Human telomerase reverse transcriptase (hTERT) overexpression modulates intracellular redox balance and protects cancer cells from apoptotic cell death

HUMAN TELOMERASE REVERSE TRANSCRIPTASE (hTERT) OVEREXPRESSION MODULATES INTRACELLULAR REDOX BALANCE AND PROTECTS CANCER CELLS FROM ROS MEDIATED CELL DEATH INTHRANI D/O RAJA INDRAN (BSc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS This thesis would not have been possible if not for the many special people who have stood by me and guided me along in my last four years. It gives me great pleasure to have this opportunity to thank these wonderful people. To Prof Shazib Pervaiz, I owe him special thanks for the unwavering faith he had placed in me especially when I was struggling in the initial phases of my project. His relentless encouragement, enthusiasm and knowledge have been truly inspiring and have added immense value to the calibre of this thesis. I will never forget the random little pep talks, the times spent in non intellectual pursuit discussing the special attributes of 3am and the reasonable and unreasonable grilling sessions we had during lab meetings. Thank you boss for these cherished times and for simply being there for me over the years. To Dr Prakash Hande, I extend my sincere thanks to him. I am truly grateful for the autonomy, freedom and timely advice he gave me in manoeuvring my project over the last four years. The efforts he expended in getting hold of the numerous plasmids and cell lines that I needed, helped open some of the crucial doors in my project. Thank you very much for all the support you have given me, sir. To my lab mates from both ROS Biology and Apoptosis lab and Genome Instability lab, all of you have made this journey an immensely memorable one with all your little quirks, random words of wisdom, motivation and constructive advice. I am privileged to have known all of you and am thankful for the valuable friendships that have been forged through the years. I will never forget our late nights in lab, our communal ranting against antibodies that never worked and our inspirational meal sessions. My special thanks to the class of 2005, Sinong, Chewy, Zhi Xiong, Greg, Lakshmi, Ai Kia, Swamy and Grace for having shared my happiness, frustrations, successes and failures and for being a huge source of support for me. Thank you all loads. I would also like to give special mention to Dr Jayshree and Kartini for taking care of the needs of the lab and being such patient and loving people whom I could turn to i anytime to relieve my frustrations. Together both of you have had a pseudo calming effect on the lab environment. In addition, I have also learnt and benefited largely from your immense wealth of scientific experience. Thank you very much. My special thanks also goes out to Dr Alan Premkumar and Dr Andrea Holmes for their kind guidance and resources that have largely shaped and developed my research ideas and inevitably contributed to the progress of my project. Thank you very much! To all my close friends Sharon, Gerry, Nurul, Ruben, Chandra, Soy, Waseem, Tahira Praveena, Prabha, Sajitha, and Vanitha thank you very much for you kindness, love and patience. Thank you for the lifts and company that have made my hour and a half long journeys between campus and home seem like nothing. Thank you for your unyielding support, the little random notes of encouragement and the colour you have added to my life. You guys are truly special and I love you all! Lastly, to my family, the people without whom, I would have never made it this far in life. To my most beloved parents, even in their toughest times, they only had words of love and encouragement for me. Their giving nature supersedes everything. To my Amma and Naina, thank you very much for your unconditional love and motivation all these years. To my sister, who has been one of the biggest pillars of strength in my life, thank you for shielding me, grooming me, inspiring me and for the confidence you placed in me. Watching you, I have learnt the true meaning of resilience. Thank you Ka! Also, special thanks to my Jega for his kind love, patience and understanding especially during some of my very stressful times. Thanks for being by my side. I love you all very much. ii TABLE OF CONTENTS Acknowledgements ii Table of contents iv Summary x List of figures xii List of abbreviations xv INTRODUCTION………………………………………………………… .1 1. CANCER.… …………………………………………………………………1 2. TELOMERES AND TELOMERASE…………………………………… .2 2.1. Telomeres and telomerase regulation….………………………………………4 2.1.1. Transcriptional regulation of hTERT. ……………………… …………… .5 2.1.2. Post transcriptional modification of hTERT………………… ……………7 2.2. Senescence and immortalization………………………………………………9 2.3. The non canonical roles of telomerase……………… ………………… ….12 3. REACTIVE OXYGEN SPECIES………………………………………….13 3.1. Different types of ROS …………………………………………………… 14 3.2. Sources of ROS ……… …………………… …………………………… .15 3.3. Functions of ROS…………………………………………………………….16 4. THE CELLULAR ANTIOXIDANT DEFENCES……………………… 18 4.1. Superoxide dismutases……….…………………………………………… 20 4.2. Catalase…………………………….……………………………………… .21 4.3. Glutathione and glutathione dependent enzymes….…………………………22 4.3.1. Glutathione …………………………… .………………………………… 22 iii 4.3.2. GSH synthesis.…………………………… .……………………………… 24 4.3.3. Glutathione reductase.…………………………… .…………………… ….25 4.3.4. Glutathione peroxidase.…………………………… .…………………….…26 4.4. Peroxiredoxins……………… ………………………………………………27 4.5. Thioredoxins……………………… ……………………………………… .29 5. hTERT AND ROS………… .…………………………………………… 30 5.1. Effects of oxidative stress on hTERT……………… ……………………….31 5.2. Effects of hTERT on oxidative stress…… ………………………………….33 6. CELL DEATH…………………………………………………………… 34 6.1. Necrosis…… .……………………………………………………………….35 6.2. Autophagy……………………………………………………………………36 6.3. Apoptosis…… ………………………………………………………………36 6.3.1. Type I – extrinsic or receptor mediated pathway…………………………….37 6.3.2. Type II – intrinsic or mitochondria mediated pathway…………………… 37 6.4. Influence of ROS on cell death signalling……………………………………38 6.5. Mitochondria membrane permeabilization….……………………………….41 6.6. Regulation of apoptosis by Bcl-2 family of protein………………………….43 7. CANCER THERAPY, ROS AND TELOMERASE…………………… .44 AIMS……………………………………………………………… .47 MATERIALS AND METHODS……….………………………………… .50 1. Cell Lines and plasmids…………………………………………………….…50 2. Antibodies…………………………………………………………………… 50 3. Chemicals………………………………………………………………….… 51 4. Cell culture…………………………………………………………………….51 iv 5. Plasmid information .………………………………………………………52 6. Amplification and purification of plasmids………………………………… .53 7. Transient transfection .……………………………………………………….55 8. Generation of stable clones……………… .……………………………… .56 9. Silencing……………………………… .…………………………………….56 10. Crystal Violet cell viability assay…………………………………………… 57 11. Analysis of DNA fragmentation by propidium iodide staining……………….57 12. Colony forming assay… .…………………………………………………….58 13. Assessment of intracellular ROS levels via CM-H2DCFDA staining……… .58 14. Assessment of mitochondrial O2.- levels via MitoSOX Red staining…………58 15. Assessment of mitochondrial membrane potential using DIOC6…………… 59 16. Assessment of intracellular reduced / oxidised glutathione levels……………59 17. Glutathione peroxidase assay… ………………………………………….… 62 18. Glutathione reductase assay……………………………………………… ….63 19. Assessment of telomerase activity using TRAP assay……………………… 64 20. Assessment of Cytochrome c oxidase activity……………………………… 65 21. Detection of hTERT gene expression by RT-PCR………………………… 66 22. Determination of protein expression by western blot analysis……………… 66 22.1. Buffers used for western blot analysis……………………………………….67 23. Isolation of mitochondrial and cytosolic fractions……………………………69 24. Isolation of nuclear and cytosolic fractions………………………………… .70 25. Measurements of protein concentration (Bradford Assay)………………… 70 26. Statistical analysis…………………………………………………………… 71 v RESULTS….…………….……….……………………………………… 72 1. hTERT AND ROS CAN CROSS REGULATE EACH OTHER … .72 1.1. hTERT expression and activity is regulated by H2O2 in a dose dependant manner…….……………………………………………………….…………72 1.2. hTERT localization can be modulated by exposure to ROS…………… .74 1.3. Transient hTERT overexpression can modulate intracellular ROS levels……………………………………………………….……………… 76 1.4. Transient hTERT overexpression resists the increase in intracellular ROS levels following treatment with H2O2………… .………………………… .79 1.5. Transient hTERT overexpression resists the increase in intracellular ROS induction following treatment with the ROS inducing compound C1……….82 1.6. Generation of stable hTERT overexpressing cells and verification of hTERT expression and activity in stable clones .84 1.7. Stable hTERT overexpression reduces basal intracellular ROS…………… 86 1.8. Stable hTERT overexpression blocks intracellular ROS induction following treatment with H2O2 or C1.…… .………………………….…….89 1.9. Stable hTERT overexpression alters hTERT expression and localization patterns……………………………………………………………………….92 2. UNRAVELING THE MECHANISMS BY WHICH hTERT EXPRESSION MODULATES INTRACELLULAR REDOX STATUS………………………………………………………………… 94 2.1. Assessment of critical antioxidant defences………………………………….94 2.1.1. Assessment of intracellular SOD and Catalase expression following hTERT overexpression………… …………………………………….….…95 2.1.2. Assessment of intracellular Peroxiredoxin and Thioredoxin levels following hTERT overexpression… .… ……… ……………….…………98 2.1.3. hTERT overexpression increases the rate of regeneration of Peroxiredoxins from the hyperoxidised forms ……………………….…100 2.1.4. hTERT overexpressing cells maintain a higher intracellular GSH/GSSG ratio following H2O2 treatment………………………………………… .…102 2.1.5. hTERT overexpressing cells maintain higher intracellular mitochondrial GSH levels following H2O2 treatment………………… 104 vi 2.1.6. hTERT overexpression results in earlier and sustained induction of GCLC levels following H2O2 treatment.……………………… ………… 106 2.1.7. hTERT overexpression results in differential regulation of Glutathione dependant enzymatic activities……….……………………………….… .107 2.2. hTERT overexpression results in improved mitochondrial function……….110 2.2.1. hTERT localizes to the mitochondria…………………………………….…110 2.2.2. hTERT expression increases Cytochrome c oxidase activity………………112 3. THE EFFECTS OF hTERT EXPRESSION IN AN ALTERNATIVE MODEL; SH-SY5Y CELLS ………………………………… ………….114 3.1. Studying the influence of hTERT expression in SH-SY5Y Cells …… .114 3.1.1. hTERT silencing in SH-SY5Y cells potentiate the increase in intracellular ROS levels following treatment with H2O2 ………………… .114 3.1.2. hTERT silencing reduces the rate of Peroxiredoxin regeneration from the hyperoxidised form in SH-SY5Y cells…………………………….……118 3.1.3. hTERT silencing results in reduced GSH/GSSG ratios in SH-SY5Y cells following H2O2 treatment .…………………………………………………118 4. hTERT EXPRESSION IMPAIRS ROS INDUCED CHANGES IN INTECELLULAR MILIEU AND PROTECTS FROM CELL DEATH…………………………………………… 120 4.1. Induction of cytosolic acidification by H2O2 and C1 is reduced in hTERT overexpressing Cells…………………………………………….….120 4.2. Mitochondrial Bax translocation and release of pro-apoptogenic factors is partially inhibited in hTERT overexpressing cells…… ………… .124 4.3. Dissipation of the mitochondrial membrane potential (m) is reduced in hTERT overexpressing cells… .…………………………………… .126 4.4. hTERT overexpression can protect cells from H2O2 induced cell death .130 4.5. hTERT overexpression can protect cells from C1 induced cell death…… .135 vii DISCUSSION…………………………………………………………….…138 1. THE USE OF DIFFERENT MODELS IN THIS STUDY…… .…138 1.1. The use of HeLa cell Line and the limitations in telomerase biology…… 138 1.2. The use of transient and stable transfection models in this study……… …139 1.3. The use of H2O2 and C1 as tools to investigate the relationship between hTERT and ROS and the different time points at which the investigations were performed.………………….………………………….………………141 2. ESTABLISHING THE RELATIONSHIP BETWEEN hTERT AND ROS………………………………………………………………… 142 2.1. ROS mediated suppression and nuclear export of hTERT can be antagonized by hTERT overexpression……… ……………………… ….142 2.2. hTERT expression significantly reduces basal intracellular ROS levels and antagonize the increase in cellular ROS levels in response to oxidative stress…………………………………………………………… .144 3. MECHANISMS THAT hTERT CAN EMPLOY TO MODULATE CELLULAR ROS LEVELS…………………………………………… 145 3.1. Cellular antioxidant defences…………………………………………… .146 3.2. hTERT expression can enhance the Glutathione antioxidant defences…….147 3.3. hTERT expression improves Peroxiredoxin regeneration…………… … .149 3.4. hTERT expression enhances mitochondrial antioxidant defences……… 150 3.5. How hTERT could modulate the antioxidant defences? .151 3.6. The role of hTERT at the mitochondria…………………………………….154 4. PHYSIOLOGICAL SIGNIFICANCE OF MODULATION OF CELLULAR REDOX BALANCE BY hTERT……………………… 156 4.1. hTERT expression creates an intracellular environment unfavourable for eliciting death triggers…………………………………… .……… .156 4.2. hTERT expression protects cells from apoptotic triggers………………… 158 viii 5. CONCLUDING REMARKS………………………………………… .…160 6. REFERENCES…………………………………………………………….164 7. APPENDIX……………………………………………………………… .182 ix Chou, W.C., Hawkins, A.L., Barrett, J.F., Griffin, C.A., and Dang, C.V. (2001). Arsenic inhibition of telomerase transcription leads to genetic instability. J Clin Invest 108, 1541-1547. Clement, M.V., Hirpara, J.L., and Pervaiz, S. (2003). Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumour cells to receptor and drug-induced apoptosis independent of the mitochondria. Cell Death Differ 10, 1273-1285. Clement, M.V., and Pervaiz, S. (1999). Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: an hypothesis. 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Hande, Shazib Pervaiz. hTERT overexpression alleviates intracellular ROS production, improves mitochondrial function and endows cells with a survival advantage against ROS-mediated apoptosis. (Manuscript in progress) CONFERENCE PAPERS Inthrani R. Indran, Manoor P. Hande, Shazib Pervaiz. (2008) Telomerase reverse transcriptase (hTERT) overexpression protects cancer cells from ROS mediated cell death. American Association for cancer research. San Diego, America. Inthrani R. Indran, Manoor P. Hande, Shazib Pervaiz. (2009) Telomerase reverse transcriptase (hTERT) overexpression protects cancer cells from ROS mediated cell death. EMBO Workshop on Mitochondria, Apoptosis and Cancer. Prague, Czech Republic. * * Received the Best Poster Award 182 [...]... et al., 2001) Telomerase is differentially regulated under normal and pathological conditions While in most human somatic cells telomerase activity is extinguished during embryonic differentiation, the highly proliferative cells such as stem cells, germ cells, activated lymphocytes, haematopoietic progenitor cells, intestinal crypt cells, endometrial cells, basal layer of skin cells and cervical keratinocytes... resistance are the major goals in developing anti cancer agents today In order to achieve these goals, exploitation of the intrinsic differences between normal and cancer cells could be a valuable approach to target cancer cells for cell death One of the glaring differences between normal cells and cancer cells is their difference in telomerase expression Telomerase is is a multi component ribonucleoprotein... transfection of HeLa cells with pBabe-Neo and pBabe-hTERT-Neo Figure 20: Stable hTERT overexpression reduces basal intracellular ROS Figure 21: Stably hTERT overexpressing cells displays lower intracellular ROS when treated with H2O2 Figure 22: Stably hTERT overexpressing cells displays lower intracellular ROS when treated with C1 Figure 23: hTERT expression and localization in Neo and HT1 cells following... of intracellular SOD and Catalase expression following hTERT overexpression and H2O2 treatment Figure 25: Regulation of Peroxiredoxins and Thioredoxin Figure 26: hTERT overexpression increases the rate of Peroxiredoxin regeneration from the hyperoxidised form Figure 27: hTERT overexpressing cells maintain a higher intracellular GSH/GSSG ratio following H2O2 treatment Figure 28: hTERT overexpressing cells. .. 1 Singapore Cancer Registry 1 strands in the telomere regions thus playing a crucial role in telomere maintenance It is active in almost all cancer cells but is inactive in most somatic cells with the exception of a few, including stem cells and germline cells, thus making it an attractive target for cancer therapy 2 TELOMERES AND TELOMERASE Telomeres are specialized structures that cap and preserve...SUMMARY The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase holoenzyme which is critical for the maintenance of telomere lengths and the enhanced replicative capacity of the cells Being silenced during embryonic differentiation in normal cells, its reactivation in over 85% of cancer cells has made hTERT an attractive therapeutic target in the field of cancer However... balance that the cells maintain for optimal survival ROS are important second messengers who regulate numerous pathways and redox regulated transcription factors like Nf-Kb and AP1 and steer cells towards cell growth and survival (Bubici et al., 2006; Clerkin et al., 2008) Furthermore, a slight increase in intracellular ROS, and specifically O2.-, has been shown to favour survival by antagonizing apoptotic. .. is assembled onto the template (b) (Neidle and Parkinson, 2002) 3 2.1 Telomerase and telomerase regulation The telomerase holoenzyme consists of a catalytic protein subunit, human telomerase reverse transcriptase (hTERT), the RNA component of the telomerase (hTR) that is used as the template by the reveres transcriptase to elongate the telomeric ends, and telomerase associated proteins such as TEP1... it has been reported that in normal ovarian tissues and uterine leiomyoma, cells have no detectable telomerase activity despite expressing both hTR and full length hTERT mRNAs (Ulaner et al., 2000) This lack of correlation between hTERT expression and telomerase activity has also been noted in peripheral T and B cells, human colon and renal tissues and tumours The discordance was attributed to the difference... genome from oxidative stress and the cells from apoptosis (Ahmed et al., 2008) Another novel role for hTERT is its involvement in modulating cellular responses to apoptotic triggers Indeed, it has been shown that targeting hTERT may help sensitize a variety of cancer cells to apoptotic triggers (Bollmann, 2008; Cong and Shay, 2008; Sung et al., 2005) Several studies have also shown that the apoptotic . HUMAN TELOMERASE REVERSE TRANSCRIPTASE (hTERT) OVEREXPRESSION MODULATES INTRACELLULAR REDOX BALANCE AND PROTECTS CANCER CELLS FROM ROS MEDIATED CELL DEATH INTHRANI. normal and cancer cells could be a valuable approach to target cancer cells for cell death. One of the glaring differences between normal cells and cancer cells is their difference in telomerase. overexpressing cells …………………………………… 126 4.4. hTERT overexpression can protect cells from H 2 O 2 induced cell death 130 4.5. hTERT overexpression can protect cells from C1 induced cell death … 135