Ionizing Radiation, DNA Damage Response and Cancer Therapy Resistance

A frequently used treatement option in cancer is ionizing radiation. Unfortunately , therapy resistance occurs in the course of treatments. The work described in this thesis aims to elucidate the role of the DNA damage response in acquired resistance to ionizing radiation and its differential regulation upon DNA damage. Chapter 2 shows that different DNA repair pathways display redundancy in radiotherapy-resistant prostate cancer cells. Chapter 3 unravels the mRNA response in radiotherapy resistance using next generation sequencing to identify novel pathways. Energy metabolism was identified as one of the main contributors to radiotherapy resistance next to the DNA damage response. Chapter 4 presents the differential response of an important cell type, embryonic stem cells, to equitoxic dosages of three genotoxic agents. Based on mRNA and small RNA sequencing we were able to identify a novel posttranslational modification, citrullination, that is involved in the proper function of homologous recombination upon induction of double strand breaks (DSBs) in DNA in cancer cells. In Chapter 5 the role of RNA-binding proteins in DSB repair in cancer cells was investigated. Unexpectedly, we found that proper homologous recombination repair of DSB is dependent on RNA-binding proteins. However, the ef

Attenuated XPC expression is not associated with impaired DNA repair in bladder cancer

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors

Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications