Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer... Show moreCellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted cellular changes, elicited by DNA damage, use of high-throughput techniques and integration with bioinformatics tools is of great value. This thesis summarizes recent advances in the field of systems biology studies of the DNA damage response and furthermore shows integrated approaches of the study of DNA damage response signaling networks in embryonic stem and cancer cells. By integration of transcriptional changes and the phosphorylation and metabolic response of cisplatin-treated embryonic stem cells, with RNAi-based knockdown screens we identify novel DNA damage response signaling networks, linking process such as Wnt signaling, translation arrest or altered metabolic pathways to the cellular response to DNA damage. Furthermore, genes, whose knockdown sensitizes embryonic stem cells to DNA damage-induced killing, are tested in cancer cells of varying genetic backgrounds identifying a small subset of genes, which represent potential drug targets for sensitization of cancer cells. Altogether, our systems approach for studying the DNA damage response identifies novel DNA damage-induced signaling networks and molecules, which modulate survival in the presence of DNA damage, potentially providing new targets for therapeutic intervention or biomarker discovery. Show less
Versieren, K.; Jeught, M. van der; O'Leary, T.; Duggal, G.; Gerris, J.; Lopes, S.C.D.; ... ; Sutter, P. de 2012
Cells in the human body have to deal with DNA damage daily, either caused by external or internal sources. The DDR is particularly strong in stem cells. Since these cells have a long life span and... Show moreCells in the human body have to deal with DNA damage daily, either caused by external or internal sources. The DDR is particularly strong in stem cells. Since these cells have a long life span and are essential for tissue homeostasis, tolerance to damaged DNA would lead to accumulation of mutations and malignant transformation. In addition, accumulation of damaged DNA would lead to loss of the stem cell pool and contribute to aging. In this thesis I investigated the role of the DNA damage response in the context of stem cells as well as cancer cells, from the response to different DNA damaging agents, to the importance of the interaction with the extracellular matrix in combination with the presence of oncogenes. In order to acquire a complete picture of the DNA damage response in mES cells, and therefore elucidate novel pathways involved in this particular response, we combined OMICS techniques such as Functional Genomics, Transcriptomics and Phosphoprotoemics, that once overlapped, allowed us to find novel pathways that where not previously described to be involved in the DNA damage response. Show less
Dekker, M.; Vries, S. de; Aarts, M.; Dekker, R.; Brouwers, C.; Wiebenga, O.; ... ; Riele, H.T. 2011
Given their self-renewing and pluripotent capabilities, embryonic stem cells (ESCs) are well-poised as a cellular source for tissue regeneration therapy. Successful in vitro differentiation of both... Show moreGiven their self-renewing and pluripotent capabilities, embryonic stem cells (ESCs) are well-poised as a cellular source for tissue regeneration therapy. Successful in vitro differentiation of both mouse (m) and human (h) ESCs into multiple somatic cell types has been reported, including cardiomyocytes, neurons and pancreatic islet cells. However, the host immune response against transplanted ESCs is not well characterized. In fact, controversy remains as to whether ESCs have immune-privileged properties. The scope of the current thesis is to gain insight into immunological aspects of transplantation of embryonic stem cells or their differentiated progeny by using molecular imaging techniques to follow cell fate. Specifically, this thesis presents evidence that: (1) molecular imaging can be used to quantify organ and ESC survival following transplantation and non-invasively follow donor graft fate; (2) ESCs express MHC and co-signaling molecules that are upregulated upon differentiation; (3) mESCs and hESCs can trigger potent cellular and humoral immune responses following allogeneic and/or xenogeneic transplantation, leading to rejection; and (4) immunosuppressive drugs can significantly mitigate the host immune response to prolong hESC survival in immunocompetent mice. These results clearly indicate that ESC immunogenicity is a significant hurdle that must be overcome before successful clinical application can be accomplished. Show less
In this thesis the role of DNA mismatch repair (MMR) in the cellular response to several genotoxic agents is described. We show that MMR plays an important role in the protection against UVC... Show moreIn this thesis the role of DNA mismatch repair (MMR) in the cellular response to several genotoxic agents is described. We show that MMR plays an important role in the protection against UVC-induced mutagenesis in mouse embryonic stem (ES) cells. UVC was shown to induce six times more mutations in mouse ES cells deficient for the mismatch recognition dimer MutSalpha compared to wild type cells. The Hprt mutational spectrum of UVC-induced mutations was similar in MutSalpha-proficient and MutSalpha-deficient mouse ES cells. We subsequently tried to gain insight into the mechanism by which MMR mediates protection against UVC-induced mutagenesis. We found that UVC induces a late S/G2-phase arrest which partially depends on the presence of MutSalpha. The MutSalpha-dependent late S/G2-phase arrest coincided with the appearance of phosphorylated Chk-1 of which the levels were higher in MutSalpha-proficient cells compared to MutSalpha-deficient cells. Importantly, abolishment of the UVC-induced late S/G2-phase arrest in both MutSalpha-proficient and MutSalpha-deficient cells did not result in a smaller difference in mutation induction between both genotypes after UVC treatment. We propose that MMR removes mismatches from UVC-induced compound lesions and that the MutSalpha-induced late S/G2-phase arrest is the result of the appearance of DNA single stranded regions arising during this process of MMR. Show less
