Radiotherapy remains one of the most important treatment modalities for cancer therapy. Malignant tumors show an extended spectrum of intrinsic radiation sensitivity even among tumors of the same entity or with similar histological features. Predicting intrinsic radiation sensitivity might improve treatment outcome and allow individualized treatment. Hence, an assay that provides a predictive information of the tumor’s intrinsic radiation sensitivity is of great need. Histone H2AX, a histone variant of histone H2A family, is rapidly phosphorylated upon DNA double strand break (DSB) induction resulting in gamma H2AX (γH2AX). Gamma H2AX accumulates at DNA DSB sites and subsequently recruits DNA damage repair factors. Formation of γH2AX is visualized by an immunohistology-based approach and detected as foci under an epifluorescent microscope. Gamma H2AX foci represent DNA DSBs, while residual γH2AX foci (foci detected 24 h post irradiation) are considered as unrepaired damages. In previous studies, the γH2AX foci assay showed a high potential as a predictive method for radiosensitivity. This thesis aims to further translate and optimize the ex vivo γH2AX foci assay for a clinical applicability. In this study, all experiments were performed using human head and neck squamous cell carcinoma (hHNSCC) xenograft models. For ex vivo investigations, tumors on the hind legs of nude mice were excised and cut into multiple pieces, or fine-needle biopsies of the tumors were taken. Tumor biopsies were reoxygenated in culture medium for 10 h or 24 h followed by radiation exposure of 0 8 Gy. Tumor biopsies were fixed and embedded in paraffin 24 h post irradiation. For the γH2AX foci assay under in vivo conditions, tumors-bearing mice were irradiated with single doses of 0 8 Gy. Tumors were excised, fixed, and paraffin embedded 24 h post irradiation. Manual quantification of γH2AX foci was performed exclusively in perfused areas, which were identified by pimonidazole (hypoxic marker) and BrdU (proliferation marker) staining. Foci number was corrected, normalized, and statistically analyzed by a linear mixed effects model (LMEM), linear regression model or analysis of covariance. To investigate tumor heterogeneity in the ex vivo γH2AX foci assay, γH2AX foci were enumerated in four equally treated tumor specimens per group i.e. unirradiated and ex vivo irradiated with 4 Gy. Strong intratumoral heterogeneity in γH2AX foci was determined with a minor intertumoral heterogeneity. No significant effect of reoxygenation between 10 h or 24 h was observed, enhancing clinical practicability of the assay. The effect of experimental settings was studied by analyzing data from this study (ex vivo) and from comparable published data (in vivo) with LMEM. Radiation induced nuclear area alteration was detected in some of the evaluated tumor models in under both experimental conditions. A greater intra and intertumoral heterogeneity were observed in the ex vivo set up compared to the in vivo set up. Radiation response determined by the γH2AX foci assay in ex vivo irradiated biopsies and in the corresponding in vivo irradiated tumors was evaluated. Between in vivo and ex vivo, four out of five tumor models showed comparable slopes of dose response curves (SDRC) of normalized and corrected γH2AX foci. SDRC of normalized γH2AX foci was able to classify tumors according to their intrinsic radiation sensitivity (TCD50). In conclusion, the ex vivo γH2AX foci assay holds a promising potential for predicting radiation sensitivity in solid tumors. The comparable radiation response assessed by γH2AX foci of in vivo irradiated tumors and the matching ex vivo irradiated tumor biopsies supports clinical applicability of the assay. Using SDRC of γH2AX foci as a predictor of radiosensitivity, radioresistant and radiosensitive tumors could be classified. The significant intratumoral heterogeneity in the ex vivo γH2AX foci assay suggests a limited representativeness of a single biopsy for radiosensitivity prediction. Additionally, the change of tumor microenvironment modulated cellular adaptation and DNA damage repair capability. The outcomes suggested that a sufficient number of cells, regions of interest, and biopsies are required to obtain a solid prediction.:Contents List of Abbreviations List of Figures List of Tables 1. Introduction 1.1 Effect of ionizing radiation on cellular level 1.1.1 Radiation induces cell death 1.1.2 Cell-cycle arrest mediated by radiation 1.2 DNA damage repair 1.2.1 Non homologous end joining (NHEJ) 1.2.2 Homologous recombination (HR) 1.2.3 Base damage repair and single strand break repair 1.2.4 Role of γH2AX in DNA damage repair 1.3 Prediction of tumor radioresponsiveness 1.3.1 Prediction of tumor radiation sensitivity by γH2AX 2 Tumor heterogeneity determined with a γH2AX foci assay: A study in human head and neck squamous cell carcinoma (hHNSCC) 2.1 Summary of the publication 3 Heterogeneity of γH2AX foci increases in ex vivo biopsies relative to in vivo tumors. 3.1 Summary of the publication 4 Comparable radiation response of ex vivo and in vivo irradiated tumor samples determined by residual γH2AX foci 4.1 Summary of the manuscript 5 Discussion 5.1 Tumor heterogeneity in γH2AX foci assay 5.2 Alteration of nuclear area post irradiation 5.3 Clinical relevance of the γH2AX foci assay 5.4 Technical challenges and limitations of the assay 5.5 Conclusion and Outlook 6 Abstract 7 Zusammenfassung 8 Bibliography Acknowledgement Appendices Part A: Materials A.1 Tumor lines A.2 Chemicals and Materials A.3 Devices and Software Part B: Supplementary materials B.1 Supplementary materials of publication I B.2 Supplementary materials of publication II B.3 Supplementary materials of manuscript

The structure, function and accessibility of epidermal skin provide aunique opportunity to study the DNA damage response (DDR) of a normaltissue. The in vivo response can be examined in detail, at a molecularlevel, and further associated to the structural changes, observed at atissue level. We collected an extensive skin biopsy material frompatients undergoing fractionated radiotherapy for 5 to 7 weeks. Several end-points inthe DDR pathways were examined before, during and after the treatment. Quantification of DNA double strand break (DSB) signalling focirevealed a hypersensitivity to doses below 0.3Gy. Furthermore, aconsiderable amount of foci persisted between fractions. The low dosehypersensitivity was observed throughout the treatment and was alsoobserved for several key parameters further downstream in the DDR-pathway, such as p21-associated checkpoint activation, apoptosisinduction and reduction in basal keratinocyte density (BKD).Furthermore, for dose fractions above 1.0 Gy, a distinct acceleration inDDR was observed half way into treatment. This was manifested as anaccelerated loss of basal keratinocytes, mirrored by a simultaneousincrease in DSBs and p21 expression. Quantifications of mitotic events revealed a pronounced suppression ofmitosis throughout the treatment which was clearly low dosehypersensitive. Thus, no evidence of accelerated repopulation could beobserved for fraction doses ranging from 0.05 to 2Gy. Our results suggest that the keratinocyte response primarily isdetermined by checkpoints, which leads to pre-mitotic cell elimination by permanent growth arrest and apoptosis. A comparison between the epidermal and dermal sub-compartments revealsa consistent up-regulation of the DDR response during treatment. Adifference was however observed in the recovery phase after treatment,where miR-34a and p21 remain up-regulated in dermis more persistentlythan in epidermis. Our observations suggest that the recovery phaseafter treatment can provide important clues to understand clinicalobservations such as the early and late effects observed in normaltissues during fractionated radiotherapy.

Ionisierende Strahlung verursacht in Abhängigkeit von den strahlenphysikalischen Eigenschaften der Radionuklide Zellschäden unterschiedlicher Komplexität. An humanen Lymphozyten wurde untersucht, ob die biologische Wirksamkeit von Alpha- und Betastrahlung sowie der Einfluss von Doxorubicin der Qualität des Strahlenschadens zugewiesen werden kann. Die DNA-Schäden und deren Reparatur wurden mit zellbiologischen Methoden quantifiziert.

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Ionisierende Strahlung verursacht in Abhängigkeit von den strahlenphysikalischen Eigenschaften der Radionuklide Zellschäden unterschiedlicher Komplexität. An humanen Lymphozyten wurde untersucht, ob die biologische Wirksamkeit von Alpha- und Betastrahlung sowie der Einfluss von Doxorubicin der Qualität des Strahlenschadens zugewiesen werden kann. Die DNA-Schäden und deren Reparatur wurden mit zellbiologischen Methoden quantifiziert.