<p>Radiotherapy is one of the most common therapeutic methods for treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (< 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use.</p><p>8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids.</p><p>Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively.</p><p>Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption.</p><p>The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG.</p><p>In summary, our results confirm that extracellular 8-oxo-dG is a sensitive <i>in vivo</i> biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.</p>

Radiotherapy is one of the most common therapeutic methods for treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (&lt; 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids. Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively. Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption. The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG. In summary, our results confirm that extracellular 8-oxo-dG is a sensitive in vivo biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.

Fifty percent of solid cancers are treated with radiation therapy (RT). The dose used in RT is adjusted to the most sensitive individuals so that not more than 5% of the patients will have severe adverse healthy tissue effects. As a consequence, the majority of the patients will receive a suboptimal dose, as they would have tolerated a higher total dose and received a better tumor control. Thus, if RT could be individualized based on radiation sensitivity (RS), more patients would be cured and the most severe adverse reactions could be avoided. At present the mechanisms behind RS are not known. The long term aim of this thesis was to develop diagnostic tools to assess the individual RS of breast cancer patients and to better understand the mechanisms behind the RS and radiation effects after low dose exposures. The approach was based on the hypothesis that biomarkers of individual RS, in terms of acute adverse skin reactions after breast cancer RT, can be found in whole blood that has been stressed by low doses of ionizing radiation (IR).  To reach this goal two different approaches to identify biomarkers of RS have been investigated. A protocol for the analysis of differential protein expression in response to low dose in vitro irradiated whole blood was developed (paper I). This protocol was then used to investigate the proteomic profile of radiation sensitive and normo-sensitive patients, using isotope-coded protein labeled proteomics (ICPL). The results from the ICPL study (paper III) show that the two patient groups have different protein expression profiles both at the basal level and after IR. In paper II the potential biomarker 8-oxo-dG was investigated in serum after IR. The relative levels of IR induced 8-oxo-dG from radiation sensitive patients differ significantly from normo-sensitive patients. This indicates that the sensitive patients differ in their cellular response to IR and that 8-oxo-dG is a potential biomarker for RS. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Genomic alterations may initiate cancer development as the consequence of endogenous or exogenous DNA damaging factors. Defects in DNA repair mechanisms may also facilitate cancer progression as well as accumulation of mutations which favor cancer cell survival. However, DNA repair pathways in cancer cells can be considered as their Achilles heel which are possible targets in order to compromise their survival. For instance, it has been demonstrated recently that inhibition of a protein called MTH1 via RNA interference (RNAi) or chemical inhibitors can stop tumor growth and triggers cell death by increasing the load of oxidative DNA damage. MTH1 is a hydrolase which converts 8-oxo-dGTP into 8-oxo-dGMP in order to prevent incorporation of oxidatively damaged nucleotides into DNA. In addition, DNA glycosylases which recognize and remove mismatched or damaged nucleotide pairs in DNA can also participate in repair of 8-oxo-dG, such as MUTYH repairing A:8-oxo-dG pair. The goal of the current study was to investigate the importance of MUTYH activity upon MTH1 depletion. The current study tried to answer whether simultaneous knock-down of MTH1 and MUTYH sensitizes cancer cells to oxidative stress and increases cell death. Both enzymes were simultaneously depleted in T cell acute lymphoblastic leukemia cells using RNAi. Then, we analyzed the efficiency of gene and protein knock-down by quantitative real-time-PCR and western blotting, respectively. Induction of cell death was also assessed by flow cytometric analysis of cell cycle. Afterwards, the effect of the treatments on DNA repair pathways was studied by analysis of gene expression of several DNA glycosylases and DNA polymerases using qRT-PCR. The results showed that concurrent depletion of both enzymes led to synergistic induction of cell death. Down-regulation of NEIL1 DNA glycosylase as well as POLQ and POLH DNA polymerases mRNAs adapted their DNA repair pathways to cope with induced damages under these conditions. Finally, the results of this study suggest that dual suppression of MTH1 and MUTYH may provide a new approach to reduce survival of T cell ALL.

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