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UBC Theses and Dissertations

UV-Induced DNA damage response in blood cells as a potential method for cancer detection Farivar, Negin 2019

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 UV-INDUCED DNA DAMAGE RESPONSE IN BLOOD CELLS AS A POTENTIAL METHOD FOR CANCER DETECTION  by  Negin Farivar  B.S., Sharif University of Technology, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2019  © Negin Farivar, 2019    ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  UV-INDUCED DNA DAMAGE RESPONSE IN BLOOD CELLS AS A POTENTIAL METHOD FOR CANCER DETECTION   submitted by Negin Farivar in partial fulfillment of the requirements for the degree of Master of Applied Science in Chemical and Biological Engineering  Examining Committee: Dr. Fariborz Taghipour Co-supervisor Dr. Mads Daugaard Co-supervisor  Dr. James Piret Supervisory Committee Member Dr. Vikramaditya Yadav Additional Examiner  Additional Supervisory Committee Members:  Supervisory Committee Member  Supervisory Committee Member  iii  Abstract  Early detection of cancer is the single best parameter predicting positive outcome for patients. Detection of cancer in the early disease stages usually requires a combination of tests that are often expensive for our health care systems and invasive for the patients. Hence, developing a simple crude non-invasive liquid biopsy test able to detect cancer would be of utmost significance. This proof of concept case-study aimed to investigate the potential of developing an assay to distinguish between cancer patients and healthy individuals based on their immune cells’ responses to ultraviolet light exposure. The new technology of ultraviolet light emitting  diodes (UV-LEDs) makes it possible to provide exact irradiating doses and wavelengths, superior to traditional UV lamp-based light sources for these types of studies. The human leukemia cell line, Jurkat,  was used as a model to develop and improve the assay. Expression and phosphorylation of the DNA repair marker histone 2AX (γH2AX), was analyzed in 18 human blood samples after UV irradiation: 9 samples from prostate cancer patients all undergoing Radical Prostatectomy treatment with no history of neoadjuvant therapy and 9 control samples from healthy individuals. The expression of γH2AX was amplified 6 hours after exposure in all samples and the dose (3 mJ/cm2) and the wavelength (285 nm) chosen for this study induced cell death in blood cells after 24 hours. The median of the normalized γH2AX expression was significantly different between prostate cancer (PCa) patients and healthy donors (p<0.01). Receiver operating characteristic (ROC) analysis of normalized γH2AX expression in peripheral blood monocyte cells (PBMCs) isolated from PCa patients vs. PBMCs isolated from healthy donors gave an area under the curve (AUC) of 0.9306 (P=0.0029). The method described in this study had sensitivities, specificities, and an AUC comparable to current cancer detection assays currently in clinical use. As such, this method could potentially provide a supplement to current gold standard early cancer detection applications for the benefit of patients and our health care economy.   iv  Lay Summary  Cancer is the second leading cause of death globally responsible for an estimated 9.6 million deaths in 2018. Precise diagnosis depend on biomarkers for specific cancers. However,  a crude first line pre-screening method able to detect cancer could facilitate earlier diagnosis. This study aimed to develop a method for identifying patients with cancer amongst healthy individuals, by exposing their immune cells to ultra violet irradiation. Evidence suggest that immune cells functionality might be impaired in patients with cancer due to tumor-associated inflammatory stress. We hypothesised that, as a result of inflammatory stress, the immune system in a cancer patient might be more vulnerable to a generic DNA damaging agent like UV. The method developed in this study could have a potential application in early cancer detection and might reduce the need for expensive cancer specific tests by identifying those who do not require further investigation.    v  Preface This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical and Biological Engineering. In this thesis, I have been the major contributor alongside my supervisors, Dr. Fariborz Taghipour and Dr. Mads Daugaard. I have conducted all of the experimental work, the statistical analysis and the write up. All the illustrations demonstrated in this thesis are originally created be me. All work contained herein is unpublished. The clinical and pathology procedures performed in this research was approved by the UBC Clinical Research Ethics Board. (Certificate number H14-03035).   vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Chapter 1: Background, hypothesis and objectives....................................................................1 1.1. Cancer Epidemiology and Detection .............................................................................. 1 1.1.1. Prostate Cancer ....................................................................................................... 1 1.1.1.1. Overview ................................................................................................................ 1 1.1.1.2. Prostate Cancer Staging and Treatment ................................................................. 2 1.2. Genomic instability and Cancer susceptibility ............................................................... 4 1.3. UV-induced DNA damage .............................................................................................. 5 1.3.1. Cyclobutane pyrimidine dimers .............................................................................. 6 1.3.2. Pyrimidine (6-4) pyrimidone dimers and their Dewar valence isomers ................. 8 1.3.3. UV-induced purine photoproducts .......................................................................... 9 1.3.4. UV induced DNA double strand breaks ............................................................... 10 1.3.5. UV-induced DNA damage detection .................................................................... 11 1.4. DNA UV-induced Damage Repair ............................................................................... 12 vii  1.4.1. Photoreactivation .................................................................................................. 13 1.4.2. Excision repair ...................................................................................................... 13 1.4.2.1. Base excision repair (BER) ........................................................................... 13 1.4.2.2. Nucleotide Excision Repair (NER) ............................................................... 14 1.4.2.3. Differences between GG-NER and TC-NER in mammalian cells ............... 15 1.4.3. Recombinational repair ......................................................................................... 17 1.4.3.1. Homologous recombination .......................................................................... 17 1.4.3.2. Non-homologous end joining ....................................................................... 18 1.4.4. UV-induced apoptosis (Other repair machineries) ............................................... 19 1.5. Research hypothesis and objectives .............................................................................. 20 1.5.1. Hypothesis............................................................................................................. 20 1.5.2. Research objectives ............................................................................................... 21 Chapter 2: Materials and Method ..............................................................................................22 2.1. Experimental setup........................................................................................................ 22 2.2. UV dose and irradiance ................................................................................................. 23 2.3. Cell culture .................................................................................................................... 24 2.4. Blood samples, and Peripheral Blood Mononuclear Cell (PBMC) separation ............. 24 2.5. UV-Irradiation............................................................................................................... 25 2.6. Western Blot ................................................................................................................. 26 2.7. Flow Cytometry ............................................................................................................ 27 2.7.1. Cell cycle analysis and PI staining........................................................................ 27 2.7.2. Fixable viability dye staining and fixation............................................................ 27 2.7.3. Permeabilization and γH2AX intercellular staining ............................................. 27 viii  2.8. Statistical analysis ......................................................................................................... 29 Chapter 3: Results and Discussion .............................................................................................30 3.1. Developing and optimizing the assay ........................................................................... 30 3.2. Cell Cycle analysis after UV exposure ......................................................................... 34 3.3.       Description of study subjects ........................................................................................ 36 3.4. Measurements of UV-induced DNA damage in PCa patients ...................................... 37 3.4.1. γH2AX detection with flow cytometry ................................................................. 37 3.4.2. Time-course analysis of Radical Prostatectomy patients...................................... 39 3.5. Comparison of post-UV γH2AX expression in normal study subjects and PCa patients 45 3.6. ROC curve analysis....................................................................................................... 48 Chapter 4: Conclusion and Recommendations .........................................................................50 Bibliography .................................................................................................................................52 Appendices ....................................................................................................................................63 Appendix A : KI/KIO3 Actinometry ......................................................................................... 63 Appendix B : UV-LED specification sheet .............................................................................. 66 Appendix C : Male vs. Female response to UV exposure ........................................................ 68 ix  List of Tables  Table 1. Prostate cancer stages . ..................................................................................................... 3 Table 2. Experimental variables and conditions ........................................................................... 26 Table 3. Antibodies Information ................................................................................................... 28 Table 4. Patient's clinical information .......................................................................................... 37 Table 5. Mann whitney test parameters. ....................................................................................... 47  x  List of Figures  Figure 1. Structures of the cyclobutane pyrimidine dimers ............................................................ 7 Figure 2. Formation of uracil containing product via hydrolysis of the C4 amino group .............. 8 Figure 3. Pathway of UV induced 6-4PPs and their Dewar isomers .............................................. 9 Figure 4. Purine photoproducts ..................................................................................................... 10 Figure 5. GG-NER and TC-NER mechanism............................................................................... 15 Figure 6. Schematic representation of recombinational pathways ............................................... 18 Figure 7. Experimental apparatus schematics ............................................................................... 23 Figure 8. Dose dependent UV-induced γH2AX formation .......................................................... 31 Figure 9. Time dependent phosphorylation of γH2AX.. .............................................................. 32 Figure 10. Simplified DNA damage response diagram ................................................................ 33 Figure 11. Histogram of Gated G0/G1/s/G2/M phase cells .......................................................... 34 Figure 12. Jurkat cell line cell cycle analysis by flow cytometry.. ............................................... 36 Figure 13. Representative FCM plots showing the gating strategy .............................................. 39 Figure 14. Time course analysis of γH2AX expression after UV exposure in patients’ samples. 42 Figure 15. γH2AX expression level in 6 hour post-UV normalized to 6 hour control expression........................................................................................................................................................ 42 Figure 16. Normalized γH2AX expression after UV exposure in regards with Clinical Data.. ... 44 Figure 17. Normalized percentage of live and γH2AX positive cells in PCa patients (A) compared to Healthy Donors (B). Box and whisker plots, comparing PCa patients with healthy donor subject group (C). ............................................................................................................... 46 xi  Figure 18. Number of cells live and positive for γH2AX at 6 hour and 24 hour post-UV irradiation time point in healthy donors (A) and prostate cancer patients (B) .............................. 48 Figure 19. ROC curve analysis ..................................................................................................... 49  xii  List of Abbreviations 53BP1 P53 Binding Protein ATM Ataxia Telangiectasia Mutated ATR Ataxia Telangiectasia Rad3-related BER Base Excision Repair BRCA1 Breast Cancer type 1 Susceptibility Protein CD4 Cluster of Differentiation 4 CPD Cyclobutane Pyrimidine Dimer DDR DNA Damage Response DP DNA Polymerase DRE Digital Rectal Examination DSB Double Strand Break EAU European Association of Urology ERCC1 Excision Repair Cross-Complementation Group1 FDA Food and Drug Administration FVD Fixable Viability Dye GAPDH Glyceraldehyde 3-phosphate Dehydrogenase GG-NER Global Genome NER HR Homologous Recombination MDC1 Mediator of DNA Damage Checkpoint 1 NER Nucleotide Excision Repair NHEJ Non-Homologous End Joining xiii  PBMC Peripheral Blood Mononuclear Cell PBS Phosphate Buffer Saline PCa Prostate Cancer PMT Photo-Multiplier Tubes PSA Prostate Specific Antigen ROC Receiver Operating Characteristics ROS Reactive Oxygen Species RP Radical Prostatectomy SSB Single Strand Break ssDNA Single-Stranded DNA SWI/SNF SWItch/Sucrose Non-Fermentable TC-NER Transcription-Coupled NER TFIIH Transcription Factor II Human TNM  Tumor, Node, Metastases Top1 Topoisomerase 1 TURP Transurethral Resection of the Prostate UV-LED Ultraviolet Light Emission Diodes UVR Ultra-Violet Radiation XPC Xeroderma Pigmentosum, complementation group C XPF(ERCC4) Excision Repair Cross-Complementation Group 4 XRCC2 X-Ray Repair Cross-Complementing Group 2 XRCC3 X-Ray Repair Cross-Complementing Group 3 xiv  Acknowledgements I would like to express my sincere appreciation to my supervisors Dr. Fariborz Taghipour for being a great mentor who guided me with patience and optimism, and Dr. Mads Daugaard for his wonderful supervision and insightful inputs over the last two years. Thank you for giving me this opportunity to pursue this master project.  I am very thankful for both lab group members for their generous help and support during this project. Dr. Ata Kheirandish of the Taghipour group, helped me a lot in designing and manufacturing my experimental apparatus. Dr. Beibei Zhai of the Daugaard group also assisted in designing the experiments and troubleshooting. It was my honor to work in the Vancouver Prostate Centre, and I wish to express my gratitude to Dr. Peter Black for his generous help in this project. I am so pleased to have worked in such a friendly and supportive environment. Finally, I would like to thank my friends and family, who are the dearest to my heart. I am so grateful for all of the support and love from my wonderful parents and my beloved sister, Niki, for always being my closest friend.             1  Chapter 1: Background, hypothesis and objectives 1.1. Cancer Epidemiology and Detection Cancer is the leading cause of death in Canada. Canadian Cancer Society projects that nearly 1 in 2 Canadians will develop cancer in their lifetime and 1 in 4 Canadians will die from cancer [1]. National Cancer Institute of Canada indicates that the number of newly registered cancer cases has increased from 159,900 cases/yr to 206,200 cases/yr during the past 10 years [2]. A very recent study by de Oliviera et al. [3] shows that the cost of cancer (e.g. diagnosis, treatment, etc. as direct costs and  resources lost due to inability to work, permanent disability and death before 65 years of age as indirect costs) has increased from $2.9 billion to $7.5 billion over the past decade in Canada. Early diagnosis of cancer can significantly increase the chance of treatment and prognosis and thus reduce the costs [4]. Cancer patients undergoing treatments such as chemotherapy, radiotherapy, hormonotherapy, etc., are subjected to severe physiological stresses. In addition to the serious physical side effects of such treatments, more than 80% of the patients report fatigue causing adverse psychological (e.g., depression, anxiety), situational (e.g., sleep deprivation) and loss of productivity leading to economic consequences which can result in undesired routine change and affect the quality of both patients’ and caregivers’ life [5]. A direct survey on 2287 newly diagnosed cancer patients shows that the labor participation of patients and their caregivers has reduced by 36% and 23 %, respectively, resulting in a wage loss of $3.18 billion [6]. Hence, there is a clinical need to develop assays to diagnose cancer in early stages to reduce these individual and socio-economic consequences of cancer.   1.1.1. Prostate Cancer  1.1.1.1. Overview Prostate cancer (PCa) is the most common non-skin cancer among men worldwide [7]. Prostate cancer incidence rate is higher in high resources areas including North America and Europe, while the mortality rate for prostate cancer is higher in low resources and developing countries such as, South America, the Caribbean, and sub-Saharan Africa [8]. Early detection of cancer is the key strategy to significantly decrease the mortality rate and enhance prognosis. After the discovery of prostate-specific antigen (PSA), and later on its approval by the U.S. Food and 2  Drug Administration (FDA), it has become the prevailing test for detection. PSA is a kallikrein-type serine protease that maintains the liquefaction of seminal fluid and is generated and secreted by the prostate epithelial cells [9]. However, over the years there has been clinical  trial evidence showing the limitations of the PSA testing [10]. One of the drawbacks of PSA testing is low specificity leading to unnecessary treatments as a result of over-diagnosis [11]. PSA testing has no definite cut-off to ensure whether the tumor exists or not, furthermore about 15% of prostate cancer cases happens in men with low serum PSA levels [12]. The majority of current diagnosis methods are laboratory-based techniques such as immunoassays which are sensitive and precise but very labor-intensive and time consuming. Other methods such as digital rectal examination (DRE) and biopsy are considered to be invasive towards the patient [13]. Therefore, with the current existing challenges in prostate cancer diagnosis we chose this cancer as a model for us to explore the possibility of detecting early stage cancer with a simple blood test which will be described in this thesis.  1.1.1.2. Prostate Cancer Staging and Treatment Currently, Gleason Grading system is used to evaluate the prognosis of prostate cancer (PCa) based on prostate biopsy and tissue histology. This system includes 5 grades (1-5) and combines the most common and the second most common pattern. Gleason scores ranging from 1-5  are no longer assigned, grade 6 is assigned to low-risk, 7 being intermediate and 8-10 considered as high risk [14], [15]. However, a contemporary grading system was suggested in 2015 which uses grade groups rather than Gleason score. It ranges from 1-5 with grade group 1 (Gleason score ≤ 6) being low-risk and grade group 5 ( Gleason score 9-10) being the most aggressive [16]. Additionally, the TNM classification of Malignant Tumor systems (a standard to classify the extent of cancer) is used.  In TNM system, T refers to the primary tumor within or adjacent to the prostate tissue, N describes metastasis to the lymph node and M refers to bone metastasis. Based on the guideline of the European Association of Urology (EAU), the primary treatment options for low-risk to intermediate PCa are: 1) Active surveillance for patients with clinically confined PCa (T1-T2), (Gleason score ≤ 6) and PSA ≤ 10 ng/ml. 2) Radical Prostatectomy (RP), is the only surgical treatment for localized PCa and refers to removal of all or parts of prostate gland. According to EAU recommendation, RP treatment is indicated for patients 3  with low to intermediate-risk localized PCa (cT1a-T2b), Gleason score 6-7, PSA ≤ 20 ng/ml and a life expectancy > 10 years. 3) Radiation Therapy and low-dose-rate brachytherapy for patients in stage cT1c–T2aN0M0 and a Gleason score of ≤ 7a on 12 random biopsies [17] (more details of TNM and Gleason score staging is reviewed in [18]). Table 1 shows a summary of PCa stages based on Gleason score and clinical stage and treatment options for each stage.   Table 1. Prostate cancer stages regarding the clinical stage, Gleason score, PSA level and treatment options.  Stage Clinical stage Gleason score PSA level Description Treatment option I T1,T2a,N0,M0 <6 <10 Cancer grows very slowly and may never cause symptoms or health problems. Active Surveillance Radical Prostatectomy  Radiation Therapy II T1-T2b-T2c-N0,M0 >6 >10 Cancer has not grown outside the prostate, but is larger with a higher Gleason Score.  Watchful waiting Radical Prostatectomy  Radiation Therapy Clinical Trial participation III T3,N0,M0 6-10 >20 Cancer has grown outside the prostate but has not reached the bladder or rectum.   External beam radiation plus hormone therapy  Radical Prostatectomy  Active Surveillance Clinical Trial participation  IV T4,N1,M1 6-10 >20 Cancer has spread to nearby areas such as the bladder or rectum, to nearby lymph nodes, or to distant organs such as the bones. Hormone Therapy External beam radiation  plus hormone therapy  Radical Prostatectomy  TURP Active Surveillance Clinical Trial participation  4  1.2. Genomic instability and Cancer susceptibility Genotoxins, genetic instability, genome integrity and probably DNA repair capacity seem to be the major factors for susceptibility to cancer in individuals [19]. Since the majority of human carcinogens are genotoxins, in terms of studying genome integrity and genotoxic insults, the World Health Organization International Programme for Chemical Safety recommends the use of lymphocytes because of the relative ease of sampling and the fact that blood lymphocytes circulate around the entire body so they are more likely to be exposed to most genotoxic insults and malignant lesions, also their subpopulations are long lived and can carry genetic mutations and abnormalities for more than 40 years [20], [21]. It has been suggested in previous studies that tumor associated cytokines and chemokines can impair lymphocyte functionality and these inflammatory processes can induce mutations leading to cancer progression and further genomic damage in lymphocytes [22].  The impaired tumor-associated lymphocyte functionality can be also probably linked to a state in T lymphocytes called ‘exhaustion’. During chronic infections and cancer that involves continuous exposure to antigens and/or inflammations, the programme of memory T cell differentiation is dramatically changed. A state termed T cell exhaustion [23]. T cell exhaustion usually exhibits several characteristics such as progressive loss of effective functions, upregulation and expression of inhibitory receptors [24]. Several pathways have been suggested to cause and develop T cell exhaustion such as, chronic exposure to antigens, inhibitory signals from cytokines, cell surface inhibitory receptors and other lymphocyte populations such as CD4+ T cell, B cells and regulatory T(Treg) cells. It has been known that depletion of CD4+ T cells contributes to defective CD8+ T cell response and increased exhaustion in these cells [25]. Although the exact role of these pathways in causing CD8+ T cell exhaustion is incompletely defined, the impact of exhausted CD8+ T and loss of CD4+ T cells is highly relevant especially in the context of DNA Damage Response (DDR). In a recent study, the ability of CD8 T cells to cope with DNA lesions has been evaluated with the presence and absence of  CD4+ T cells [26]. It has been shown that CD8 T cells manifest an exclusive DDR in their exponential division phase. In dividing CD8 T cells all DDR pathways and cell cycle check points are activated while in other cell types only a single DDR pathway is affected. They further showed in this study that in the absence of CD4 help, all secondary responses fail in CD8 T cells. Therefore, these T cells are not able to sense 5  DNA damage and all DNA repair mechanisms fail. In addition, they are not capable to stop cycling to repair the DNA lesion, thus, they are driven to compulsive suicidal division [26].  Overall, the fact that cancer impacts immune system and lymphocytes functionality has been proven in many studies, and there are moves toward classifications of malignant tumors based on immune system infiltration and ‘Immunoscore’ [27].   1.3. UV-induced DNA damage Ultraviolet radiation (UVR) is a well-known mutagen and one of the most powerful carcinogenic exogenous agents. It has the ability to interact with DNA and cause genetic instability which can affect the normal reproduction, development and life process of all organisms from prokaryotes to mammalian cells [28], [29]. UVR (wavelength 100–400 nm) consists of UVA (315–400 nm), UVB (280–315 nm), and UVC (100–280 nm). Most of the UV radiation that reach the earth’s surface is the less-energetic UVA, while the more energetic UVB and UVC are actively absorbed by atmospheric ozone [30]. The nature and the mechanism of UV-induced DNA damage mainly depend on the wavelength of the UVR. In UVB/C spectrum, direct absorption of radiation by DNA leads to dimerization reactions between adjacent pyrimidine bases, whereas in UVA radiation due to extremely weak direct radiation absorption by DNA, the excitation of endogenous chromophores causes the formation of singlet oxygen and DNA damage by indirect photosensitizing reactions [31], [32]. UV-induced DNA damages result in misincorporated bases in the replication process, deamination, deprurination, depyrimidination, and oxidative damages caused by UV radiation-induced free radical and reactive oxygen species [33], [34]. UVR exposure can lead to single as well as double DNA strand breaks where both strands are affected, they are the most destructive among all types of DNA damage and can result in loss of genetic material [35]. Reactive oxygen species (ROS), at high concentrations, can harm cell structures, proteins and DNA and lead to oxidative stresses which are associated with a number of human diseases [36]. The hydroxyl radicals (OH) can also damage all DNA components as well as the deoxyribose backbone, and restrain the normal functionality of the cells [34].    6  1.3.1. Cyclobutane pyrimidine dimers UVB is one of the more energetic components of the UV spectrum and is responsible for the majority of damages caused by direct light absorption by cellular structures. The three largest DNA lesions that UVB radiation can cause are cyclobutane pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone dimers(6-4PPs) and their Dewar valence isomers [35]. The first characterized bypyrimidine product was thymine (T <> T) cis-syn cyclobutane pyrimidine dimer, which led to an enormous increase and improvement in DNA photochemistry, photocarcinogenesis and DNA repair studies [37].  Cyclobutane pyrimidine dimers (CPDs) which can also appear in other main C-C, T-C and C-T bipyrimidine sequences are formed from a [2+2] photocycloaddition of C5-C6 double bonds of neighboring bases (Figure 1(A)). In an aqueous solution of isolated pyrimidine nucleobases, a mix of diastereoisomers (Figure 1 (B)) are formed upon UVB/C irradiation. They vary in the orientation of the two pyrimidine rings relative to the cyclobutane ring, and the relative orientations of the C5–C6 bonds in each pyrimidine base [38]. In oligonucleotides and DNA, in its natural configuration, the dimer contains two pyrimidine bases on the same strand, and because of the steric constrains, only the syn can be generated, therefore, the cis- syn isomer is greatly preferred over the trans-syn diastereomers [39]. In a denatured or single strand DNA, the backbone of the DNA has more flexibility, hence, the trans-syn isomer is more prevailing. The formation of these dimers has also been found in between double–stranded DNA upon UVC irradiation [31].  7      The saturation of the C5-C6 bond in P<>P dimers containing cytosine can lead to hydrolytic deamination of cytosine and formation of uracil (Figure 2). This uracil formation can have potential mutagenic properties and alter the coding during DNA transcription and RNA translation, resulting in changed base pairs in the genome [38], [40].    Figure 1. Structures of the cyclobutane pyrimidine dimers (P<>P) (A) and possible diastereoisomers of TT (B) 8   Figure 2. Formation of uracil containing product via hydrolysis of the C4 amino group   1.3.2. Pyrimidine (6-4) pyrimidone dimers and their Dewar valence isomers The second class of pyrimidine products are pyrimidine (6-4) pyrimidone [(6-4)PP] adducts (Figure 3). Although the process of their formation is similar to P<>P dimers, they arise from a cyclization involving the C5–C6 double bond of the 5՛-end pyrimidine and the C4 carbonyl or imino groups (thymine or cytosine) of its 3՛ neighbor. Consequently, causing the formation of an unstable oxetane and azetidine intermediates. These intermediates are spontaneously rearranged to yield (6-4)PP adducts, whereat the 3՛ base’s carbonyl or imino group is transferred to the C5 position of the 5՛ base (reviewed in [39]). The absorption band of (6-4)PP dimers show a 50 nm shift from their native bases into the near UV region, therefore, upon UVB or UVA irradiation they are easily converted into their Dewar valence isomers [41]. The Dewar isomers are not highly photoactive but they can reverse into (6-4)PP dimers upon UV irradiation of shorter wavelengths [42]. Indeed, when cytosine is in the 3՛ position (the transfer of the amino group to the 5' base during (6-4) PP adducts formation) the photoproduct cannot be hydrolyzed. Therefore, deamination reaction to uracil-containing adducts only occurs when the cytosine residue is on the 5' side of the dimer. However, it should be noted that the deamination process in both (6-4)PP and their Dewar valence isomers, is 100 times slower than the cis-syn cyclobutane pyrimidine dimer [43].    9   Figure 3. Pathway of UV induced 6-4PPs and their Dewar isomers   1.3.3. UV-induced purine photoproducts Although dipyrimidine products are the preferential consequences of UVB radiation, UV induced transformations of DNA purine bases is also biologically important [44]. The first type of photoproducts that comprises, at least, one adenine residue is formed upon exposure to UVB from either photocycloaddition to an adjacent thymine or dimerization with a vicinal adenine [45]. A [2+2] cycloadduct undergoing rearrangement reactions is the first intermediate in both cases. This intermediate photoproduct undergoes competing reaction pathways to form either adenine dimer (A=A) (Figure 4(A)) or to a structure where the ring in one of the adenine residues has opened (The Porschke photoproduct) (Figure 4(B)) [46]. For the TA photoproduct, rearrangement of the cyclobutane intermediate results in a product with an 8-membered ring structure (Figure 4(C)) [38].The quantum yield of adenine-containing photoproducts (A-T) are very low compared to different pyrimidine dimers [47]. However, since the A-T adduct has been shown to be mutagenic, these lesions may be contributing to the biological effect of UV [48].  Overall, CPDs, 6-4PPs, strand breaks and oxidative products are the leading and most consistent type of lesions. If left unrepaired they can cause severe distortion in the DNA molecule, impacting DNA replication and transcription, cellular viability and functionality yielding mutagenesis, tumorigenesis and cell death [33], [49]. 10    Figure 4. Purine photoproducts, Adenine dimer (A), Porschke photoproduct (B), and TA photoadduct (C)  1.3.4. UV induced DNA double strand breaks The formation of DNA double-strand breaks (DSBs) upon UV radiation has been known for years. These strand breaks are found broadly in the UVB range [50]. But it seems that UV radiation is not capable of inducing DNA double strand breaks directly and instead it is the pyrimidine dimers that can cause replication arrests and DSBs. These lesions have been reported to create DSBs at the location of collapsed replication forks of DNA including CPDs [51]. One of the possible pathways that has been studied to clarify how DSBs are generated at a stalled replication fork, suggests that when the replication machinery of DNA comes across a lesion that is blocking the replication, DNA polymerase (DP) is stopped at the blocked site leading to the formation of a Y-shaped DNA structure. A particular endonuclease could recognize this DNA structure and is capable of successfully making a nick to the other template strand and inducing a DSB close to the replication-blocking site [52]. In addition, topoisomerase I (Top1) cleavage complexes can be trapped by replication stresses and DSBs are formed by inhibiting Top1-mediated DNA ligation [53]. It has also been shown that radiation-induced single strand breaks (SSBs( can also promote generation of replication-induced DSBs [54].   11  1.3.5. UV-induced DNA damage detection Rapid phosphorylation of H2AX, the minor histone H2A variant, at the position of  Ser-139 to form γH2AX is one of the initial cellular responses to DSBs [55]. This immediate phosphorylation is one of the most well-known chromatin alterations linked to DNA damage and repair [56].  DNA damage response (DDR) includes several complex pathways to recognize, signal and repair the DSBs in order to recover and maintain the chromatin structure, and H2AX phosphorylation plays a significant role to signal and initiate the repair process in  DDR [57]. It has been suggested that the γH2AX modification in chromatin boosts accessibility to DNA and recruits DDR proteins at DNA ends [58]. The epigenetic signal produced upon H2AX phosphorylation is recognized by specific domains on DDR proteins [59]. Furthermore, γH2AX has the ability to anchor broken ends of DNA together by nucleosome repositioning and reducing chromatin density to help facilitate the DSB rejoining [60]. γH2AX also keeps the ends in close vicinity by recruiting cohesins during the repair process to avoid the loss of large chromosomal regions [61].  ATM (ataxia telangiectasia mutated) kinase is known to be one of the main physiological intermediator of H2AX phosphorylation. ATM undergoes auto phosphorylation at Ser1981 which result into disassociation of inactive ATM dimers into single molecules and increased kinase activity [62]. MRN (MRE11-RAD50-NBS1) protein complex recognized the damage, recruits ATM to the location of the DNA damage and targets ATM to start phosphorylating respective substrates [63]. Besides H2AX, other substrates such as BRCA1, 53BP1 and MDC1 as well as Chk1 and Chk2, checkpoint proteins, are phosphorylated by ATM. These phosphorylation processes aim to stop the normal cell cycle and start the DNA repair process. H2AX can also be phosphorylated by ATR. ATR-dependent H2AX phosphorylation occurs in response to replication stresses including replication fork arrest and also UV-induced DNA damages [64].  γH2AX formation in S-phase which is mainly ATR-dependent, is an indicator of DSBs formed from replication stresses when replication forks collide UV-induced lesions. γH2AX generation in other phases possibly corresponds to the initial cellular response to UV-induced DSBs or their repair [65], [66].   12  The loss of γH2AX at DSB sites is thought to be an indication of repair happening in DNA [67]. It is not clear yet when a DSB is repaired, some studies suggest when the two strands are rejoined and some think when the chromatin is returned to its original compact state [58], [68]. Several mechanisms have been studied to clarify how γH2AX is removed from chromatin. One possible mechanism is dephosphorylation of γH2AX by phosphatase 2A and phosphatase 4C [69]. Another mechanism is histone exchange and redistribution in chromatin, substituting γH2AX with H2AZ in the course of chromatin remodeling  where acetyltransferase acts as a mediator [70].  Since γH2AX can be detected away from DSB sites, its removal might involve both chromatin remodeling and histone substitution at the DSB site and dephosphorylation at remote sites. γH2AX removal prevents further segregation of DDR and repair proteins[70]. Chromatin regions which were affected by DSB repair are restored and cell cycle progresses, assuring the genetic information is preserved [71].  However, it has been reported that γH2AX removal can be associated with repair activity only at relatively low levels of DNA damage, usually lower than 150 DSBs per genome, and in DNA repair proficient cells [67].   Detecting γH2AX phosphorylation and consequently DNA damage and repair in individual cells is now possible with the development of antibodies specific to γH2AX. The presence of nuclear foci containing γH2AX can be measured by microscopy, flow cytometry, and Western blotting of cell/tissue lysates, normalized to the total H2AX level [72]. Measuring γH2AX using multiparameter flow cytometry can be very helpful since γH2AX expression can be determined with high sensitivity and accuracy in cell populations and further be correlated with DNA content and apoptosis induction [73].   1.4. DNA UV-induced Damage Repair The precise transmission of information from one cell to its daughters is essential for the survival of an organism. High precision in DNA replication, accurate chromosome distribution, surviving natural and induced DNA damages and minimizing the amount of heritable mutations are crucial for an exact transmission to occur. Therefore, organisms have developed  efficient DNA repair mechanisms to resist the lethal effect of DNA lesions [74].  13  Specific repair proteins scan the genome constantly for DNA lesions and once it recognizes a mismatch base, an apurinic/apyriminic site, or structurally altered bases, it activates an efficient DNA repair, leading to the recovery of genetic information [75]. Some of the important DNA repair mechanisms are described in the following sections.   1.4.1. Photoreactivation This is possibly one of the oldest repair systems that includes only one enzyme: photolyase. To remove the lesions in DNA caused by UV, this enzyme binds specifically to CPDs and 6-4 PPs and reverses the DNA damage using light energy through a process called photoreactivation  [76]. CPD photolyases have been reported in many organisms such as bacteria, fungi, plants, invertebrates and some vertebrates. Whereas the 6-4 PP photolyases have been identified in certain organisms such as Drosophila, silkworm, rattlesnakes and Xenopus laevis [77]. However, in placental mammals like humans, these photolyase enzymes seem to be absent or non-functional[78], [79].    1.4.2. Excision repair Excision repair, in contrast to photoreactivation, is a multiple step, complex dark repair in which the damaged base is removed via two main sub-pathways: 1) Base excision repair (BER) and 2) Nucleotide excision repair (NER).  1.4.2.1. Base excision repair (BER) BER is the leading repair pathway against DNA base lesions caused by hydrolytic deamination, reactive oxygen species, alkylating agents and other intracellular metabolites that alter the DNA base structures [80]. The specificity of the BER repair pathway is determined by the several  types of DNA glycosylase that are involved, and each type removes different kinds of damages by splitting the N-glycosidic bond between the abnormal base and deoxyribose [81].  When the modified base is removed, the apurinic/apyrimidinic (AP) site is cleared out by an AP endonuclease along with phosphodiesterase that excises the DNA strand from 5 or 3 to the AP site, respectively. Afterwards, a repair DNA polymerase fills the gap and the strand is sealed 14  by a DNA ligase [81], [82]. It has been reported that the repair Pol. Β has the capability to break the 5 deoxyribose phosphate residues, that is generated by the combined actions of DNA glycosylase and Class II AP endonuclease [83]. A single nucleotide lesion is removed by short-patch BER (SP-BER) while long-patch BER(LP-BER) is involved when two/more nucleotide lesions need to be repaired [84].   1.4.2.2. Nucleotide Excision Repair (NER) In terms of UV-induced DNA lesions, NER is the most crucial repair system. It is one of the most variable and flexible systems that exists in many organisms but mostly preserved in eukaryotes. NER removes a wide range of distorted DNA lesions, including CPDs and 6-4 PPs, DNA intra-strand crosslinks and some forms of oxidative damages. Even though both CPDs and 6-4 PPs are probably removed by the same NER proteins, the repair efficacy of these lesions differs relatively in mammalian cells [85]. It has been reported that the removal of 6-4PPs is five times faster than CPDs in human and hamster cells [86].  NER was first found and described in E. Coli [87], which recruits 6 proteins including UvrA, B, C (ABC-complex), UvrD (helicase II),  DNA polymerase I (pol. I), and DNA ligase to complete the repair [88]. In eukaryotes, the biochemical scheme of NER is known to be the same as prokaryotes but varies extensively in both the type and the number of proteins involved [85].  The NER pathway has been broadly studied at the molecular level in human cells and its schematic representation is demonstrated in Figure 5.  NER is divided into two sub-pathways of global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which are regulated differently. The global genome repair (GGR) repairs lesions over the entire genome whereas the lesions blocking the transcription in DNA strand are repaired by transcription-coupled repair(TCR) [85]. Both pathways clear out the UV-induced DNA lesions subsequently by recognizing the damage, opening the DNA double-helix and splitting the DNA on both sides of the lesion, succeeded by resynthesizing and ligation [89]. 15   Figure 5. GG-NER and TC-NER mechanism   1.4.2.3. Differences between GG-NER and TC-NER in mammalian cells GG-NER is a slow and random process whereas the TC-NER, which is linked to RNA polymerase II (RNA Pol II), is more precise and effective. It is supposed that TC-NER is activated when RNA pol II encounters a lesion. The stalled polymerase displacement is done by CSA and CSB proteins. The CSA protein (44 kDa) is from ‘WD repeat’ family of proteins and has structural and regulatory roles. The CSB protein (168 kDa) is from SWI/SNF protein family and performs 16  DNA-stimulated ATPase activity [90], [91]. As mentioned earlier, active RNA pol II elongation is required for effective TCR; the CSA and CSB genes are required only in the course of elongation stage of RNA pol II transcription [92]. It has also been reported that in order to assist the accessibility of repair machinery to the location of the lesion, the RNA pol II backs up some nucleotides once it encounters a lesion [93].  The elongation of RNA pol II is extremely impaired at higher doses of UV radiation, which affects the TCR efficiency. Therefore, at higher UV doses the GGR dominates the TCR pathway [94]. The lesions are recognized by hHR23B-XPC protein complex in the GG-NER pathway. The rate of GGR depends on the type of lesion, for example, 6-4PPs are repaired much faster than the CPDs and that is probably due to differences in affinity of the hHr23B-XPC damage sensor. XPC is the only factor that is restricted to GG-NER [86].  New mechanism and regulation of mammalian GG-NER has been discovered which reports association of UV-damaged DNA-binding (UV-DDB) with a cullin-base ubiquitin ligase. In addition, it is also suggested that XPC and UV-DDB emerge to help optimized recognition of UV-induced photolesions [95]. It has been shown that on moderately deformed DNA helix, DDB complex is recruited to the CPD lesion site; on the other hand, large distortions like 6-4 PPs are probably recognized by XPC directly [95]. However, the mechanism by which the XPC recognizes the lesion in the mammalian genome is not elucidated yet [96]. hHR23B-XPC complex or XPC alone show the same affinity for both UV-induced single stranded and double stranded DNA, but prefers binding to DNA that has multiple lesions [97]. hHR23B-XPC complex is necessary for DNA incision on both sides of the lesion. Therefore, RNA pol II with CSA and CSB leads to TCR and the hHR23B-XPC complex triggers the GGR[98]. Following the damage recognition stage, the next step’s pathways are alike in both TCR and GGR. Multi-subunit transcription factor-IIH (TFIIH), opens up the DNA double helix at the location of the lesion using two subunits including XPB (3 to 5 helicase) and XPD (5 to 3 helicase) [99]. Once the DNA double helix is opened, RPA, XPA and XPG proteins are recruited. XPA and RPA proteins validate the existence of DNA damage and help materialize a stable complex prior to incision [100]. However, XPG which is from the flap endonuclease-1 (FEN-1) family of endonucleases is essential for the 3 incision, stabilization of open DNA bubble structure and the permission of the 5 incision by ERCC1- XPF [101]. Afterwards, XPG and XPF/ERCC1 complex cuts the damaged part of DNA from 3 to 5 of 17  the lesion, creating a 24-32 base oligonucleotide fragment [102]. Lastly, DNA polymerase δ or ε fills the gap and DNA ligase seals it.  1.4.3. Recombinational repair NER and BER repair pathways remove lesions in one strand of DNA in a ‘cut-and-patch’ mechanism and use the other strand of DNA as a complementary strand. However, DNA double strand breaks that are a result of ionization radiation, UVR, ROS and chemotherapeutic genotoxic chemicals are far more cytotoxic and hard to repair, since cells cannot easily copy information from the undamaged strand. These lethal lesions are removed by two distinct pathways, such as homologous recombination (HR) and non-homologous end joining (NHEJ). Multiple protein complexes are required for these pathways and deficiencies in this mechanism may lead to hereditary diseases. For example mutation in one important protein in this pathway, BRCA1, can cause hereditary breast cancer [103]. HR pathway requires a wide range of DNA homology between the damaged and the template strand, therefore it is considered to be an error free mechanism. On the other hand NHEJ is error prone because it is independent of sequence homology and basically joint two broken chromosomal ends [104]. A simplified schematic representation of these pathways and their mechanisms are shown in Figure 6.  1.4.3.1. Homologous recombination In eukaryotes, RAD52 epistasis group of proteins carries out the HR. In human cells, this group of proteins include the MRN complex (MRE11/RAD50/NBS1), RAD51, RAD51B, RAD51C, RAD51D, XRCC2 (X-ray repair cross-complementing group 2), XRCC3), RAD54 and RAD54B [105]. Breast cancer susceptibility genes, BRCA1 and BRCA2, also play a role in HR pathway [106]. The MRN complex and ATM initiate the damage response in cells once a DSB is detected.  The first step of HR is 5 end resection to generate a 3 single-stranded DNA (ssDNA) tail by an exonuclease such as MRN complex. The central protein in HR is RAD51, it binds to the ssDNA overhang and forms a nucleoprotein strand and this step is promoted by Rad55/Rad57 protein heterodimer [107]. It has been reported that in eukaryotes HR, γH2AX plays an important role in recruiting RAD 51 protein [108]. The RAD 51 nucleoprotein filament along with other repair 18  proteins scan the genome to find an undamaged copy of the broken DNA on the sister chromatid and form a D-loop that is later on matured in to a Holliday junctions (HJs) [104]. This joint molecule uses the intact sister chromatid as a template for DNA polymerases, and the genetic information is restored. Afterwards, two complete DNA copies are produced from DNA strand ligation and the joint molecule separation [109].    Figure 6. Schematic representation of recombinational pathways  1.4.3.2.Non-homologous end joining NHEJ is the simpler pathway for repairing DSBs compared to HR. It does not require a template and essentially re-ligates broken DNA end. NHEJ is very significant for repairing DSBs in G1 phase where there is no sister chromatid and HR is inefficient [110]. The Ku70/80 19  heterodimer bind to damaged DNA ends and facilitates the recruitment of the DNA-PKcs (the DNA-PK catalytic subunit) to the location of DSB. This process leads to activation of the DNA-PKcs phosphorylating function that is able to phosphorylate itself, the Ku heterodimers and some other cell cycle regulating proteins [111]. Cells that are deficient in DNA-PK components functionality are expected to be more sensitive towards UV irradiation [112].  It has been suggested that Ku70/80 possibly improves accessibility and stimulates the functioning of DNA ligase IV (Lig4)-XRCC4 complex which highly increases the NHEJ efficiency. The juxtaposed DNA ends are later ligated by this (Lig4)-XRCC4 complex [113]. Measurements of the frequency of DSBs based on H2AX phosphorylation shows that 10% of the radiation-induced DSBs are repaired by a sub-pathway of NHEJ that requires the nuclease Artemis. This pathway requires several proteins such as NSB1, MRE11, DNA-PK, and other intermediate proteins including H2AX and P53B [114]. It has been found recently that a third protein called XLF or Cernunnos that is similar to XRCC4 is also involved in NHEJ pathway and  interacts with XRCC4 to stimulate Lig4 activity [115]. It has been reported that in mammalian cells, DSBs are repaired by NHEJ more frequently than HR [104].    1.4.4. UV-induced apoptosis (Other repair machineries) Apart from the repair mechanisms mentioned above, other repair machineries including mutagenic repair, programmed cell death (PCD) and apoptosis can also be effective. When the repair mechanisms fail in repairing UV-induced photolesions and DNA double strand breaks it can lead to permanent cell cycle arrest (cellular senescence), oncogenesis or apoptosis [116]. P53 tumor suppressor protein plays a crucial role in the regulation of cell response to DNA damage. P53 stimulates DNA repair, delays cells cycle progression and induces apoptosis and senescence. The P53 protein ability to delay the cell cycle is mediated by various genes such as p21 which derives G1/S arrest by inhibiting the cyclin-dependent kinase complex and suppressing S phase entrance [117]. P53 is also capable of transactivation of pro-apoptotic genes and inducing cell death. However, other transcription-independent pathways also are proposed to contribute to the induction of cell death [118]. Previous studies on human fibroblasts and CHO cells has suggested that UV-induced apoptosis might be resulting from cell progression to S phase. In fact signaling 20  from the formation of DSBs, replication of unrepaired damages and collapse of replication fork contribute to explain the induction of cell death in UV-irradiated cells [119], [120].  1.5. Research hypothesis and objectives 1.5.1. Hypothesis We hypothesized that patients with somatic malignancies could be distinguished from healthy individuals based on their immune cells’ response to DNA damage caused by UV irradiation. In a recent study by Anderson et al., it was reported that the lymphocyte genome stability (LGS) blood test, using high doses of UVA irradiation, showed higher DNA damage degree (Olive tail moment measurement) in cancer and precancerous compared to healthy individuals blood samples [121]. However, direct measurement of DNA damage using Comet assay can be subject to background noise and probable false results, specifically in the case of UVA radiation which is not absorbed directly by the DNA. Moreover, high degree statistical analysis was required to distinguish between cancerous and healthy cells in this method.                As mentioned earlier, direct radiation absorption happens in the UVB/C  region which will result in the formation of various photolesions in DNA [31], [32], [39]. These lesions, if left unrepaired, will lead to replication stresses and consequently DNA single or double strand breaks [122].We suggested that the direct DNA damage caused in cells upon UVB/C exposure, is the main factor to identify tumor-associated immune cells from healthy ones. In this study, we aimed to establish a robust procedure to distinguish cancerous patients from healthy donors by direct measurements of DNA damage caused after UV exposure. This was accomplished by exposing PBMCs isolated from blood samples to an optimized UV radiation setting with a specific wavelength at UVB region, measuring the degree of DNA damage within various time intervals. Various UV exposure conditions are now feasible due to recent advances in semiconductor technology leading to the development of UV-LEDs. The small form factor, efficiency, narrow spectrum and the possibility to turn them on/off at any frequency make them a better alternative over traditional UV lamp-based light sources. The amount of DNA damage after UV exposure was measured using a specific bio marker for DNA double strand break, γH2AX. The exact number of cells positive for γH2AX after UV exposure at different time intervals was investigated using flow cytometry. Utilizing this method, in addition to the initial damage caused, the removal of γH2AX 21  could also be investigated as a sign of repair, and the DNA repair capacity could be compared between healthy individuals and cancer patients.   This was a unique approach from previous studies as the radiance power, wavelength, and the operation mode of UV irradiation were highly controlled, which had the advantage of providing accurate damage and repair conditions for cells compared to UVA lamps with broad spectrum and no control over the dose/operation mode. The damage was caused using UVB/C region wavelength to provide direct radiation absorption and induce double strand breaks in the genome. Furthermore, DNA damage was measured using a biomarker for double strand break and flow cytometry. Using flow cytometry made it possible to sort and quantify every single cell based on their positive signal for double strand breaks, reducing the need for extensive statistical analysis.   1.5.2. Research objectives The overall objective of this study was to investigate the possibility of developing an assay to distinguish between cancer patients and healthy individuals based on their immune cell’s responses to ultraviolet exposure. The specific objectives of this study included: 1. Designing and manufacturing an experimental apparatus capable of providing relatively uniform radiation distribution, multiple wavelengths and multiple UV doses.  2. Studying the degree of DNA damage caused by various doses of UV irradiation, and determining the adequate dose of UV radiation to create double strand breaks in peripheral blood mononuclear cells (PBMCs).  3.  Developing an assay to perform time-course analysis of UV-exposed cells to investigate the repair process and possible removal of double strand breaks. 4. Using the developed assay to investigate the response of immune cells obtained from a proof of concept cohort of prostate cancer patients undergoing Radical Prostatectomy surgery to UV irradiation.   5. Using the developed assay to test healthy donor samples and determined if the assay was able to distinguish between study subjects and had the potential to be used for diagnostic purposes.  22  Chapter 2: Materials and Method 2.1.  Experimental setup An experimental setup capable of adjusting the UV-LED dose and uniform radiation was designed as shown in Figure 7. The UV-LED used was a 3.5 x 3.5mm smd flat top type series purchased from DOWA Electronics Materials Co., Ltd. (Tokyo, Japan) with a peak wavelength of 280 nm and a power of 25 mW (details about the UV-LED and it’s specification can be found in Appendix B).  The LED was mounted on the bottom of an air cooler heatsink. The center of the LED was aligned with the center of both the heatsink and the sample plate. Two height adjustable clamps were used to hold the heatsink in order to change the distance of the LED from the samples. After examining various distances, 5 cm was chosen and kept constant during all the experiments. An air cooler heatsink is used to dissipate the heat produced by LEDs. Optical black anodized breadboards and rails obtained from Thorlabs, Inc. (Newton, NJ, USA) were used to provide parallel light beams and minimum light reflection. The whole setup was enclosed in a black box made of black fabric to block any stray light as well as ensuring the Safety Operation Procedure during the irradiation of samples. 23   Figure 7. Experimental apparatus schematics, the UV-LED used is a 285 nm from DOWA electronics.   2.2. UV dose and irradiance The measurement of UV fluence (irradiance) was done by chemical actinometry. Chemical actinometer is a chemical system (fluid, gas, solid, etc.) that undergoes a light-induced reaction that has a determined Quantum yield. Quantum yield is defined as the number of events divided by the number of photons absorbed. Here we used a common chemical actinometer to determine the UV dose inside our samples, the KI/KIO3 actinometer. The overall photochemical reaction is as follow  8I− +  IO3− + 3H2O + hν → 3I3− + 6OH− Where measuring I3− photoproduct leads to the determination of the total UV fluence.  24  The same protocol as described by Goldstein et al. [123] was used for the KI/KIO3 actinometry. Briefly, a solution of 0.6 M in KI, 0.01 M in KIO3, and 0.01 M in Na2B4O7.10H2O was prepared and because of the thermal reaction between iodide and iodate the solution was used within 4 hours of preparation. The test can be safely done in room light and determination of I3− photo-product was done using a Nanodrop 2000 spectrophotometer. Results of these tests, detailed calculations and experimental procedure can be found in Appendix A. The actinometry was performed regularly during all the experiments.   2.3. Cell culture The human T cell leukemia cell line Jurkat, purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) was used for optimizing the experimental procedure. Jurkat cell line is  an immortalized line of human T lymphocytes obtained from the peripheral blood of a 14-year-old boy with T cell leukemia in the late 1970s [124]. These cells are frequently used in studies to determine the mechanism of differentiation and susceptibility of cancer to drugs and radiation. Jurkat cells were routinely cultured in T75 tissue-culture treated flasks (Corning® cell culture flasks) at 37 °C under a 5% CO2 humidified atmosphere in RPMI 1640 medium (GE Healthcare Hyclone) supplemented with 10% fetal bovine serum (FBS).  2.4. Blood samples, and Peripheral Blood Mononuclear Cell (PBMC) separation In this study we used two different group of subjects. The first group was our normal healthy donors group with no physical sign of disease. The healthy donors were laboratory students and personnel from the Vancouver Prostate Centre. The second group were all Prostate cancer patients undergoing Radical Prostatectomy treatment, whose blood was collected prior to surgery. All the patients’ samples were recruited from Dr. Black’s prostate group in Vancouver General Hospital. Complete description of the subjects is given in the result section (Table 4).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Peripheral blood (~12ml) was collected by venipuncture in to lithium heparin vacutainers at room temperature and samples were kept on ice until the isolation procedure. Under sterile condition, the blood was diluted 1:1 with calcium/magnesium free Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich) supplemented with 2% FBS. The SepMate PBMC isolation kit from Stem Cell Technologies was used. 15ml of Lymphoprep density gradient medium (Stem Cell Technologies, 25  BC, Canada) was transferred into a 50 ml SepMate tube. Then the diluted blood was transferred carefully and layered over the density gradient. The SepMate tube was centrifuged at 1200g for 20 minutes at room temperature with breaks on. Then the top layer which contained PBMCs as well as platelets was transferred to another tube and washed twice with DPBS+2%FBS once at 350g and once at 125g each for 10 minutes to remove the platelets. The washed cells were resuspended in 1 ml of RPMI 1640 medium (Gibco) and cells were counted using a hemocytometer. After counting the cells, they were resuspended in the growth medium (RPMI 1640 supplemented with 10% FBS) to a final concentration of 2 − 5 × 105cells/ml. The PBMCs were activated using 20 ng/ml of Interleukin-2 (IL2) which is a cytokine that is implicated to stimulate of immune responses for its ability to act as a growth factor.   2.5. UV-Irradiation Briefly, 5 − 10 × 105cells were plated on 60 mm diameter petri dishes in about 2.7 ml of the growth media so that the depth of solution did not exceed 1 mm to ensure the UV radiation would penetrate through the whole sample and all cells receive the same dose of UV. Samples were irradiated using various exposure times (different UV dose).  In all experiments, there were two control conditions. One negative control, which was not irradiated with UV, and one positive control using a chemotherapeutic agent named VAL-083. VAL-083 is currently in phase 2 of clinical trial sponsored by the U.S. National Cancer Institute ("NCI") and has not been approved by FDA yet, but it is very promising against a range of tumors including lung, brain, cervical, ovarian and hematologic cancer. Studies on the mechanism of this drug which is being carried out in our research group at the Vancouver Prostate Centre (VPC) confirms that even very low concentration of the drug (5μM) can result in very high amount of DNA double strand breaks after 24 hr treatment [125]. For each dose of UV used, cells were incubated for various time points, from 10 minute up to 96 hours after irradiation, to observe and optimize the time point that the cells will be able to remove the DNA double strand breaks upon UV irradiation. A summary of all the variables and experimental conditions is demonstrated in Table 2, all the incubation times were tested for each exposure time (UV dose).    26  Table 2. Experimental variables and conditions Variables  Exposure time (s) 0 5 10 20 40     UV irradiance (mJ/cm2) 0 2 4 8 16     Post-exposure incubation time (h) 0 0.2 0.5 1 6 24 48 72 96   2.6. Western Blot After collecting samples at their relevant time point, cells were washed and centrifuged. Pelleted cells were lysed using the RIPA Lysis and Extraction Buffer (Thermo Fischer) supplemented with protease and phosphatase inhibitors (Sigma) in 1.5 ml Eppendorf tubes. The cell lysates were briefly sonicated using water bath sonication and the protein lysates were collected from the supernatant layer after centrifuging at 12,000g for 20 minutes at 4C. Protein concentrations were measured using BCA Protein Assay Kit (Thermo Fischer) according to the manufacturer’s protocol. Approximately equal amounts of total protein (20 − 30 g) where then mixed with sodium dodecyl sulfate (SDS) and boiled at 95C for 5 minutes. Protein samples were loaded on 4 − 15% SDS precast polyacrylamide gel (Bio-Rad), separated by electrophoresis and then transferred onto 0.2 m nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo RTA transfer kit (Bio-Rad) according to the manufacturer’s protocol. Upon completion of protein transfer onto nitrocellulose membrane, they were blocked using 5% dried non-fat milk for at least 30 min and probed with Phospho-Histone H2AX (Ser139) antibody (Cell Signaling Technology) (1:1000 dilution) at 4C overnight. Blots were then washed with tris-buffered saline plus TWEEN-20 (TBST) buffer 3 times, 10 min each, and probed with the secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, Cell Signaling Technology) (1:2000 dilution) for at least 1 hour at room temperature with gentle shaking. After three additional 10 min TBST washes, the blot was developed using SuperSignal West Femto Max Sensitivity Substrate (Thermo Fischer) and exposed to Syngene G:BOX XT4: Chemiluminescence and Fluorescence Imaging System machine. Gel and western blots image capturing was performed with Genesys software interphase.  27  Each experiment was repeated three independent times and one representative result was shown. Image J software was used to perform quantification analysis of protein bands.   2.7.  Flow Cytometry 2.7.1. Cell cycle analysis and PI staining Cells collected at each time point were centrifuged at 1000 rpm for 5 minutes. Cell pellets were resuspended in 1 ml of phosphate buffer saline (PBS) and 2.5 ml of ice-cold 100% ethanol was added (final concentration 70% ethanol) to fix the cells. The ethanol fixed cells were stored at -20 °C.  After all the time points samples were collected and fixed the cells were taken out of -20 °C and pelleted by centrifugation at 1500 rpm for 5 minutes. The cells were then suspended in 500 µl of PI-Solution prepared by adding 500 µl of stock 1 mg/ml PI, 10 µl of RNase A and 5 µl of 100% Triton X-100 to 10 ml of PBS. The cells were incubated for 40 minutes at 37 °C in dark.  Then 3 ml of cold PBS was added and the cells were pelleted at 1500 rpm for 10 minutes at 4 °C, at which point the supernatant was discarded. Cells were resuspended in 500 µl of PBS. For flow cytometry analysis the cell suspension was filtered by a 40 µm filter before data acquisition.  2.7.2. Fixable viability dye staining and fixation Upon completion of each incubation time, the cultured cells were washed twice with PBS and surface staining was performed with fixable viability dye (eBioscience Fixable Viability Dye eFluor 506, Thermo Fischer) (1:500 dilution) for 30 min in dark and on ice. Then, cells were washed once with a wash buffer ( 1X PBS, 2.5 mM EDTA, 2% FBS, 0.05% Sodium azide) and fixed with freshly prepared ice-cold 70% Ethanol for at least 1 hour in -20C (fixed cells can be kept at -20C for up to two weeks before intercellular staining).  2.7.3. Permeabilization and γH2AX intercellular staining  The ethanol fixed cell suspensions were centrifuged at 700g for 5 minutes and ethanol was removed. Cell pellets were washed once with 2 ml of BSA-T-PBS (1% BSA, 2% Triton X-100 in PBS) permeabilization buffer and then permeabilized in 2 ml of BSA-T-PBS buffer for 5 minutes 28  at room temperature in the dark. The fixed and permeabilized cells were centrifuged at 800g for 5 minutes and the supernatant discarded. Cells were then resuspended in 100 l of Human BD Fc-Block (BD Bioscience) (1:300 dilution in permeabilization buffer) and incubated for 15 minutes on ice in the dark. After blocking the Fc receptors, 100 l of a fluorochrome-conjugated monoclonal antibody solution specific for H2AX (Alexa Fluor 647 anti-γH2AX; BioLegend, UK; cat # 613408) (1:100 dilution in BSA-T-PBS) was added. A list of all the antibodies used in this study is demonstrated in Table 3. After the addition of the antibody, cells were incubated for at least 30 minutes at room temperature in the dark. The tubes were washed once with wash buffer and the supernatant was discarded. The tubes were then gently vortexed to loosen the cell pellet and 0.3-0.5 ml of wash buffer was added to each tube and the samples were analyzed for flow cytometry right away. In each flow cytometry analysis, a sample with no staining was used to determine the background signal, and a sample with single staining for each fluorescence was used to determine any type of compensation.   Table 3. Antibodies Information Name Description Dilution Cat. No. Supplier γH2AX Phospho-Histone H2A.X (Ser139) 1:1000 2577 Cell Signaling Alexa Fluor 647 anti-H2A.X Phospho (Ser139) 1:100 613408 BioLegend H2AX Rabbit polyclonal to Histone H2A.X 1:1000 11175 Abcam Fc Block Human BD Fc Bloc 1:300 564219 BD Biosciences Anti-Rabbit Anti-rabbit IgG, HRP-linked 1:2000 7074 Cell Signaling GAPDH GAPDH (D16H11) XP Rabbit mAb 1:1000 5174 Cell Signaling  29    The flow cytometer used in this study was a BD FACSCanto™ II (BD Biosciences, CA, USA). Data acquisition and daily routine of start-up, shut down and cleaning procedures were automated by BD FACSDiva™ software interphase, which allows the user to preview and record data from multiple samples with an automated acquisition process. The recorded data were saved as FCS files and later analyzed using Flow Jo software (version 10.4.2).    2.8. Statistical analysis The statistical analysis was performed using GraphPad Prism 6 (version 6.07) for two-tailed student’s unpaired t-test as well as Mann Whitney non-parametric test, with statistical significance set at p<0.05. Also, receiver operating characteristic (ROC) analysis was performed to assess the effectiveness of the assay to distinguish subject category (Healthy vs. Patient). The sensitivity and specificity was modeled using different cut-off values to be able to identify cancer patients using the following formulas: Sensitivity = TPR =TPTP + FN Specificity = TNR =TNTN + FP  Where TP is true positive, TN is true negative, FN is false negative, and FP is false positive.      30  Chapter 3: Results and Discussion 3.1.  Developing and optimizing the assay An experimental apparatus capable of providing exact wavelength and UV dose using the recent UV-LED technology was designed and manufactured as described in the methodology section (Figure 7). The UV-LED used for this study was in the UVB region with peak wavelength of 285 nm )please refer to Appendix B for the UV-LED specification sheet). In this range, direct absorption of radiation by DNA leads to dimerization reactions between adjacent pyrimidine bases and formation of photolesions in DNA structure such as cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidone. If these lesions remain in the genome unresolved, the transcription stress leads to formation of DNA double strand break as a result of collapsed replication fork [35]. One of the very first responses to DSBs is the phosphorylation of H2AX (γH2AX or pH2AX), the minor histone H2A variant, at the position of Ser-139. There are previous studies suggesting that the loss of γH2AX at DSB sites can be an indicator of repair [60]. Therefore, in this study we used γH2AX as the DNA double strand break marker because we could analyze both the initial damage and later the possible repair process by only measuring the amount of γH2AX induction and its dephosphorylation, respectively.  To determine the required UV dose threshold  for DSB formation, we used Jurkat cell line which is an immortalized T-cell leukemia line of human T-cell lymphocytes [126]. In the optimization experiments we irradiated the samples in 60 mm plates, for various durations from 5 to 40 seconds and later incubated them from 0 up to 24 hours. Then the γH2AX as DSB marker was detected using an antibody specifically for phospho-histone H2AX (Ser 139) and western blotting. The γH2AX signal was first detected after 10 minutes in the cells treated with 16 mJ/cm2 which was the highest dose applied (Figure 8). Whereas in the cells treated with 2 mJ/cm2 the first band appears after 1 hour. Usually it takes 6-8 hours for Jurkat cells cultured in the presence of required growth factors to enter S-phase and pass the remainder of the cell cycle. γH2AX signal was detectable in all samples 6 hours after irradiation in agreement with previous findings on UV- induced DSB being a result of replication stress rather than the direct UV irradiation itself [51-54]. The weak bands in negative controls suggest that the antibody is specific to phosphor-histone-H2AX but, two different samples were used as negative controls and the difference in the 31  intensity of the band in the control sample was probably due to noise in the background signal of the antibody itself. The strong signal of γH2AX detected in the positive control sample treated with 5 M of VAL-083 drug, suggests that the assay we developed to quantify the amount of γH2AX and DSB formation was accurate and precise.     Figure 8. Dose dependent UV-induced γH2AX formation. Cells were exposed to 1-16 mJ/cm2 of UV radiation in 285 nm and incubated for 0-24 hours after exposure in 37  incubator humidified with 5% CO2. At each time point cells were collected and lysed with RIPA buffer, supplemented with protease and phosphatase inhibitors. Western blot analysis was performed after all the time points were collected. Membrane was probed once for γH2AX antibody and later for total H2AX as a loading control.   Collectively, our results indicate that γH2AX formation after UV irradiation is both time-dependent and dose-dependent. Our objective in this section was to determine the UV dose threshold that is achievable considering the device limitations and is also capable of inducing DSBs in cells. Based on our results in this part, even 2 mJ/cm2 of UV irradiance (285 nm), induced a detectable DSB and γH2XA signal. However, this UV dose with our UV-LED power and actinometry data (Appendix A) was equivalent to only 5 seconds of irradiation. The experimental apparatus designed for this study had to be entirely enclosed in a black box for safety considerations and for that reason, 5 seconds of irradiation was challenging to keep constant and 32  was prone to increase random human errors. Couple of other exposure times were tested and amongst them 4 mJ/cm2 UV irradiance proved to be the applicable considering the experimental apparatus limitations and also induced adequate DSB signal in the Jurkat cell line (Figure 9). Therefore, 10 seconds of irradiation was chosen as the optimized UV irradiance and it was kept constant throughout the entire experiments. However, it should be noted that the reported doses here are UV fluence rate on the surface of the solution of cells in the culture media (RPMI + 10% FBS) and the depth of the solution was calculated to be 1mm. Therefore, changes to compositions of the media, the serum content or the volume would change the UV transmittance and consequently the UV dose.    Figure 9. Time dependent phosphorylation of γH2AX. Jurkat cells were exposed to 4 mJ/cm2 of UV radiation in 285 nm and incubated for 0-96 hours after exposure in 37  incubator humidified with 5% CO2. After each time point cells were collected and lysed with RIPA buffer, supplemented with protease and phosphatase inhibitors. Western blot analysis was performed after all the time points were collected. Membrane was probed once for γH2AX antibody and later for GAPDH as a loading control.   The analysis of H2AX phosphorylation over a time course of 96 hours after exposure to 4 mJ/cm2 UV irradiance showed a peak after 6 hours and the peak was sustained almost throughout the whole 96 hour time point. Upon formation of DSB, ATM and ATR, the key components in DDR-signaling are activated (Figure 10). Two of the targets of ATM/ATR are CHK1 and CHK2 protein kinases which in collaboration with ATM and ATR inhibit cyclin-dependent kinase (CDK) activity and slow down the progression of cell cycle G1-S, intra-S and G2-M “cell-cycle checkpoint”. This pathway is thought to provide time for DNA repair before replication or mitosis starts. In addition the ATM/ATR signaling improves the repair by recruiting repair proteins and 33  modulating their phosphorylation/acetylation at DSB sites [127]. If these events result into DNA repair, the DDR gets deactivated. But, if the damage remains unrepaired, chronic DDR signaling leads to cell death by apoptosis or cellular senescence (permanent cell-cycle withdrawal) [128]. In the time-course analysis, the γH2AX band appeared with the same intensity repeatedly from 24 to 96 hour (Figure 9), the weaker band in 24h was a result of lack of total protein in the sample and when quantified and normalized using ImageJ software it had similar intensity to 48, 72 and 96h timepoint (normalization data not shown). Hence, no definite  sign of γH2AX removal or dephosphorylation was observed during the extended timepoint. Although our cell counts from each time point (data not shown) showed some cell death in samples, further investigation of the cell cycle was necessary to indicate whether this was a result of a possible cell cycle arrest or cell death induction.    Figure 10. Simplified DNA damage response diagram     34  3.2.  Cell Cycle analysis after UV exposure The complete cell cycle consists of a replication phase (S phase), distribution of genetic materials and other cell components to daughter cells (M phase) and two intermediate gap phases (G1 and G2) (for a more detailed review of cell cycles please refer to [129]). In flow cytometry analysis of DNA content in cells three major phases can be recognized: G1, S, and G2/M. Since G2 and M-phase have the same DNA content they cannot be discriminated in this method. A normal cell cycle histogram with the number of cells in each cycle is represented in Figure 11. The first peak is the G0/G1 phase and corresponds to DNA content in each cell. The middle segment is the replication phase (S) and the second peak represents the G2/M phase in which the PI intensity is twice that of the first peak and corresponds to two times the DNA content of the first peak. This method also makes it possible to distinguish apoptotic cells with fractional DNA content.   Figure 11. Histogram of Gated G0/G1/s/G2/M phase cells  Jurkat cells were exposed to 4 mJ/cm2, and then incubated for 0 to 96 hours. Propidium iodide (PI) was used to stain the DNA and cells were analyzed for flow cytometry. A histogram of PI relative fluorescence intensity versus the cell count for all the time points, plus negative and positive control is demonstrated in Figure 12. The cell cycle looked similar to the non-treated sample after 1 hour post exposure. A small fractional DNA signal appeared after 6 hours where 35  we had the peak γH2AX signal in the simultaneous western blot analysis. However, after 24 hours, a dramatic increase was seen in the population of cells accumulating in the G0/G1 phase. The fractional DNA signal, which is an indicator of cell death, intensified 24 hour after exposure and continued in a similar trend up to 96 hours after irradiation. In the VAL-083 treated Jurkat cells, our results showed a cell cycle arrest in the S–phase which was in agreement with the previous result from our group investigating the mechanism of this drug [125]. There was a small population of cells that survived the UV exposure and got back to their normal cell cycle after 96 h, but because of the dead cell population, it was not feasible to say for sure whether these cells were capable of repairing  the UV-induced DNA damage or not. While, it has been previously reported using traditional UV lamps, higher doses is needed to induce cell death response in cells [130], in this study our results shows that using a 285 nm UV-LED with 4 mJ/cm2 irradiance induces cell death after 24 hour.   36   Figure 12. Jurkat cell line cell cycle analysis by flow cytometry. After exposure 4 mJ/cm2 UV-irradiation, at each time point cells were fixed using 70% ethanol. After all the times were collected, cells were stained with 500 µl of PI-Solution and run for flow cytometry.  3.3.  Description of study subjects Group 1 (Healthy Donors (HD1-9)) consisted of 9 laboratory personnel (5 females and 3 males) with mean age of 37 years (range 27-54) with no history of cancer and no physical sign of illness. Group 2 (Prostate cancer patients (P1-9) was composed of 9 prostate cancer patients diagnosed with needle biopsy, all were going under Radical Prostatectomy surgery treatment with 37  no history of previous neoadjuvant therapy. To test the efficiency of our assay to distinguish between subject groups, we chose our study group mostly from low-intermediate risk PCa patients with Gleason scores of 7 and clinical stages of T1c or T2a, we also included some high risk patients with Gleason scores of 9 and clinical stages of T3a. A complete explanation of the study groups and their clinical information is given in Table 4.    Table 4. Patient's clinical information Patients ID Age Primary Gleason Score Secondary Gleason score Clinical Stage Treatment P1 62 3 4 N/A* Robotic Prostatectomy P2 54 4 5 T3a Robotic Prostatectomy P3 61 4 3 T3a Robotic Prostatectomy P4 61 4 4 T2a Robotic Prostatectomy P5 62 3 3 T1c Robotic Prostatectomy P6 61 3 3 T1c Robotic Prostatectomy P7 68 3 4 T1c Radical  Prostatectomy P8 67 3 4 T1c Robotic Prostatectomy P9 64 3 4 T1c Robotic Prostatectomy *: No record of clinical stage information was found for this patient.   3.4.  Measurements of UV-induced DNA damage in PCa patients  3.4.1. γH2AX detection with flow cytometry In order to shorten the procedure time, reduce variations and apply apoptotic cells exclusion we further enhanced our previous assay to measure γH2AX signal. Here, we analyzed Jurkat cell after UV irradiation of 4 mJ/cm2 with a fluorochrome-conjugated antibody against γH2AX, further, a fixable viability dye (eBioscience Fixable Viability Dye (FVD) eFluor) was used to sort cells based on their viability.  38  When a cell suspension is run through the cytometer, light scattered from the cells is detected as they go through the laser beam one at a time. A detector in front of the light beam measures forward scatter (FS) which correlates to cell size and several detectors to the side measure side scatter (SS) which is proportional to the granularity of cells. The gating strategy to analyze FSC files is outlined in Figure 13. First, all cells were gated on the SSC-FSC (Side Scatter vs. Forward Scatter) plots as shown on the left panel (figure 13 (A)). For the single cell discrimination, we used FSC-H vs. FSC-A or Area Scaling method, shown in the middle panel in figure 13(B).  In a flow cytometer, when a cell passes through the laser, the generated pulses are identified by the height (H) of the peak, the area (A) underneath the peak and the width (W). Most flow cytometry instruments have a function that allows the user to correlate A and H so that when the PMT voltage changes, they present the same variations and they will have the same reported value (refer to [131] for a more detailed review of flow cytometry principals). If these parameters are set correctly, a single cell will have the same value in both axis, therefore, the single cells will be presented in a diagonal display compared to doublets. Thereafter, live and apoptotic single cells were gated on γH2AX-APC vs. FVD-AmCyan dot plot as shown in the left panel in figure 13(C).  Live/dead fixable dead cell stain used in this study is based on the reaction of the fluorescent reactive dye with cellular proteins (amines). The reactive dye can permeate the damaged membranes of dead cells and stain both the interior and exterior amines, resulting in 50-fold fluorescence intensity, thereby allowing complete discrimination between the two cell populations [132] . The FVD used, was conjugated with a fluorochrome with maximum emission of 405 nm and can be detected with AMCyan channel in FACS Conto II (BD Biosciences, USA) flow cytometer (Figure 13 (C)). Fixing and permeabilization of the cell membrane allows the antibody to enter the cell and label the intracellular target of interest. The antibody used against the γH2AX was conjugated with Alexa Fluor 647, which is a fluorochrome that has a maximum emission of 668 nm when it is excited at 633nm / 635nm and can be detected using APC channel in FACS Conto II (BD Biosciences, USA) flow cytometer (Figure 13 (C)). In each experiment a set of samples with no fluorescent staining and two with single staining for each antibody used ( γH2AX only and FVD only) was included to determine and eliminate the background noise. A 39  shift in the fluorescent intensity (higher than 103) from negative controls was considered to be a positive sample for both γH2AX signal and the Fixable viability dye.  The percentage of live cells expressing γH2AX signal was obtained as the final readout from the dot plot and was used for most of the graphs presented in the study.   - Figure 13. Representative FCM plots showing the gating strategy. (A) SSC-FSC dot plot. (B) FSC-A-FSC-H dot plot to select single cells. (C) γH2AX-APC vs. Viability dye-AMCyan dot plot to gate γH2AX signal in live cells, the four gated populations are: Q1) dead and γH2AX (-), Q2) dead and γH2AX (+), Q3) live and γH2AX (+) and Q4) live and γH2AX (-)  3.4.2. Time-course analysis of Radical Prostatectomy patients Figure 14 demonstrates the time-dependent expression of intercellular γH2AX over 48 hours after irradiating the PCa patients PBMCs. The PBMCs were collected from PCa patients going under Radical Prostatectomy surgery treatment, and the blood was collected prior to surgery.  The time-course appearance of γH2AX was somewhat similar to what was shown in Jurkat cell line western blot analysis previously, the expression of γH2AX peaked at 6 hours after UV exposure. The percentage of γH2AX positive and live cell population dropped from 6 hour to 24 and 48 hour and the percentage of dead cells increased in these two timepoints (Figure 14 (C)). In the control samples (not-irradiated), the level of γH2AX positive cells elevated in a time-dependent 40  manner over the 48 hour time frame which could be explained with in vitro culture-induced DNA damage and stress in the human PBMCs primary culture.  A cell’s genome continuously gets damaged and this can be a result of various causes, from normal cell processes and metabolism by-products, to environmental exposure to radiation and chemical agents [133]. Therefore, the initial γH2AX expression level is expected to be different in each individuals. Since we were using patient’s blood in this study and we only had access to a limited amount of blood for each patient, we were not able to produce technical replicates for each measurement at various time point. Hence, we used normal healthy donor blood samples and repeated each time point measurements in triplicates to calculate the standard deviation and consequently evaluate the experimental variability. Figure 14 (B) demonstrates the variations of experimental procedure error (random error) in a technical replicate study. Very little deviation in each time point for the percentage of γH2AX positive live cells was observed in these experiments, for not treated sample the mean γH2AX positive cell percentage was 25.67± 0.8 SEM, for 6 hour incubated control sample (not-treated) was 14.83 ± 0.68 SEM and for 3 mJ/cm2 UV-treated and 6 hour incubated sample was 61.17 ± 1.19 SEM. This result confirmed that variations in measurements in each time point including the initial γH2AX signal in figure 14 (A) was not due to random errors and instead, it was a result of true individual variations  between patients. To reduce the impact of these initial variation  between individuals and analyze the effect of UV irradiation on γH2AX expression elevation, the data needed to be normalized. The 6 hour post-UV incubation timepoint was a more reliable data point for normalizing the data. It had the lowest percentage of dead cells as well as the highest amount of γH2AX positive response (Figure 14(C)) point. The normalization enabled us to study how each patient’s immune cells responded to UV exposure. Figure 15 shows the γH2AX expression level in 6 hour post-UV sample normalized to the 6 hour incubated control (not-irradiated) sample. The percentage of γH2AX positive cells doubled compared to the non-irradiated cells in all patients (Figure 15). However, some patient’s immune cells were more sensitive to the UV exposure and got as high as 20 times more γH2AX positive cells after 6 hours post-UV exposure incubation.    41    42   Figure 14. Time course analysis of γH2AX expression after UV exposure in patients’ samples. A) PBMCs were isolated from 12 ml of blood and plated in 𝟐 − 𝟓 × 𝟏𝟎𝟓 cells/ml in 60 mm culture plates. Then cells were irradiated with 3mJ/cm2 of UV. At each time point cells were labeled with fixable viability dye and Alexa 647 anti-γH2AX antibody and analyzed with flow cytometry. The percentage of live and γH2AX positive cells was plotted for each time point as the final read out. B) Blood was collected from healthy individuals and the same procedure as A was repeated in triplicates for each time point. C) Dot plot of AMCyan-A (Fixable viability dye-exclusion assay) versus APC-A (Alexa 647-anti-γH2AX).   Figure 15. γH2AX expression level in 6 hour post-UV normalized to 6 hour control expression. PBMCs were plated in 𝟐 − 𝟓 × 𝟏𝟎𝟓 cells/ml in 60 mm culture plates and irradiated with 3 mJ/cm2 of UV. At each time point cells were labeled with fixable viability dye and Alexa 647 anti-γH2AX antibody and analyzed with flow cytometry. The percentage of live and γH2AX positive cells 6 hour after exposure was divided and normalized to the percentage of live and γH2AX positive cells in the non-treated sample.    We investigated some relevant clinical background information that might have contributed to this dramatic variation in between patients. As stated earlier, many factors can contribute to an individual’s immune cells’ initial level of DNA damage as well as their response to the UV exposure. We chose some cancer related variables such as, patient’s age, PSA level, Gleason score and TNM stages and drew multiple variable graphs to compare the normalized γH2AX level after UV exposure with these variables (Figure 16). The normalized percentage of γH2AX positive and live cells is presented with regards to individual patient’s Gleason score and clinical stage in figure 16 (A). Most patients in our cancer patient group fell into the category of low-intermediate risk PCa (clinical stage T1c and Gleason score of 7), but we also had high-risk patients with Gleason 43  scores of 9 (Patient #2(P2)). P2 had an above average response to the UV exposure, it was not the highest level of γH2AX expression. On the other hand, the sample that had 20 times more elevation in the percentage of cells expressing γH2AX (P9), had a Gleason score of 7. Figure 16 (B) and (C) are the normalized γH2AX expression after UV exposure and clinical stage in comparison with age and PSA level respectively. Similar to the Gleason score, no correlation could be seen between post-UV γH2AX expression and age/PSA levels. Overall, the sample size of this study was rather small and this made it difficult to find a correlation between how each individual patient responded to the UV exposure and their relevant clinical history.    44   Figure 16. Normalized γH2AX expression after UV exposure in regards with Clinical Data. A) Normalized γH2AX expression after UV exposure vs.  PCa Gleason score and PCa clinical stage. B) Normalized γH2AX expression after UV exposure vs. Age and PCa clinical stage. C) Normalized γH2AX expression after UV exposure vs. PSA and PCa clinical stage.  45  3.5.  Comparison of post-UV γH2AX expression in normal study subjects and PCa patients Results presented in Figure 17 shows the normalized γH2AX expression in PCa patients PBMCs (Figure 17(A)) and healthy donors PBMCs (Figure 17(B)). The percentage of cells positive for γH2AX signal in the healthy donor subject group was lower in all cases than the PCa group. The percentage of positive cells in most cases was less than double compared to the non-irradiated cells whereas in the PCa group it was at least doubled and some cases as high as almost 20 times more. The highest response in healthy donor group was 2.7 and in PCa group was 19.2. The median of the normalized γH2AX expression in PCa group was 4.678, and for healthy donor group was 2.035 (Figure 17(C)). The median between subject group was tested using Mann-Whitney non-parametric test and they were significantly different (p<0.001) (refer to Table 5. Mann whitney test parameters. for Mann-Whitney non-parametric test results and parameters). Although we included female blood samples in our healthy donors control group, our statistical analysis showed that there were no significant differences between the γH2AX expression after UV exposure in males and females in this study (Appendix C).  The expression of γH2AX which is an indicator of DNA double strand breaks and the degree of the UV-induced DNA damage was higher in PBMCs isolated from cancer patient blood samples. The results shows that the assay developed in this study has the potential to be used for diagnostic purposes distinguishing between cancer patients and healthy individuals. As stated before, the impaired immune system functionality in cancer patients could be a consequence of tumor associated cytokines and T-cell exhaustion due to chronic exposure to antigen. However, the exact mechanism leading to increased DNA damage in cancer patients’ PBMCs remains unclear. We know that it could not be a result of therapeutic treatment since none of the patient samples used were under neoadjuvant therapy. Based on the empirical nature of this study, this uncertainty does not have an effect on the functionality of the developed assay.  The possibility that cancer type, status and disease progression could have an effect on cancer patients’ responses can only be ascertained by long-term study of a larger cohort, with various cancer types.  46   Figure 17. Normalized percentage of live and γH2AX positive cells in PCa patients (A) compared to Healthy Donors (B). Box and whisker plots, comparing PCa patients with healthy donor subject group (C).         47   Table 5. Mann whitney test parameters. Mann Whitney test P value 0.0008 Exact or approximate P value? Exact P value summary ** Significantly different (P < 0.05)? Yes One- or two-tailed P value? Two-tailed Sum of  ranks in column A,B 21 , 57 Mann-Whitney U 0 Difference between medians Median of column A 2.035, n=9 Median of column B 4.678, n=9 Difference: Actual 2.644 Difference: Hodges-Lehmann 2.644  In our hypothesis, we suggested that in addition to differences in the initial DNA damage, the repair process could also be impaired in these cells. In all subject groups, the expression of γH2AX was reduced in live cells from 6 hour to 24 hour time point (Figure 18). Yet, as mentioned in the optimization part, the dose and the wavelength chosen for this study induced the cell death signal in cells after 24 hour (Figure 12 and Figure 14). As a result, this reduction in γH2AX signal could not be considered as a definite sign of dephosphorylation or repair. The focus of this study was to develop an assay to identify cancer patients from healthy individuals based on their responses to UV exposure, therefore, further investigation in the molecular level to determine possible repair pathways was out of the scope of this study. However, studying these possible mechanisms in the future would definitely help clarify the increased γH2AX signal in cancer patients. 48   Figure 18. Number of cells live and positive for γH2AX at 6 hour and 24 hour post-UV irradiation time point in healthy donors (A) and prostate cancer patients (B)  3.6.  ROC curve analysis The diagnostic accuracy of a laboratory test, or the ability of the test to correctly distinguish study subjects can be evaluated using Receiver operating characteristic (ROC) curve analysis. In a ROC curve the true positive rate (Sensitivity) is plotted as a function of the false positive rate (Specificity) for different cut-off values and each point represents a sensitivity/specificity pair corresponding to a particular threshold value. The area under the ROC curve (AUC) is a measure of how effective the test is in distinguish between two diagnostic groups.  A test with perfect discrimination has a ROC curve that passes through the upper left corner (100% sensitivity, 100% specificity).   The ROC curve analysis was undertaken in GraphPad Prism 6 (version 6.07) comparing individuals to see if PBMC responses to UV exposure could be used for diagnostics purposes. ROC analysis was performed comparing normalized γH2AX expression in live cells in PBMCs 49  isolated from PCa patients vs. PBMCs isolated from healthy donors (Figure 19). The area under the ROC curve was 0.9306 (95% CI: 0.8123 to 1.049) (P=.0029).  Cancer assays depend on being able to identify cancer-specific markers. Many cancer marker-specific assays have good sensitivities/specificities, with areas under the ROC curves of 0.80–0.9. However, to test for early-stage cancer would require a combined use of these assays. Many of these assays require identification of gene sequences and bio marker proteins which could be expensive, time-consuming and often invasive methods towards the patients. Developing a simple, inexpensive and single assay to predict susceptibility to cancer can highly reduce the need for further examinations in many cases. Although the number of individuals whose samples were collected for this study was rather small, the ROC analyses shows that the assay can identify patients with cancer from healthy volunteers with high specificity/sensitivity and narrow 95% confidence interval. However, to develop this into an assay used in clinic, future work is needed to explore various types of cancer and larger cohorts to enhance this study into a universal cancer-specific diagnostic test.     Figure 19. ROC curve analysis of normalized γH2AX expression in live cells in PBMCs isolated from PCa patients vs. healthy donors. Area under the curve was 0.9306 (95% CI: 0.8123 to 1.049). 50  Chapter 4: Conclusion and Recommendations This proof of concept case-study aimed to develop an assay to distinguish between cancer patients and healthy volunteers using their immune cell’s response to UV exposure. The objectives of the study were to evaluate and compare the degree of UV-induced DNA damage in between cancer patients and healthy individuals. Jurkat cell line was used as a model to develop and optimize the assay. To test the functionality of the assay, blood samples were collected from a proof of concept cohort study group including prostate cancer patients and healthy volunteers and irradiated using the experimental apparatus designed for the study. The analysis of γH2AX expression in live cells suggested that there were differences between the initial degree of UV-induced DNA damage in PCa patients and healthy individuals. The median of the normalized γH2AX expression in PCa group was 4.678, and for healthy donor group was 2.035 and they were proved to be statistically significant using Mann-Whitney non-parametric test (p<0.01). Our results showed an apoptosis cell response 24 hour after exposure to 3 mJ/cm2, 285 nm UV-LED.  The outcome of the study indicated that the immune system functionality and DNA damage response was more vulnerable to the UV-induced DNA damage. It is well documented that the immune system function is affected in cancer patients. However, the exact mechanism leading to increased response in PCa patients remains an open question. Since, this study was aimed to develop an assay to distinguish between study groups, it was out of the scope of this study to look at the DDR response at the molecular level and further investigation is necessary to clarify the pathway leading to the amplified γH2AX expression in cancer patients. Despite the fact that the numbers of individuals from whom samples were derived for this exploratory study was rather small, the ROC analyses showed that the assay could identify patients with cancer from healthy volunteers at high specificities and sensitivities. This view is supported by the high area under the curve value and the narrow 95% confidence intervals. Methods for the early detection of cancer are of utmost importance and progress in the identification of new biomarkers for specific cancers will continue to improve specific cancer detection. The challenge in detection of cancer in early stages is that usually a combination of cancer specific assays are required which are considered to be expensive and invasive towards the patients. Studies have found that the level of anxiety in patients waiting for their biopsy results becomes chronic, cortisol secretion goes into continuous overdrive and other body functions will 51  be affected [134]. Therefore, developing a pre-screening assay to determine which patients require further investigation with cancer specific assays would be of great significance.  The results presented in this study and the ROC curve analysis indicated that it is possible to use this assay to identify cancer-free individuals with few false negatives and individuals with cancer with few false positives. The simple and generic nature of this method makes it particularly valuable as it is not a measurement of a single causative mechanism and instead it evaluates a surfeit of mechanisms at the same time. With this being said, to evolve this method in to a simple, inexpensive, empirical and potential universal cancer pre-screening test future work is needed with a greater cohort of patients with various types of cancer. Further the effect of UV irradiation at shorter wavelengths, various operation modes, pulsation and combination of UV ranges can be investigated to have enhanced and possibly more targeted DNA damage. Overall, the test described in this study has sensitivities and specificities comparable to those of cancer-specific assays, and, can potentially identify patients with any cancer, but not a specific cancer. This assay could have a potential application in the diagnostic workup and in many cases reduce the need for expensive cancer specific tests by identifying those who do not require further investigation.    52  Bibliography [1] Canadian Cancer Statistics Advisory Committee., “Canadian Cancer Statistics 2018,” Toronto, ON, 2018. 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The overall photochemical reaction is:  8I− +  IO3− + 3H2O + hν → 3I3− + 6OH−  The actinometer solution is 0.6 M KI and 0.1 M KIO3 in 0.01 M Na2B4O7 buffer to keep the solution pH constant at 9.2 and avoid oxidation of I-. The photoproduct of this reaction is triiodide ion which has a strong absorption in UV range and can be quantified at λ=352 nm (molar absorption coefficient ε = 27636 M−1cm−1 in a 0.6 M KI/0.1 M KIO3 solution).  The following procedure was used to measure incident irradiance: 9.96 g of KI, 2.14 g of KIO3 and 0.381 g of sodium tetra-borate was weighed out and transferred into a 100 ml flask. About 60 ml of dH2O was added and the solution was stirred until all solids were dissolved. Then the solution was transferred into a volumetric flask and brought up to 100 ml mark with dH2O. The solution was prepared fresh each time and used within 4 hours of preparation.  The absorbance of freshly prepared actinometer stock was measured at 300 nm and 352 nm using Nanodrop 2000, UV-Vis option as a spectrophotometer. The NanoDrop 2000/2000c can measure samples with 10 mm path length absorbance equivalent of 300 A when using the auto path length feature. 5.0 ml of the actinometer solution and an 8 mm x 15.9 mm Teflon-coated stir bar was added to a 60 x 15 mm Corning tissue culture treated plates. The plate was placed in the centre of the LED beam as determined for all the samples throughout this thesis and the platform was raised to 64  have the same 5 cm distance as determined as the desired distance between the LED and the samples. The stir speed was set so that the stirring is gentle and avoid the formation of vortex. The solution was stirred for 7 minutes with the LED being off and then the absorbance was measured at 352 nm (A blank). The temperature of the solution was measured using a thermocouple before exposure to UV (tbefore). Then the LED was turned on and left to stabilize for at least 10 minutes and it was kept on throughout the entire process. Again 5.0 ml of the actinometer solution was added with the stir bar to the 60 mm plate, but this time it was exposed to UV for 7 minutes with the same stirring speed. The absorbance was measured at 352 nm (A sample). The temperature of the solution was measured again (tafter).The same steps were repeated for three replicates in each actinometry test performed.  The following calculations show how the incident irradiance is calculated:  [I3−] = [A352(sample) − A352(blank)]/27636 = 3.53 × 10−4 M moles I3− = [I3−] × V(L) = 3.53 × 10−4  ×5/1000= 1.7628 × 10−6 moles enstines (moles of photon) = moles (I3−)/Φ = 1.7628 × 10−6 0.37⁄=  4.76432 × 10−6 ensteins The internal diameter of the plate was measured using a caliper and the cross-sectional area (Area).  photon irradiance (Ep) =ensteins(Area × time)⁄ = 4.76432 × 10−6 22.05065 × 420s⁄= 5.14434 × 10−10ensteins s−1cm−2  Irradiance(E) = Ep × photon energy at 280 nm = 5.14434 × 10−10 × 4.72 × 105= 2.43 × 10−4 W cm−2 = 0.243 mWcm−2  The irradiance should be corrected for the 2.5% reflection from the water surface, so the incident rate on the surface is:  Irradiance(E) =0.2430.975= 0.249 mWcm−2 65   The actinometry test was performed regularly before each set of experiments to measure the UV irradiance, the results of all the tests performed is demonstrated in Figure A1. As expected, the LED efficiency showed a linear drop over the period of almost 2 years.    Figure A 1. The actinometry results over the course of the study                66  Appendix B  : UV-LED specification sheet The model used in this study is 280nm, product ID# DF8XA-0F001.  67    68  Appendix C  : Male vs. Female response to UV exposure Our healthy donor group was consisted of 9 individuals, 5 female and 3 males. In order to investigate if there was differences in the γH2AX expression after UV irradiation in these two groups, we did the Mann-Whitney statistical analysis on the normalized γH2AX expression in the 6 hours post-UV incubation timepoint (Figure C 1). The results showed that there were no significant differences between males’ and females’ immune cells response to UV exposure in our control group (p > 0.05).    Figure C 1. Comparison of γH2AX expression level in females and males in healthy donors    

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