Mycobacterium bovis, BCG modulation of gene expression in bladder cancer

MYCOBACTERIUM BOVIS, BCG MODULATION OF GENE EXPRESSION IN BLADDER CANCER JUWITA N RAHMAT B.Sc (SCIENCE), NUS DEPARTMENT OF SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2010 MYCOBACTERIUM BOVIS, BCG MODULATION OF GENE EXPRESSION IN BLADDER CANCER JUWITA N RAHMAT B.Sc (SCIENCE), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements "If I have seen further than others, it is by standing upon the shoulders of giants." -Sir Isaac Newton It would be impossible to survive a PhD experience without friends by your side I would like to sincerely express my gratitude to them First and foremost, this thesis would be impossible without the guidance, patience and encouragement from my supervisor, Dr Ratha Mahendran, who gave me the opportunity to pursue a PhD program No words can express my gratitude for her invaluable advice and support throughout the course on my study I hope that she knows she is greatly appreciated by all her students And to Prof Kesavan, who never fails to always encourage us to go further in research To my fellow labmates, Rachel, Shih Wee, Shirong and Mathu, thank you for all the stimulating discussions and for sharing all the weals and woes of working in a lab I feel blessed to have friends like all of you as colleagues I would also like to thank Jason and Azhar for their advice and the help they have given me especially during the course of writing my thesis Special thanks goes out to Rathiga, who provided a listening ear at a time when I needed it the most Jan, Meera and Priya, whose friendship and love have kept me going Thank you for forcing me to have a night out once in awhile What use is your life if you not live it, right? Special thanks to Thomas, for being an exceptional ex-colleague and friend, who continued to provide advice and help throughout the years Last but not least, I would like to thank my family for their love and support To my parents, Asiah and Rahmat, thank you for having faith in me To my sister, Diana, who really pushed me throughout the writing process and made me feel better when I was feeling really low And my brother, Redzuan, who never fails to show his concern all the time I would have never made it through one of the most testing periods without all of you Thank you i Table of Contents Table of Contents Page Acknowledgement ……………………………………………………………………………… i Table of content…………………………………………………………………………………… ii List of figures……………………………………………………………………………………… x List of Tables……………………………………………………………………………………… xii List of publications………………………………………………………………………………… xiv List of abbreviations……………………………………………………………………………… xvi Abstract…………………………………………………………………………………………… xviii Chapter Introduction……………………………………………………………………… 1.1 The prevalence and challenges of bladder cancer………………………………… 1.1.1 The stages of bladder cancer……………………………………………………… 1.1.2 Superficial bladder cancer………………………………………………………… 1.1.3 Advanced bladder cancer………………………………………………………… 1.2 BCG immunotherapy of superficial bladder cancer……………………………… 1.2.1 Mechanisms of action……………………………………………………………… 1.2.1.1 Interactions of BCG with the bladder wall………………………………………… 1.2.1.1.1 Adhesion of BCG with the urothelium…………………………………………… 1.2.1.1.2 Internalization of BCG by urothelial cells………………………………………… 1.2.1.1.3 Secretion of cytokines and chemokines by urothelial tumour cells……………… 1.2.1.1.4 Antigen presenting properties of uroepithelial tumour cell………………………… 1.2.1.2 Cell mediated anti-tumour effects in BCG immunotherapy……………………… 1.2.1.3 TH1 versus TH2 dynamics………………………………………………………… 1.2.2 The role of neutrophils in BCG immunotherapy of bladder cancer……………… 1.3 Reactive Oxygen Species (ROS)…………………………………………………… 11 1.3.1 The generation of ROS during cellular respiration………………………………… 11 1.3.2 The detrimental effects of ROS…………………………………………………… 13 1.3.2.1 Lipid peroxidation ………………………………………………………………… 13 1.3.2.2 Cross linking and inactivation of proteins………………………………………… 14 ii Table of Contents 1.3.2.3 Oxidative DNA damage…………………………………………………………… 14 1.3.3 Defence mechanisms against ROS………………………………………………… 15 1.3.3.1 Non enzymatic ROS scavenging mechanisms…………………………………… 15 1.3.3.2 Enzymatic ROS scavenging mechanisms………………………………………… 16 1.3.3.3 Glutathione-S-transferases……………………………………………………… 16 1.3.4 ROS in tumour progression and signal transduction……………………………… 20 1.3.5 BCG and ROS……………………………………………………………………… 22 1.4 Epidermal growth factor receptor [EGFR] and bladder cancer………………….… 22 1.5 Mycobacterial secreted factors…………………………………………………… 23 1.5.1 BCG secreted proteins……………………………………………………………… 23 1.5.2 Mycobacterial protein tyrosine phosphatases (Mptps)…………………………… 24 1.6 Live versus lyophilized BCG……………………………………………………… 27 1.7 Aims of this study………………………………………………………………… 28 Chapter Material and Methods…………………………………………………………… 29 2.1 Materials………………………………………………………………………….… 30 2.1.1 Cell Lines/Cells/Bacteria…………………………………………………………… 30 2.1.2 Cell and bacteria Culture Reagents………………………………………………… 30 2.1.3 Chemicals………………………………………………………………………… 30 2.1.4 Antibodies and Enzymes…………………………………………………………… 32 2.1.5 Kits, Materials and Reagents……………………………………………………… 33 2.1.6 Equipments………………………………………………………………………… 35 2.1.7 Softwares…………………………………………………………………………… 35 2.1.8 Buffer compositions……………………………………………………………… 36 2.2 Methods…………………………………………………………………………… 37 2.2.1 Cell Culture and BCG preparations………………………………………………… 37 2.2.1.1 Growth and Maintenance of bladder cancer cell lines…………………………… 37 2.2.1.2 Preparation of Lyo BCG and live BCG…………………………………………… 37 2.2.1.3 Preparation of FITC labelled BCG………………………………………………… 38 iii Table of Contents 2.2.2 Investigating genes that are up-regulated in MGH cell line after hours Lyo BCG treatment using Representational Differential Analysis (RDA)…………………… 40 2.2.3 α5β1 analysis and BCG internalization assay…………………………………… 41 2.2.3.1 Integrin α5β1 receptor analysis…………………………………………………… 41 2.2.3.2 BCG internalization assay………………………………………………………… 41 2.2.3.3 Treatment of bladder cancer cell lines with live BCG and Lyo BCG for ROS and cytokine comparisons……………………………………………………………… 42 2.2.3.4 Cycloheximide treatment, BCG internalization and cytotoxicity studies………… 43 2.2.3.5 Cell proliferation assay with BrdU………………………………………………… 43 2.2.4 Animal experiments and SuperArray analysis…………………………………… 44 2.2.4.1 Live BCG instillation in mice……………………………………………………… 44 2.2.4.2 BCG treatment of MGH cells for RNA isolation and SuperArray analysis……… 44 2.2.4.3 Harvesting bladder and iliac lymph nodes for Immune cells recruitment analysis… 44 2.2.4.4 RNA isolation……………………………………………………………………… 45 2.2.4.5 Gene expression studies with SuperArray’s pathway specific OligoArrays……… 46 2.2.4.5.1 Preparation of poly A+ mRNA from bladder and MGH samples………………… 46 2.2.4.5.2 Linear amplification of poly A+ mRNA and preparation of Biotin-16-UTP labelled cRNA……………………………………………………………………… 47 2.2.4.5.3 Purification of synthesized cRNA………………………………………………… 47 2.2.4.5.4 Array Hybridization………………………………………………………………… 48 2.2.4.6 cDNA conversion………………………………………………………………… 49 2.2.4.7 Polymerase chain reaction………………………………………………………… 49 2.2.5 GSTT2 silencing and its effects on Lyo BCG treatment of MGH cells…………… 53 2.2.5.1 GSTT2 siRNA transfection and lyo BCG treatment……………………………… 53 2.2.5.2 Real time validation of GSTT2 silencing………………………………………… 54 2.2.6 Oxidative stress: ROS, Nitrite/Nitrate and Lipid Peroxidation Assay…………… 55 2.2.6.1 ROS measurement………………………………………………………………… 55 2.2.6.2 Nitrate/Nitrite assay………………………………………………………………… 55 2.2.6.3 Preparation of cells for lipid peroxidation assay…………………………………… 56 2.6.4 Lipid Peroxidation Assay………………………………………………………… 57 iv Table of Contents 2.2.7 Isolation and characterization of mycobacterial MptpA…………………………… 58 2.2.7.1 Expression and purification of MptpA…………………………………………… 58 2.2.7.2 Rapid Coomasie Staining………………………………………………………… 61 2.2.7.3 Preparation of phosphorylated Myelin basic protein……………………………… 61 2.2.7.4 Phosphatase Assay with Myelin Basic Protein…………………………………… 61 2.2.7.5 Treatment of MGH cells with epidermal growth factor (EGF) and MptpA……… 62 2.2.7.6 Treatment of MGH cells with purified MptpA for immunoblotting……………… 62 2.2.7.7 Immunoprecipitation of EGFR…………………………………………………… 63 2.2.7.8 Immunoblotting with phosphotyrosine, EGFR, MBP, actin and phospho-specific antibodies…………………………………………………………………………… 63 2.2.7.9 Measuring protein concentration with MicroBCA Assay Kit……………………… 64 2.2.8 In vitro effects of MPTPA on bladder cancer cell line and ex vivo on mouse Neutrophil-DC interactions………………………………………………………… 65 2.2.8.1 The signalling and cell cycle regulatory effects of MPTPA on MGH cell line…… 65 2.2.8.1.1 Cell Cycle analysis………………………………………………………………… 65 2.2.8.1.2 Effects of MPTPA on MGH gene expression of GSTT2 and TNFα ……………… 66 2.2.8.1.3 Preparation of cells for Human Phosphokinase Array…………………………… 66 2.2.8.1.4 Human Phosphokinase array assay………………………………………………… 66 2.2.8.2 Effects of MPTPA on Neutrophil-DC interactions………………………………… 67 2.2.8.2.1 Mouse bone marrow cells preparation……………………………………………… 67 2.2.8.2.2 Purification of Neutrophils and Dendritic cells (DC) from bone marrow cells preparation………………………………………………………………………… 68 2.2.8.2.3 BCG internalization comparisons between purified neutrophils and generated DCs 69 2.2.8.2.4 Mouse Neutrophil-DC co-cultures………………………………………………… 69 2.2.8.2.5 DC surface marker expression analysis…………………………………………… 70 2.2.8.2.6 Cytokine analysis…………………………………………………………………… 70 2.2.9 Statistical analysis………………………………………………………………… 71 Chapter Studying the effects of intravesical live BCG instillations in mice…………… 72 Introduction……………………………………………………………………… 73 BCG treatment induces phenotypic changes in the iliac lymph nodes…………… 74 3.1 v Table of Contents 3.2 Expression of immune related genes induced by BCG instillations in the bladder 75 3.2.1 Pathway specific array analysis of mouse bladder specimens…………………… 75 3.2.2 RT-PCR analysis of BCG induced gene up-regulation in the mouse bladder……… 76 3.3 Increased lymphocytes were observed in the bladder and iliac lymph nodes (ILN) after BCG instillation……………………………………………………………… 80 3.4 Discussion 1……………………………………………………………………… 82 Chapter Comparisons of live and lyophilized BCG treatment on gene expression and ROS production in human bladder cancer cell lines…………………………… 85 Introduction……………………………………………………………………… 86 4.1 Integrin expression and BCG internalization profiles of human bladder cancer cell lines………………………………………………………………………………… 87 4.1.1 Expression of Integrin α5 on human bladder cancer cells correlates with the ability to internalize BCG………………………………………………………… 87 4.1.2 BCG internalizing cells are more susceptible to BCG induced cytotoxicity……… 88 4.1.3 MGH cells internalize cultured BCG better at hours than Lyo BCG but there are more Lyo BCG on the surface at 24 hours………………………………………… 88 4.1.4 Blocking mammalian protein synthesis does not affect BCG internalization but abrogated BCG induced cytotoxicity of MGH cells……………………………… 89 4.2 Comparisons of cultured BCG and Lyo BCG treatment on ROS in human bladder cancer cell lines…………………………………………………………… 90 4.2.1 Lyo BCG and cultured BCG differentially regulate ROS levels in human bladder cancer cell lines…………………………………………………………………… 90 4.2.2 The BCG induced ROS changes in MGH cell line corresponds to lipid peroxidation levels………………………………………………………………… 93 4.2.3 RDA analysis of up-regulated transcripts showed that Glutathione-S-Transferase mRNA levels increased in MGH cells after hours of Lyo BCG treatment……… 94 4.2.4 Comparisons of gene expression regulation in MGH cell line after treatment with cultured BCG or lyo BCG for hours……………………………………………… 96 4.2.4.1 Pathway specific microarray analysis of genes induced in MGH cells after BCG treatment…………………………………………………………………………… 96 4.2.4.2 Lyo BCG induced increased expression of GSTT2, TNFα and IL1β whereas cultured BCG did not up-regulate any of the genes significantly………………… 98 4.2.4.3 RT4 cells up-regulates Tollip after cultured BCG treatment for hours………… 99 vi Table of Contents 4.2.4.4 MGH and RT4 cells displayed significantly different basal gene expression profiles……………………………………………………………………………… 99 4.2.4.5 RT-PCR analysis of GSTT2, TNFα and IL-1β expression in J82 cell line……… 102 4.2.5 Blocking direct BCG interaction with a transwell device………………………… 102 4.2.5.1 Blocking BCG interaction with MGH cells for hours reduced GSTT2 and TNFα expression………………………………………………………………………… 103 4.2.5.2 Inhibiting tyrosine phosphatase activity with sodium orthovanadate abrogated the GSTT2 transcript reduction in BCG blocked samples…………………………… 103 4.3 Effects of GSTT2 silencing on lyo BCG treatment of MGH cells………………… 107 4.3.1 Transfection with GSTT2 SMARTpool siRNA successfully reduces GSTT2 expression……………………………………………………….………………… 107 4.3.2 Lyo BCG treatment for hours after GSTT2 knockdown significantly reduced ROS levels compared to Dotap control…………………………………………… 108 4.3.3 GSTT2 knockdown increased basal NO levels and reduced NO after lyo BCG treatment…………………………………………………………………………… 109 4.3.4 GSTT2 knockdown increased TNFα production after hours of lyo BCG treatment…………………………………………………………………………… 110 4.4 Discussion 2…………………………………………………………………… 111 4.4.1 Integrin α5–role in BCG internalization, cytotoxicity and ROS production……… 111 4.4.2 Live BCG and Lyo BCG differentially regulate cellular ROS…………………… 113 4.4.3 Increase in ROS leads to increase in lipid peroxidation end product, MDA……… 113 4.4.4 Lyo BCG induced more gene up-regulation than live BCG at hours…………… 114 4.4.5 Higher endogenous ROS levels in MGH cell line leads to basal expression of genes……………………………………………………………………………… 115 4.4.6 Silencing GSTT2-DOTAP MBC mediated changes in MGH cells……………… 116 4.4.7 GSTT2 knockdown enhanced basal NO levels but reduced NO and increased TNFα after lyo BCG treatment…………………………………………………… 117 4.4.8 RDA vs microarray………………………………………………………………… 118 Chapter Characterization of the phosphatase activity of purified MptpA in vitro and its effects on MGH human bladder cancer cell line…………………………… 119 Introduction……………………………………………………………………… 120 5.1 Purification and isolation of MptpA……………………………………………… 121 5.2 Phosphatase activity of MptpA with phosphorylated Myelin Basic Protein in vitro 121 vii Table of Contents 5.2.1 Purified MptpA dephosphorylates tyrosine on myelin basic protein (MBP)……… 121 5.2.2 The tyrosine phosphatase activity of MptpA is observed as early as minutes in vitro………………………………………………………………………………… 123 5.3 Treatment of MGH cell line with MptpA in culture……………………………… 123 5.3.1 MptpA does not induce cell cycle changes in MGH cell line……………………… 123 5.3.2 MptpA does not cause a global change in phosphotyrosine residues in MGH cell line but it reduces tyrosine phosphorylation of Epidermal growth factor receptor (EGFR) after EGF stimulation…………………………………………………… 125 5.4 Investigating the cellular phosphokinase regulation by MptpA treatment in EGF stimulated MGH cells……………………………………………………………… 127 5.4.1 Combination treatment of MptpA and BCG up-regulates more phosphorylated targets than individual treatments alone…………………………………………… 127 5.4.2 Western blot validation of phosphokinase array…………………………………… 128 5.5 MptpA does not have ROS regulatory functions but may contribute to the downregulation of GSTT2 expression at hours of treatment…………………… 132 5.6 Discussion 3…………………………………………………………………… 133 5.6.1 5.6.2 MptpA is not the secreted factor responsible for ROS increase and TNFα downdownregulation…………………………………………………………………… MptpA exerts its phosphatase function on the EGFR by possibly dephosphorylating inhibitory Y1045 signal……………………………………… 133 133 5.6.3 MptpA with BCG treatment has more signalling modulatory potential…………… 134 5.6.4 Phosphokinase array vs western blot……………………………………………… 135 Chapter Modulatory effects of MptpA on neutrophil-DC interactions………………… 137 Introduction……………………………………………………………………… 138 6.1 Characterization of isolated D generated from GM-CSF conditioned media……… 139 6.1.1 Generated DCs are semi-mature phenotype and expressed low activation markers 139 6.1.2 Neutrophils internalized BCG more efficiently than DCs………………………… 139 6.2 Investigating the effects of MptpA on neutrophil-DC cooperation………………… 140 6.2.1 MptpA does not induce cytokine production in DC and neutrophil nor affect cytokine production in DC-neutrophil co-culture samples………………………… 141 6.2.2 DC activation and CD40 up-regulation induced by BCG treatment is not affected by MptpA…………………………………………………………………………… 141 6.3 Discussion 4……………………………………………………………………… 143 viii Chapter 79 Kruman 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Studies involving bladder cancer patients undergoing BCG immunotherapy reveal dynamic increases in cytokine production in the urine and bladder of these patients The cytokines detected include... Expressed genes induced in BCG treatment of a bladder cancer cell line Rahmat JN, Esuvaranathan K, Mahendran R 5th Combined Scientific Meeting incorporating the 4th GSS-FOM Scientific Meeting Singapore,