The influence of ionising radiation on intracellular pathways of the mitogen activated protein kinase (MAPK) family
Final Report Abstract
Cancer is still one of the major causes of death in humans and dogs. More than 50% of patients diagnosed with a solid malignant cancer will be treated with radiation therapy during the course of their disease. For the treatment of radioresistant tumor types combined treatment modalities are needed to increase overall cure rates. Therefore, the effects of ionising radiation on tumor cells and on tumor endothelial cells are studied. Nowadays, it is well known that ionizing radiation itself induces a multi-layered stress response within the cell. We are now beginning to understand how these changes are influencing radiation response. To analyse the effect of several factors on tumor response, two cell lines from spontaneous canine tumors were generated and characterised. First, a radiosensitive squamous cell carcinoma cell line (DM702) and second a radioresistant fibrosarcoma cell line (TG903). In this study, we focused on mitogen activated protein kinase (MAPK) pathways known to be involved in stress response to radiation therapy. Especially the c-jun NH2- terminal kinase (JNK). This stress activated protein kinase has been implicated in cell survival and cell apoptosis. Interestingly, JNK activity differed tremendously in both cell lines. A prolonged activation was found in the radiosensitive DM702 cell line. The radioresistant TG903 cell line had a short, intense JNK activity directly after dosd application. Inhibition of JNK by the small molecular JNK-inhibltor SP600125 resulted in increased radlosensitivity and apoptosis induction in the TG903 cell line, while in contrast the DiV1702 cells were protected from radiation induced apoptosis. Thus, from this study it can be concluded, that fine tuned JNK activation by ionizing radiation can be differently regulated and thereby co-determine the cellular response to ionizing radiation. The fine tuned regulation of JNK is an additional example for the complexity of cell signalling. This fine tuned signaling makes it difficult to use modulation of activation of those pathways as therapeutical target. The second part of this thesis focused on the protective effect of erythropoietin (Epo) on tumors in vivo and in vitro. Epo is produced physiologically in the kidney and it regulates the production of red blood cells in the bone marrow. Under physiological conditions, hypoxia is a good stimulus for Epo synthesis. In the last years attention has been drawn on the additional cell protective functions on several tissues such as brain and heart. This area of research was developed based on the finding of recent clinical trials where a worsened prognosis was found in human cancer patients treated with Epo in combination with standard radiation therapy. This result was highly unexpected and it was hypothesised that tumor cells themselves might express Epo and Epo receptor and that Epo might have a tissue protective effect in tumors comparable to heart and neuronal tissue. The in vivo analysis showed that spontaneous canine tumors express Epo and EpoR. The expression of Epo in tumor tissue strongly correlated to directly polarographically measured tumor hypoxia. These findings indicated that the Epo-EpoR pathway exists in certain canine tumors. The synthesis and release of Epo seem to be induced by hypoxia, via HIF-1 as it is known under physiological conditions. These results pointed towards the possibility that recombinant human Epo might have a radioprotective effect on tumors. To further elucidate this question, an in vitro study was designed. Since one of the clinical trails with the poor outcome performed in patients diagnosed with squamous cell carcinoma of the head and neck region, the radiosensitive DM702 cell line was used for this study. This cell line expresses Epo and EpoR: The concentration of Epo in cell lysate and cell culture medium increased under hypoxic conditions. The treatment of the DM702 cells prior to ionizing radiation had a clear radioprotective effect. This effect was also seen with endogenously produced Epo by using conditioned media and a comparable radioprotection was present. Taking the results from, both studies together, we concluded that Epo is expressed and released from tumor cells. The released Epo has a protective effect on tumor cells, and this Epo might also protect vessels from radiation induced apoptosis. This pin points towards a possible negative effect of rHuEpo in the treatment of both, human and canine cancer patients when it is attempted to be corrected for cancer induced anaemia. This study elucidated a small piece of a very complex puzzle, and further work is needed for detailed understanding of the molecular interactions in a solid tumor.
Publications
- Interrelation of directly measured oxygention levels, erythropoietin and erythropoietin receptor expression in spontaneous canine tumors. Eur J Cancer 2007;43(5):963-967
C. Rohrer Bley and M. Wergin, M. Roos, B. Grenacher, B. Kaser-Hotz
