation based on the litterature 156 Mechanisms of LY30-mediated transcriptional regulation of survivin 158 LY30 induces Egr-1 159 Egr-1 represses survivin expression 161 Hsp27 and survivin as key targets of LY30 163 Potential use of LY30 and TRAIL combination in chemotherapy 164 Conclusion 166 References 169 Appendix A 206 Supplemental figures 206 Appendix B 208 Publications 208 Conferences 208 viii SUMMARY In the last decade, TRAIL has been highlighted as a tumor selective molecule capable of engaging, depending upon the cell type, both the intrinsic and extrinsic apoptotic pathway, making it a promising therapeutic candidate However, the observation that tumor cells were resistant or could acquire resistance to TRAIL treatment sparked the search for molecules capable of enhancing or restoring sensitivity to TRAIL One such molecule, LY303511, an inactive analogue of the PI3K inhibitor LY294002, has previously been shown by our group to sensitize different type of tumor cells to TRAIL-induced apoptosis This sensitization was linked to ROS production, MAPK activation, up-regulation of death receptors expression and clustering, and overall enhancement of DISC formation Based on the premises that both quercetin and LY29, from which LY30 is derived, could affect the small heat shock protein Hsp27 and the IAP survivin, we set out to investigate the possibility for LY30 to have the same effect Firstly, the model of LY30 sensitization to TRAIL was assessed in our cell system We show that pre-incubation of HeLa cells with LY30 significantly amplifies TRAIL signaling as evidenced by decrease cell viability and reduction in the cancer cells colony formation ability This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the release of cytochrome c and Smac/DIABLO, and activation of caspase-3, -8 and -9 Secondly, this study shows that LY30 rapidly induces sustained phosphorylation of Hsp27 dependent on both p38 activity and PP2A inhibition In addition, Hsp27 phosphorylation is concomitant with a drastic shift in its oligomeric size from large to small oligomers ix Antibodies Mouse monoclonal anti-Hsp27 (Assay designs/Stressgen, Ann Arbor, MI, USA) Mouse monoclonal anti-!B-Crystallin (Assay designs/Stressgen, Ann Arbor, MI, USA) Mouse monoclonal anti-Hsp70 (BD, San Jose, CA, USA) Mouse monoclonal anti-Hsp90 (BD, San Jose, CA, USA) Mouse monoclonal anti-survivin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) Mouse monoclonal anti-PARP (BD Pharmingen, San Jose, CA, USA) Mouse monoclonal anti-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) Mouse monoclonal anti-Smac (BD Pharmingen, San Jose, CA, USA) Mouse monoclonal anti-Caspase-3 (Upstate/Millipore Corporation, Billerica, MA, USA) Mouse monoclonal anti-Cytochrome c (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) Mouse monoclonal anti-CuZnSOD Mouse monoclonal anti-VDAC (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) Rabbit polyclonal anti-phospho-Hsp27 (ser82) (Assay designs/Stressgen, Ann Arbor, MI, USA) Rabbit polyclonal anti-Egr-1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) 69 Goat Anti-mouse IgG HRP-conjugated secondary antibody (Pierce Chemical Co Rockford, IL, USA) Goat Anti-Rabbit IgG HRP-conjugated secondary antibody (Pierce Chemical Co Rockford, IL, USA) Plasmids and siRNAs The plasmid pcDNA-Hsp27 containing the full-length human hsp27 gene was a generous gift from Doctor Borelli (somewhere) The pcDNA3.1 empty vector was obtained from Invitrogen (Invitrogen, Carlsbad, CA) siRNAs for Hsp27 and Survivin (BIRC5) as well as the negative control siRNA were obtained from Dharmacon Technologies (Thermo Scientific, MA) Transient transfection of plasmids For transient expression, HeLa cells (seeded at 106 cells per 100 mm Petri dish the day before) were transfected with 10 &g of pcDNA-Hsp27 or pcDNA3.1 using the calcium phosphate transfection method Briefly, 10 &g of DNA were diluted in 500 &L of a 125 mM CaCl2 solution Then, 500 &L of 2xHBS solution (50 mM HEPES pH 7.05, 10 mM KCl, 280 mM NaCl, 1.5 mM Na2PO4) was added drop-wise to the DNA solution while vortexing at low speed The transfection mix was then incubated at room temperature for 30 minutes before being added drop-wise onto the cells Cells were cultured for 24h before trypsinization and seeding for subsequent experiments 70 Transient silencing of protein expression For knockdown of gene expression, 50 nM siRNA (Hsp27 siRNA, or Survivin siRNA, or negative control siRNA) was transfected into cells in Optimem medium using the Dharmafect reagent (Dharmacon) according to the manufacturer’s instructions Briefly, µl Dharmafect1 reagents was added to 200 µl of Optimem1 and the mixture was gently mixed and incubated for minutes at room temperature In another tube, 50 nM of siRNA was resuspended in 200 µl of Optimem Similarly, this mixture was also gently mixed and incubate Both mixtures were then combined, gently mixed and incubated at room temperature for 20 minutes Finally, the 400 µl was diluted in 2.6 mL of antibiotic-free DMEM The final transfection mix was added drop-wise onto the cells (0.5x106 in a 60 mm Petri dish) Cells were cultured for 24h before trypsinization and seeding for subsequent experiments Cell viability assay In a typical survival assay, HeLa plated in 24 well plates cells (0.5'105 cells/well, 24h prior treatment) were pre-incubated for hr with 25 &M LY30 and then treated with 20 ng/ml of TRAIL for varying length of time depending on the experiment Cytotoxicity was determined by the crystal violet assay After drug exposure, cells were washed once with phosphate buffered (PBS) before addition of 0.3 ml of crystal violet solution to each well and incubation for 20 The excess crystal violet solution was washed away using distilled water where non-viable cells gets detached and washed off in the process, leaving the attached viable cells that retained the crystal violet stain Remaining crystals were dissolved in a 1% SDS in 1X PBS solution and viability was determined by reading absorbance at wavelength 595 nm using a TECAN spectrophotometer 71 (Crystal Violet Solution was prepared as a 1% (w/v) crystal violet solution in 20% (v/v) methanol and stored at room temperature) Colony forming assay HeLa cells (1'106) were pretreated for hr with 25 &M LY30 followed by 20 ng/ml of TRAIL in a 60 mm Petri dish for 6h As this treatment period did not induce any visible cell death, cells were then washed and 10,000 cells for each treatment were re-plated into 100 mm Petri dishes and left to form colonies over a period of 10 to 14 days Culture dishes were stained with crystal violet and colony number and size were determined using the image analysis software ImageJ (http://rsbweb.nih.gov/ij/) to assess colony forming abilities of cells Only populations containing more than 20 cells were considered as colonies Laser Scanning Cytometry (LSC) Cell cycle, PI uptake HeLa cells were treated with 20 ng/mL TRAIL for 4, 8, 12 or 16h with or without pre-incubation with 25 µM LY30 for 1h Cells were then incubated with µg/mL propidium iodide 20 minutes at room temperature Cells were then fixed in 4% para-formaldehyde for 10 minutes at room temperature and permeabilized in 0.1% Triton-X for 10 minutes at room temperature Cells were washed before analysis by LSC 72 Mitochondrial membrane potential measurement and mitochondrial aggregation HeLa cells were treated with 20 ng/mL TRAIL for to 6h with or without preincubation with 25 µM LY30 for 1h Living cells were then stained with 100 nM TMRE and 10 µg/mL Hoechst for 20 minutes at 37°C and analyzed by LSC Caspase activity Caspases 3, and activities were assayed using AFC and AMC-conjugated substrates HeLa cells (1x105 cells/ml) were preincubated with specified concentration of LY30 for hr, followed by treatment with indicated doses of TRAIL for the relevant time points Cells were then harvested and washed with 1X PBS, resuspended in chilled cell lysis buffer (BD Pharmingen, San Diego, CA, USA), and incubated on ice for 10 before incubation with their respective substrates and real-time measurements of enzyme-catalyzed release of AFC or AMC were obtained using TECAN spectrophotometer reader operating with Magellan software Final fluorescence values obtained after 1hr incubation at 37ºC were normalized against the protein concentration of samples and plotted as x fold increase in caspase activity over the untreated control 73 PP2A activity assay Following treatment, HeLa cells were lysed in a buffer containing mM EDTA, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) and 1% Triton X-100, supplemented with proteases inhibitors The cell lysates were then sonicated and centrifuged for 15 minutes at 16,000g PP2A activity in the supernatants was assayed using the phosphatase kit V2460 Briefly, endogenous free phosphate was removed from the supernatants using the provided columns, and the extracts were normalized for protein content &g of protein were incubated with a chemically synthesized phosphopeptide [RRA(pT)VA], which is an optimal substrate for PP2A, PP2B and PP2C, but not for PP1 This was done in a buffer optimized for PP2A activity but which inhibited cation-dependent PP2B and PP2C (protocol provided by the manufacturer) for 20 minutes at room temperature Phosphate released from the substrate was detected by measuring the absorbance of a molybdate-malachite greenphosphate complex at 600 nm The activity of PP2A was evaluated by the release of phosphate per &g protein per minute (pmol/&g/min) Western blot analysis Whole cell protein extracts were isolated after drug treatments using 1X RIPA lysis buffer Lysates were incubated on ice for 15 minutes with intermittent vortexing and freeze/thawed twice with another round of vortexing in-between Whole cell lysates, cytosolic, mitochondrial or nuclear fractions were obtained using standard procedures (see each related method) Protein concentration was quantified using the Coomassie Blue reagent Briefly, &l of the supernatant was added to 200 &l of Coomassie Blue reagent into a 96-well plate and read for absorbance at 595 nm using 74 TECAN spectrophotometer Equal amounts of proteins from the lysates were mixed with 5X Laemlli loading dye and boiled for The samples were then subjected to different concentrations (10% or 15%) of SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) depending on the molecular weight of the target proteins and transferred onto PVDF membranes (BioRad Laboratories) subsequently The membrane was blocked with 5% (w/v) fat-free milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 TBST for 1hr and washed thrice with TBST to remove excess milk The membrane was probed for the target protein with the relevant primary antibodies (refer to antibodies list above) overnight at 4ºC The membrane is washed thrice to remove unbound primary antibody and probed again using appropriate HRP-conjugated secondary antibody in TBST containing 1% (w/v) fat-free milk at room temperature for 1hr After three washes with TBST to remove any excess unbound secondary antibody, the proteins of interest were detected with Kodak Biomax MR X-ray film (Eastman Kodak Co., Rochestor NY) by enhanced chemiluminescence using the SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL, USA) Cellular fractionation Soluble / Insoluble fractions HeLa cells were lysed using 1X RIPA buffer Lysates were incubated on ice for 15 minutes with intermittent vortexing and freeze/thawed twice with another round of vortexing in-between The lysates were then centrifuged at 16,000g for 15 minutes The supernatant was kept and the pellet was resuspended in 1X RIPA buffer Both fractions were analyzed by Western blotting 75 Mitochodrial fractionation HeLa were harvested and washed once with ice-cold 1X PBS before incubation on ice for 30 in Extraction buffer A (50 mM PIPES-KOH pH 7.4, 200 mM mannitol, 68 mM sucrose, 50 mM KCl, mM EGTA, mM MgCl2, mM DTT, pH 7.4) After incubation, the cells were homogenized with a dounce homogenizer and homogenized with 20 strokes before being centrifuged at 300g for 10 minutes at 4°C The pellet yields a nuclei-enriched fraction used in Figure A The supernatant was centrifuged again at 25,000g for 15mins at 4°C and the resulting pellet was used as the mitochondrial fraction and the supernatant as the cytosolic fraction The fractions were then subjected to SDS-PAGE and western blotting as described Nuclear fractionation Following drug treatments, HeLa cells were harvested from Petri dish by tripsinization and subjected to centrifugation at 1,500g for minutes The pellet was resuspended in 400 µl of Nuclear Buffer and incubated on ice for 15 minutes Then, 25 µl of NP-40 was added to the cell suspension and vigorous vortexing was carried out for 10 seconds The cell suspension was then being centrifuged at 16,000g for 30 seconds at °C The supernatant (cytosolic fraction) was transferred to a clear centrifuge tube and stored at -80 °C The pellet (nuclear fraction) was resuspended in 50 µl of ice cold Buffer C and incubated for 15 minutes at °C before storage at -80 °C The fractions were then subjected to SDS-PAGE and western blotting as described 76 Non-radioactive Electro-Mobility Shift Assay (EMSA) Nuclear extract from HeLa cells (1x106 cells) treated for different times were obtained using the NE-PER Nuclear extraction kit (Pierce) according to the manufacturer protocol Synthetic complementary oligonucleotides were 3’biotinylated which were purchased from 1stBase (Singapore) Binding reactions were incubated at room temperature for 20 minutes in the presence of 50 ng/&l poly(dI-dC), 0.05% Nonidet P-40, mM MgCl2, 10 mM EDTA,and 2.5% glycerol in 1' binding buffer (LightShiftTM chemiluminescent EMSA kit, Pierce) using 20 fmol of biotinend-labeled target DNA and &L of nuclear extract Unlabeled target DNA (4 pmol) was added to the binding reaction mixture (20 &l) for competitive binding Assays were loaded onto native 5% polyacrylamide gels pre-electrophoresed for 60 in 0.5' Tris borate/EDTA and electrophoresed at 100 V before being transferred onto a positively charged nylon membrane (Biodyne B, PALL Lifesciences) at 100 V for 30 Transferred DNAs were cross-linked to the membrane at 120 mJ/cm2 and detected using horseradish peroxidase-conjugated streptavidin (LightShift chemiluminescent EMSA kit) according to the manufacturer's instructions Chromatin Immuno-Precipitation (ChIP) Six 100 mm Petri dishes of HeLa cells ((80%confluent) were used per samples Cells were fixed in 1% formaldehyde for 10 minutes at room temperature on a shaker After discarding the formaldehyde, the fixation was stopped by incubating the cells in 0.125 mM glycine for minutes at room temperature on a shaker The cells were then washed twice with ice-cold PBS, harvested by scraping and centrifuged for 10 minutes at 720g at 4°C The pellet was resuspended in mL of ChIP Lysis Buffer (50 mM HEPES-KOH pH7.5 140 mM NaCl, mM EDTA pH8, 77 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS) supplemented with proteases inhibitors and incubated on ice for 30 minutes The cells were then quickly homogenized with 10 gentle strokes in a dounce homogenizer and centrifuged 10 minutes at 2,400g at 4°C to pellet the nuclei The pellet was resuspended in mL of ChIP Lysis Buffer supplemented with proteases inhibitors and the samples were sonicated using the Sonics Vibracell VC 130 (optimized settings for HeLa cells: mm probe, 20 seconds pulse at 25% amplitude followed by 30 seconds rest of ice – 10 times) The sheared chromatin was then centrifuged at 16,000g at 4°C for 12 minutes The supernatant was stored at -80°C At this point, 50 µL of each samples were reverse cross-linked overnight at 65°C in a reaction containing 0.2 M NaCl and 160 µg/mL RNase A (250 µL final volume) After addition of 0.1 mg/mL Proteinase K, the samples were incubated at 42°C for 2h DNA was extracted by a classic phenol/chloroform method and DNA concentration was determined by spectrophotometry These samples were used as input samples for the PCR The equivalent of 50 µg DNA of the sheared chromatin samples diluted 1:10 in ChIP RIPA buffer (50 mM Tris-HCl pH8, 150 mM NaCl, mM EDTA pH8, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) were pre-cleared with 20 µL of protein A beads for 1h at 4°C on a rotator The primary or control antibodies were then added (2 µg of rabbit anti-Egr-1 IgG or rabbit control IgG) along with 20 µL of protein A beads and the samples were incubated overnight at 4°C on a rotator The beads were then retrieved and washed thrice with mL of Wash Buffer (0.1% SDS, 1% Triton X-100, mM EDTA pH8, 150 mM NaCl 20mM Tris-HCl pH8) and once with mL of Final Wash Buffer (0.1% SDS, 1% Triton X-100, mM EDTA pH8, 78 500 mM NaCl, 20 mM Tris-HCl pH8) The DNA was eluted twice by resuspending the beads in 100 µL of Elution Buffer (1% SDS, 100mM NaHCO3) and incubating them for 15 minutes at 30°C on a rotator The DNA was reverse cross-linked and extracted as described earlier µL of DNA (diluted 1:10 for the input samples) for each sample were used as PCR templates Reverse-Transcriptase Polymerase Chain Reaction Total RNA extraction HeLa cells (60mm Petri dishes) were lysed in 1mL of TRIZOL (Invitrogen, Carlsbad, CA, USA) Homogenized samples were incubated for minutes at room temperature before addition of 0.2 mL of chloroform Samples were mixed by vigorous shaking and incubated for minutes at room temperature Following centrifugation at 12,000g for 15 minutes (4°C), the top aqueous phase was retrieved, mixed with 0.5 mL of isopropyl alcohol Samples were incubated at room temperature for 10 minutes and centrifuged at 12,000g for 10 minutes After discarding the supernatant, the RNA precipitate was washed once with mL of 70% ethanol The RNA pellet was briefly air-dried before resuspension in RNAse-free water and determination of RNA concentration RT-PCR Each reverse transcription reaction was done on µg of total RNA and in a final reaction volume of 20 µL Each reaction also contains 1X RT buffer, 0.5 mM 79 dNTP mix, 25 µg/mL oligo(dT)12-18, 10 mM DTT and 200 units of SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) The reaction carried out at 65°C for minutes and chilled on ice before adding the RT buffer and DTT and incubating at 42°C for minutes Following addition of the reverse transcriptase, the reaction was incubated at 42°C for 50 minutes and heat-inactivated for 10 minutes at 70°C The resulting cDNAs were stored at -20°C µL of cDNAs were used as a template for the polymerase chain reaction in a final reaction volume of 20 µL Each reaction contains 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 µM of each primers and 0.8 unit of Taq DNA polymerase (New England Biolabs) PCR products were analyzed by electrophoresis (100V) on 1.2% agarose gel containing 1X gel red (as a non-toxic substitue to ethidium bromide) and visualized under UV in a gel-doc apparatus Hsp27 oligomerization analysis HeLa cells (3x100 mm confluent) were lysed by brief sonication in 25 mm HEPES buffer, pH 7.4, containing 3.33% glycerol, mm EDTA, mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride at 4!°C The lysate were cleared by centrifugation at 17,000 ' g for at 4!°C The supernatant were used directly for glycerol gradient centrifugation The cell lysates (0.5 ml) were loaded on top of a 5.2ml non-linear gradient of glycerol (10, 15, 20,25,30,35,40%) made in 25 mm HEPES buffer, pH 7.4, containing mm EDTA and mm dithiothreitol The tubes were centrifuged for 18 h at 35,600 80 rpm in a SW55Ti rotor (Beckman) at 4!° C The gradients were fractionated in 24 fractions Aliquots were diluted in Tris/glycine/SDS buffer (25 mm Tris, 192 mm glycine, 0.01% SDS) and slot-blotted on nitrocellulose membrane Hsp27 was revealed by immunoblotting using an antibody against the total-Hsp27 In vivo chaperone activity assay The in vivo chaperone activity assay was adapted from Nollen et al (20) HeLa cell lines transiently expressing luciferase were seeded in triplicates for each treatment, at the same confluence (0.6x106 cells) in three different 60 mm culture dishes On the next day, media from the three plates was replaced by mL of DMEM media supplemented with 20 &g/ml cycloheximide Cells were put back for 30 minutes in the 37°C incubator to ensure complete inhibition of translation by cycloheximide Subsequently, two dishes were heat shocked for 30 minutes at 44°C while one was kept at the 37°C incubator as a non-heat-shocked control After the heat shock, one of the dishes was allowed to recover at 37°C for 180 minutes while cells from the other dishes were harvested immediately and the lysates were frozen at -80°C After the 180 minutes recovery time, cells from the third dish were harvested and the lysate was frozen at -80°C Renilla luciferase activity was measured using the dual-luciferase reporter assay system, using the STOPGlo solution only (Promega) following manufacturer instructions Measurements were normalized against the protein concentration for each sample The luciferase activity was expressed as ratio of the luminescence between the heat-shocked conditions and the non-heat shocked control 81 Densitometry All densitometry measurements (Western blot, Slot-blot, PCR) were done using the image analysis software ImageJ (http://rsbweb.nih.gov/ij/) When necessary, normalization was done against the loading control (actin) Statistical analysis All experiments were performed at least three times for statistical significance Numerical data were expressed as mean + SD Statistical analysis was performed using the paired Student’s t-test considering the variances unequal P values < 0.05 were considered significant 82 AIMS OF THE STUDY This study originated from the fact that both quercetin and LY29, from which LY30 is derived, had an effect on the small heat shock protein Hsp27 as well as on the IAP family member survivin As both proteins play important roles in cancer cells survival, we were interested in finding out whether LY30 could have retained quercetin and LY29 ability to negatively regulate these proteins Interestingly, our initial studies showed that LY30 did affect Hsp27 cellular localization (but not its expression) LY30 also induced a time-dependent down-regulation of survivin at the protein level Encouraged by these results, we set out to investigate the significance of Hsp27 and survivin regulation in LY30 sensitization to TRAIL-induced apoptosis The specific aims of this study were to determine: • the mechanism(s) of Hsp27 regulation with regards to its protective functions • the mechanism(s) of LY30-mediated survivin down-regulation • the significance of Hsp27 and survivin regulation in LY30 sensitization • the importance of Hsp27 and survivin in resistance to TRAIL 83 ... cleaving lamins [39] and membrane blebbing by targeting proteins such as Rho-associated kinase (ROCK1), p- 21 activated kinase (PAK) and gelsolin [40-42] 1. 2 .1. 2 Inhibitors of apoptosis The IAP... DISC [11 8] and results in the autocatalytic activation of the initiator caspases [11 9] in accordance with the induced proximity model Activated caspase-8/ -10 in turn targets the effector caspase-3... domains [11 5] Alternatively, ligation of TNF-! to TNF-R1 leads to the recruitment of the TNF-R1 Associated Death Domain protein (TRADD) [11 6] TRADD can then recruit FADD Acting as an adaptor