The work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1... Show moreThe work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1 components ATF3, Fra1, c-Jun, ATF-2 and c-Fos. Chapter II provides information on the signaling pathways involved in the activation of ATF-2 and ATF3 in the response of primary human fibroblasts to ionizing radiation. In chapter III c-Jun and ATF3, the MAPK JNK and the MAPK-phosphatase MKP-1 are identified as important sensors of UV-induced-DNA damage in transcribed genes. Chapter IV shows that ATF3 acts as an antiapoptotic JNK target in T98G glioblastoma cells, whereas Fra1 seems to act as a proapoptotic effector of both JNK and ERK. In addition, it is shown that ATF3 and Fra1 have opposite effects on cisplatin-induced S phase arrest. Chapter V shows that Fra1 also can exhibit a pro-apoptotic function in UV-irradiated fibroblasts. Furthermore, this chapter reports an as yet unknown function of JNK: repression of the transactivating activity of c-Jun/Fos(- like) dimers, mediated via hyper-phosphorylation of the c-Jun transactivation domain. The data further emphasizes that c-Jun/Fos(-like) and c-Jun/ATF dimers and their respective target genes can exhibit opposite functions in DNA damage responses. Show less
One of the major problems in the treatment of patients with acute leukemia is failure of therapy due to acquired resistance, which may partially be caused by defects in the apoptotic machinery of... Show moreOne of the major problems in the treatment of patients with acute leukemia is failure of therapy due to acquired resistance, which may partially be caused by defects in the apoptotic machinery of these leukemic cells. Apoptosis is induced and regulated by a complex network of proteins connected via various signal transduction cascades, eventually leading to death of the target cell. The results described in this thesis demonstrate that apoptosis induction in leukemic cells after treatment with chemotherapy or (cellular) immunotherapy is very complex and frequently dependent on the target cell studied, and on the interaction between target and effector cell. G0 cells derived from patients with B-CLL compared to G0 cells from patients with acute leukemia responded differently to Ara-C treatment, which may be partially explained by the different mechanisms of action exerted by Ara-C. The role of the death receptor pathway in both chemotherapy-induced and CTL-induced apoptosis of leukemic cells was also unraveled in more detail. It will be worthwhile to focus future strategies in the treatment of leukemia on modulation or activation of proteins that are common in multiple apoptotic pathways, like caspase-8, since this approach will sensitize the leukemic cells simultaneously to chemotherapy and cellular immunotherapy. Show less
Several associations between 14-3-3 proteins and members of the p53 family have been revealed. However, numerous questions regarding 14-3-3 proteins, p53 family members and the relationships... Show moreSeveral associations between 14-3-3 proteins and members of the p53 family have been revealed. However, numerous questions regarding 14-3-3 proteins, p53 family members and the relationships between thetwo families remain. This thesis contributes to answer these questions. Downregulation of 14-3-3_ increased the propensity for DNA-damage induced apoptosis and increased cell-cell contacts, two characteristics that are often lost in cancer cells. Differences between 14-3-3_ and 14-3-3_ in nucleocytoplasmic shuttling have been described. The difference innucleocytoplasmic shuttling likely forms a basis for specialized biological functions of the 14-3-3 isoforms. Keratinocyte specific p53 regulated G2cell-cycle arrest and the lack of a contribution for 14-3-3_ in this cell-type has been observed, whereas 14-3-3_ induction clearly plays a distinct role in G2 arrest in other cell-types. An improved method for detecting p53 induced apoptosis by flow cytometry is portrayed by expressing a duplicated GFP protein. This double GFP protein can be used for future research in the fields of 14-3-3 and p53. A widely used method to detect and quantify p73 splice-variant expression is critically assessed. It turns out that, using this method it is not possible to accurately quantify p73splice-variants. Show less
For a tumor cell to propagate, it must survive extremely stressful conditions that would normally trigger the cell to die. Cancer cells however survive, probably due to evasion of the apoptotic... Show moreFor a tumor cell to propagate, it must survive extremely stressful conditions that would normally trigger the cell to die. Cancer cells however survive, probably due to evasion of the apoptotic cell death pathway. It follows that a detailed understanding of the regulation of the apoptotic pathways in cancer cells can improve the anti-cancer treatments. Part 1 of this thesis describes our in vitro studies regarding the regulation of apoptosis in melanoma cells, since melanoma is a form of cancer that is highly resistant to anti-cancer therapies. c-Myc enhances the apoptosis sensitivity of the cells. The protein Apaf-1 is not involved in this sensitivity. A yet unidentified serine protease plays an important role in the initiation of apoptosis upon DNA damage. Part 2 of this thesis describes our studies regarding both the regulation of apoptosis in rectal carcinoma and its prognostic value for rectal cancer patients. To evaluate the impact of (radiation-induced) tumor cell apoptosis on clinical outcome of cancer patients, the level of apoptosis have been determined in non-irradiated and irradiated rectal carcinoma samples. The level of tumor cell apoptosis is scored by immunohistochemical stainings of the carcinoma samples, and by measuring caspase-3 activity. Both studies show that high levels of apoptosis is associated with a low local recurrence risk. A genetic approach is used to identify factors that play a role in the regulation of apoptosis in rectal carcinoma in vivo. After evaluation two microarray procedures, the most convenient procedure is used to compare the gene expression profiles of tumors with high levels of apoptosis with low-apoptotic tumors. The difference in expression of several of the identified genes are confirmed on protein expression level by immunohistochemistry, and show two subsets of high-apoptotic tumors. These data suggest two different regulations of apoptosis in vivo. The prognostic value of one of the identified proteins, HLA-DR, has been studied in more detail and epithelial HLA-DR expression is significantly associated with lower recurrences and better survival for rectal cancer patients. Show less
