Investigation into the roles of ataxia telangiectasia mutated gene product, in multiple BRCA backgrounds

INVESTIGATION INTO THE ROLES OF ATAXIA TELANGIECTASIA MUTATED GENE PRODUCT, IN MULTIPLE BRCA BACKGROUNDS HIONG KUM CHEW NATIONAL UNIVERSITY OF SINGAPORE 2007 INVESTIGATION INTO THE ROLES OF ATAXIA TELANGIECTASIA MUTATED GENE PRODUCT, IN MULTIPLE BRCA BACKGROUNDS HIONG KUM CHEW (B.Sc., NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to take this rare opportunity to express my deepest gratitude to my supervisor, Dr Srividya Swaminathan for her guidance, support, and encouragement during the course of my study and all the help rendered in completion of the thesis It wouldn’t be possible without her My sincere appreciation goes to the present and ex-staff members of Oncology Research Institute (ORI): Tada, Tomoko, Tun Kiat, Tiling, Angela, Fenyi, Peiyi, Baidah and Diyanah, for their wonderful friendship and continuous support in me Special thanks go to my pretty group members: Dianne, Jawshin, Deepa and Joyce for their partnership and help It is really a great pleasure to be working with them This also extends to the Breast Cancer Group especially to Weiyi, Emily and Huiyin for their great company I want to thank Prof Yoshiaki Ito of ORI for his support in my graduate studies and also allowing me to use the facilities and reagents I also thank A/Prof Prakash Hande for providing me the AT-Tert cells for my project I am grateful for the research scholarship provided by NUS and giving me this opportunity to pursue graduate study Finally, I am most grateful and indebted to my parents for their unconditioned love and concern for me all these years Most importantly, they believe and gave me all the support I need to pursue my dreams Once again, Thank You i TABLE OF CONTENTS ACKNOWLEDGEMENTS i LIST OF FIGURES vi LIST OF TABLES viii LIST OF ABBREVIATIONS ix SUMMARY xi INTRODUCTION 1.1 Ataxia Telangiectasia Mutated 1.2 ATM mediated signaling 1.3 ATM and DNA damage responses 1.4 ATM and telomere stability 1.5 Clinical significance of ATM loss 1.6 Objectives 1.7 Approaches 1.7.1 Bacterial Artificial Chromosomes (BAC) recombineering 10 1.7.2 RNA interference 11 MATERIALS AND METHODS 2.1 Reagents 13 2.2 Cell Lines 13 2.3 Vectors 14 2.4 BAC engineering 2.4.1 Targeting vector design 14 2.4.2 Preparation of competent cells and electroporation 15 ii 2.4.3 Screening for recombinants 16 2.5 Transfection 17 2.6 RNA extraction and first stand cDNA synthesis 17 2.7 Quantitative PCR (qPCR) 18 2.8 SuperArray analysis 18 2.9 Western Blot 19 2.10 Immunoprecipitation 20 2.11 Genotoxin sensitivity assays 20 2.12 Soft agar assay 21 2.13 Immunohistochemistry 21 RESULTS 3.1 Generation of altered alleles of ATM deletion 23 3.2 Missense transfected AT-Tert cells exhibit altered growth 30 characteristics 3.3 Screening and verification of ATM knockdowns 33 3.4 Gamma-irradiation reduces cell viability in HeLa and 34 Capan-1 ATM knockdowns but not in HCC1937 background 3.5 ATM knockdowns are more susceptible to damage by 37 alkylating and crosslinking agents 3.6 Gamma irradiation induced Chk2 phosphorylation is 42 compromised in the ATM knockdowns 3.7 Etoposide induces Chk2 expression and phosphorylation in 45 untransfected HeLa, HCC1937 and Capan-1 cells iii 3.8 ATM knockdowns in HeLa and Capan-1 but not HCC1937 46 show reduced Chk2 expression and phosphorylation after etoposide treatment 3.9 ATM knockdowns exhibits different gene expression profiles 50 in different backgrounds in comparison to control 3.10 Interaction of ATM with BRCA2 51 3.11 Knockdown of ATM promotes anchorage-independent growth 53 in soft agar assay only in a BRCA2 null background 3.12 Gamma-irradiation reduces colony forming ability in 59 HeLa but not Capan-1 ATM knockdowns DISCUSSION 4.1 Generation of altered alleles of ATM 62 4.2 Generation of ATM knockdowns in multiple BRCA backgrounds 64 4.3 Effects of ATM knockdown on cell survival 65 4.4 Effects of drug treatment on the ATM KD 66 4.5 Effects of ATM loss on irradiation induced regulation 67 and phosphorylation of Chk2 4.6 Effects of etoposide treatment on the regulation of Chk2 68 4.7 Effects of KD on cellular transformation in a soft agar assay 69 4.8 Cellular involvements of ATM 70 4.9 Possible interaction of ATM with BRCA2 72 CONCLUSION 74 iv FUTURE DIRECTIONS 76 REFERENCES 77 APPENDIXES Appendix Relative expression profile of ATM knockdown 81 clones in various backgrounds Appendix Effects of etoposide treatment on ATM expression Appendix 3A BRCA1 expression in HeLa and HCC1937 assessed 82 83 by immunofluorescence Appendix 3B BRCA2 expression in HeLa and Capan-1 assessed 84 by immunofluorescence Appendix Chemical composition of media 85 Appendix Images of AT-Tert BACAtm (missense) cells 86 during and after selection v LIST OF FIGURES Figure Representation of ATM protein depicting various regions of possible function or interaction Figure Assessment of transformation efficiency after electroporation in the missense clones by step PCR 25 Figure Results of mismatch PCR on row and column pools (missense) 26 Figure Sequencing of BACAtm to confirm the missense generation 27 Figure Assessment of transformation efficiency after electroporation in the deletional clones by step PCR 28 Figure Results of mismatch PCR on row and column pools (deletion) 29 Figure Sequencing of BACAtm to verify the deletion of the FATC domain 30 Figure Multiple representative images of control and transfected AT-Tert cells 31 Figure Growth of AT-Tert and AT-Tert (BACAtm deletion) cells 32 Figure 10 Screening for ATM knockdowns by quantitative PCR and western blotting 34 Figure 11 MTT assay in various cell lines and their ATM knockdowns after γ-irradiation 36 Figure 12 MTT assay in various cell lines and their ATM knockdowns after BCNU treatment 39 Figure 13 MTT assay in various cell lines and their ATM knockdowns after MMS treatment 40 Figure 14 MTT assay in various cell lines and their ATM knockdowns after MMC treatment 41 Figure 15 Western blot detection in HeLa and AT-Tert cells before and after γ-irradiation 43 Figure 16 Western blot detection in HeLa, HCC1937, Capan-1 and their ATM knockdown clones after γ-irradiation 44 vi Figure 17 Western Blot for detection in HeLa, HCC1937 and Capan-1 cells after etoposide treatment 46 Figure 18 Western blot detection in HeLa and the ATM knockdowns after etoposide treatment 47 Figure 19 Western blot detection in HCC1937 and the ATM knockdowns after etoposide treatment 48 Figure 20 Western blot detection in Capan-1 and the ATM knockdowns after etoposide treatment 49 Figure 21 Expression profile of ATM knockdown on different BRCA backgrounds 51 Figure 22 Immunoprecipitation of ATM and BRCA2 53 Figure 23 Representative images various cell lines and their ATM knockdowns in a soft agar assay 55 Figure 24 Colony formation assay for ATM knockdowns after exposure to γ-irradiation 61 vii LIST OF TABLES Table Number of colonies in the controls and ATM knockdowns 54 observed in a soft agar assay viii transformation of these cells A more comprehensive screen for the expression profile of these cells is thus warranted Chen et al (2001) suggested that ATM is responsible for Rad9 hyper-phosphorylation in response to IR This phosphorylation of Rad9 is important for checkpoint activation Our data suggests that loss of ATM in a HeLa background causes significant decrease in Rad9 expression but not in other cell lines tested This reduced expression induced escape from cell cycle arrest may explain the colonizing ability of these cells on IR treatment 4.9 Possible interaction of ATM with BRCA2 To date there are no suggestions of ATM interaction with BRCA2 Studies on ATM interaction with BRCA1 (Wang et al., 2000) and interaction between BRCA1 and BRCA2 are documented (Yoshida and Miki, 2004) Based on our data discussed earlier, it is likely that ATM together with BRCA2 is important for cellular transformation Thus, we assessed possible interaction by immunoprecipitation Indeed, our data indicates such an interaction does exist under certain conditions Cellular BRCA2 and ATM interact with each other weakly in the absence of DNA No interaction was observed in chromatin bound proteins (high salt elution) Samples immunoprecipitated with ATM antibody when reprobed for BRCA2 (Fig 22; Panel C, lane and 4), did not show any co-ip of BRCA2 This could be due to the antibody used to immunoprecipitate ATM which may sterically hinder the interaction with BRCA2 Our results also indicate that this 72 interaction may be transient and weak It may be more prominent under special conditions of drug treatment or irradiation 73 CONCLUSION AT carriers are susceptible to breast cancer notably in young women Missense mutations on the ATM gene are often found in these patients Unclassified missense mutations outside the PI3-kinase domain are predicted to function like normal ATM However, the missense mutation in exon 64 generated in this study appears to have a dysregulatory effect on telomerase function, causing AT-Tert cells to die in culture This alteration thus has transformation potential possibly in other compromised backgrounds Yet, this potential does not exist on deletion of the FATC domain, a conserved region adjacent to the missense The deletion likely does not destabilize the protein structure, allows for basal kinase function to continue, and does not regulate telomerase function The level of ATM knockdowns correlates with IR induced ATM mediated Chk2 phosphorylation in all cell lines tested However, we did not detail all ATM mediated signaling responses due to paucity of funds to run a genome wide screen Drug treatments on ATM knockdown in various BRCA backgrounds exhibited differential sensitivity and survival responses It seemed that even a partial compromise of multiple functions in a single cell led to dramatically altered responses This was also true for irradiation induced survival (short and long term) The colony forming assay and soft agar assay shows that HCC1937 ATM knockdown forms fewer colonies These cells however, would prove more aggressive if established as they show an increase in Bcl2 and decrease in Rad9A Partial functional loss ATM 74 along with the loss of BRCA2 function (in Capan-1 ATM knockdowns) leads to aggressive growth in soft agar assays and heightened resistance to irradiation treatment This is of significance as cells lacking BRCA2 or ATM alone exhibit radiosensitivity Both proteins are believed to be involved in the DNA double strand break repair pathway However simultaneous loss (even partial) causes radioresistance Induction and phosphorylation pattern varies with BRCA background and the type of treatment Cells with ATM loss on multiple backgrounds are most sensitive to MMC treatment and BCNU may be effective in compromised BRCA backgrounds This indicates that the cellular transformation event and responses to IR are unrelated to compromised ATM mediated signaling We conclude that the interplay of ATM with different BRCA gene products may lead to different sensitivity to treatments and suggest that in the absense of information on BRCA status, tumors should be better managed by treatment with DNA crosslinker mitomycin C 75 FUTURE DIRECTIONS The use of BAC recombineering provides a useful platform to create mutations and study effects of genomic alterations at endogenous expression levels in a cell In order to further elucidate the functions of the altered missense allele, an over-expression model system may be utilized By using this system, it is likely possible to drive cellular transformation in multiple tumor suppressor gene backgrounds Applying this system to AT-Tert cells may help to understand how ATM mutations can affect cellular functions Cancer is a progressive disease that exhibits multiple genetic alterations A genome wide screen would thus be useful in characterizing the ATM knockdowns and developing a signature for effects in different tumor suppressor gene background This signature could be used to predict response to genotoxins, direct drug choices and develop universally successful treatment strategies 76 REFERENCES Abraham RT (2004) PI 3-kinase related kinases: 'big' players in stress-induced signaling pathways DNA Repair (Amst) (8-9): 883-887 Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation Nature 421: 499–506 Barlow C, Hirotsune S, Paylor R, Liyanage M, Eckhaus M, Collins F, Shiloh Y, Crawley JN, Ried T, Tagle D and Wynshaw-Boris A (1996) Atm-deficient mice: a paradigm of ataxia telangiectasia Cell 86: 159-171 Bosotti R, Isacchi A, Sonnhammer FL (2000) FAT: a novel domain in PIK-related kinases Trends in Biochemical Sciences 25 (5): 225–227 Chen MJ, LinYT, Lieberman HB, Chen G and Lee EY (2001) ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation Journal of 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Clinical Pathology 58: 1009-1015 Thompson D, Duedal S, Kirner J, McGuffog L, Last J, Reiman A, Byrd P, Taylor M and Easton DF (2005) Cancer risks and mortality in heterozygous ATM mutation carriers Journal of the National Cancer Institute 97: 813–822 Thorstenson YR, Roxas A, Kroiss R, Jenkins MA, Yu KM, Bachrich T, Muhr D, Wayne TL, Chu G, Davis RW, Wagner TMU and Oefner P (2003) Contributions of ATM mutations to familial breast and ovarian cancer Cancer Research 63: 3325-3333 Wang Q, Zhang H, Fishel R and Greene MI (2000) BRCA1 and cell signaling Oncogene 19 (53): 6152-6158 Wu Y, Xiao S and Zhu XD (2007) MRE11–RAD50–NBS1 and ATM function as co- mediators of TRF1 in telomere length control Nature Structural and Molecular Biology 14 (9): 832-840 79 Xu Y, Yang EM, Brugarolas J, Jacks T and Baltimore D (1998) Involvement of p53 and p21 in cellular defects and tumorigenesis in Atm-/- mice Molecular Cell Biology 18 (7): 4385-4390 Yoshida K and Miki Y (2004) Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage Cancer Science 95 (11): 866– 871 80 Appendix Gene HeLa KD ABL1 ANAPC2 ANAPC4 DIRAS3 ATM ATR BAX BCCIP BCL2 BIRC5 BRCA1 BRCA2 CCNB1 CCNB2 CCNC CCND1 CCND2 CCNE1 CCNF CCNG1 CCNG2 CCNH CCNT1 CCNT2 CDC16 CDC2 CDC20 CDC34 CDK2 CDK4 CDK5R1 CDK5RAP1 CDK6 CDK7 CDK8 CDKN1A CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CHEK2 CKS1B CKS2 CUL1 1.08 1.13 1.27 1.27 -1.35 -1.09 -1.15 -1.23 1.36 -1.36 1.06 1.10 -1.04 -1.19 -1.02 -1.11 -1.04 -1.01 1.01 1.13 -1.02 -1.10 1.09 1.36 1.05 -1.01 -1.39 -1.11 1.01 -1.14 -1.20 -1.04 -1.55 -1.26 -1.02 1.75 -1.24 -1.79 -1.92 1.02 -1.05 -1.11 -1.17 -1.18 -1.16 HCC1937 KD 1.00 -1.47 -1.13 -1.04 -1.26 -1.05 1.03 1.20 4.03 1.07 -1.13 -1.23 -1.26 -1.22 -1.13 -1.53 1.57 1.24 -1.08 1.72 -1.12 -1.52 1.28 -1.22 -1.32 1.01 -1.24 -1.19 -1.29 -1.25 1.44 1.18 -1.03 -1.14 1.03 1.35 -1.45 1.63 -1.87 -1.18 -1.55 -1.25 -1.07 -1.40 -1.27 Capan-1 KD 1.15 -1.17 -1.04 1.27 -1.31 -1.15 -1.14 -1.17 1.23 -1.19 -1.17 -1.13 1.04 -1.32 -1.27 -1.26 1.04 1.02 -1.14 -1.06 1.28 -1.54 -1.17 -1.36 1.34 -1.29 1.05 1.06 -1.14 -1.35 -1.18 -1.13 1.65 1.13 1.01 1.93 -1.14 1.04 1.04 -1.14 1.05 -1.20 -1.14 -1.51 -2.40 HeLa HCC1937 Capan-1 Gene KD KD KD CUL2 1.65 -1.55 1.05 CUL3 -1.20 -1.23 1.41 DDX11 1.46 -1.48 1.05 DNM2 -1.07 -1.26 -1.07 E2F4 -1.24 -1.17 -1.41 GADD45A 1.08 1.52 -1.09 GTF2H1 -1.08 1.20 -1.06 GTSE1 -1.11 -1.29 1.49 HERC5 -1.66 1.22 1.03 HUS1 -1.11 1.19 -1.71 KNTC1 1.13 1.06 -1.42 KPNA2 -1.24 -1.07 -1.45 MAD2L1 -1.09 -1.39 -1.22 MAD2L2 -1.18 -1.01 -1.38 MCM2 1.01 -1.12 1.02 MCM3 -1.03 -1.18 -1.36 MCM4 1.02 -1.97 -1.27 MCM5 1.07 -1.64 -1.02 MKI67 -1.25 1.05 1.06 MNAT1 1.01 1.01 -1.31 MRE11A -1.03 -1.55 -1.33 NBN -1.22 -1.19 -1.15 PCNA 1.05 -1.19 1.03 RAD1 -1.46 -1.46 -1.17 RAD17 -1.24 -1.16 -1.13 RAD51 -1.31 -1.43 -1.18 RAD9A -2.49 -1.36 1.29 RB1 1.09 -1.10 -1.14 RBBP8 1.22 -1.28 -1.01 RBL1 1.08 -1.10 -1.62 RBL2 -1.25 -1.38 -1.06 RPA3 1.29 -1.18 -1.11 SERTAD1 -1.14 -1.18 1.32 SKP2 -1.40 -1.29 -1.21 SUMO1 -1.06 -1.30 1.17 TFDP1 -1.19 1.10 -1.02 TFDP2 1.06 -1.08 -1.14 TP53 -1.02 -1.36 -1.07 UBE1 -1.04 1.20 1.01 B2M 1.34 1.11 -1.29 HPRT1 -1.07 -1.65 1.04 RPL13A 1.16 1.14 1.05 GAPDH -1.03 1.28 1.17 ACTB -1.41 1.02 -1.01 Shaded regions are genes selected for analysis Relative quantification of various genes in ATM knockdowns of different BRCA background 81 Appendix Untreated H-U HeLa H3 H1 2h posttreatment H-U H3 H1 Size (kDa) ATM 350 β actin 43 2h posttreatment Untreated Size (kDa) HCC-U HCC3 HCC-U HCC3 HCC1937 ATM 350 β actin 43 Untreated C-U Capan-1 C1 2h posttreatment C-U C1 Size (kDa) ATM 350 β actin 43 Western blot detection for ATM and β actin expression in HeLa, HCC1937, Capan-1 and their respective ATM knowndown clones before and after hours of etoposide treatment H-U, C-U and HCC-U represent control HeLa, Capan-1 and HCC1937 cells H1 and H3, HCC3 and C1 represent ATM knockdown clones of HeLa, HCC1937 and Capan-1 respectively ATM expression remained unaltered on etoposide treatment in all cells tested 82 Appendix 3A BRCA1 expression in HeLa and HCC1937 cells was probed with an antibody against the C- terminal of BRCA1 Alexa 488-labeled Goat anti-mouse IgG (H+L) (Molecular Probes, USA) secondary antibody was used to visualize expression DAPI was used to stain the nucleus of the cells HeLa cells express full length BRCA1 in their nucleus while HCC1937 cells not express functional BRCA1 83 Appendix 3B BRCA2 expression in HeLa and Capan-1 cells was assessed with a wild type antiBRCA2 antibody Alexa 568-labeled Goat anti-rabbit IgG (H+L) (Molecular Probes, USA) secondary antibody was used to visualize expression DAPI was used to stain the nucleus of the cells BRCA2 is localized in the nucleus of HeLa cells (positive control) Capan-1 expresses a truncated dysfunctional form of BRCA2 in the cytoplasm 84 Appendix Chemical composition of media Luria-Bertani broth (LB), pH 7.0 Bacto-tryptone 10 g Bacto-yeast extract 5g NaCl 5g Double distilled water 1000 ml SOC medium, pH 7.0 Bacto-tryptone 20 g Bacto-yeast extract 5g NaCl 0.5 g KCl 0.186 g Double distilled water 1000 ml Add 20ml of sterile 1M glucose before use 85 Appendix Multiple representative images (20x) of AT-Tert BACAtm (missense) cells during (a-d) or after release from selection (e-h) 86 .. .INVESTIGATION INTO THE ROLES OF ATAXIA TELANGIECTASIA MUTATED GENE PRODUCT, IN MULTIPLE BRCA BACKGROUNDS HIONG KUM CHEW (B.Sc., NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE... investigate the contribution of simultaneous loss of ATM and BRCA function in multiple cell culture based models We utilized various approaches to investigate the role of ATM in multiple BRCA backgrounds. .. various BRCA backgrounds exhibited differential sensitivity and survival responses Assessment of the expression profile of the knockdown and control cells using a PCR array of 84 genes involved in the