Genetisch bedingte Variabilität der Expression von Genen des TGFß-Signalwegs und deren Bedeutung für die Zellvitalität: Untersuchungen in T-Lymphozyten, immortalisierten B-Lymphozyten und Fibroblasten
Genetically determined variability in the expression of TGFbeta signalling pathway genes and relation to cell vitality: Investigations in T-lymphocytes, immortalized B-lymphocytes and fibroblasts.
by Michael Neumann
Date of Examination:2013-01-23
Date of issue:2013-01-29
Advisor:Dr. Markus Schirmer
Referee:Prof. Dr. Jürgen Brockmöller
Referee:Prof. Dr. Andrea Hille
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EnglishThe TGFbeta pathway is involved in a huge number of biological functions and, hence, relevant for a plethora of clinical disorders. A major function of this pathway is its contribution to fibrotic conditions as it may occur, for instance, upon radiotherapy. The aim of this thesis was to investigate gene expression patterns of the TGFbeta pathway following exposure to irradiation and to test for associations with germline polymorphisms pertinent to the respective genes. As biomaterial human fibroblasts, lymphoblastoid cell lines (LCLs) and isolated T lymphocytes of healthy volunteers were used. Radiotherapy was simulated as single dose irradiation. Specific stimulation was performed with human recombinant TGFbeta and compared with the effects of an antibody against TGFbeta. As functional read-outs mRNA gene expression changes and cytotoxic effects were assessed using quantitative real time PCR and fluorescence-activated cell sorting with cell viability assays, respectively. Dense data on germline polymorphisms of the LCLs were available and regarding fibroblasts and T-lymphocytes selected polymorphic sites were assayed and impact on the determined functional parameters was tested. Regarding the gene expression analyses, in addition to the known actions of TGFbeta on SMAD3 and SMAD7 vice versa effects could now be demonstrated for an antibody directed against TGFbeta. Gene expression modulation by irradiation was mainly seen in the fibroblasts but without any relation to cell viability. The genetic analysis revealed a an association of an intronic SMAD3 polymorphism rendering increased radioresistance in T-lymphocytes thereby confirming the findings of two predecessor studies. A second putatively relevant polymorphism was deduced form the comprehensive genetic data available for the LCLs affecting both sensitivity toward irradiation and TGFbeta-mediated effects on SMAD3 expression. The TGFBR1*6A allele, which was frequently discussed in literature, did not affect cell viability but appeared to modify TGFbeta signalling. In summary, germline polymorphisms in SMAD3 genetic region seem to modulate radiosensitivity and may serve, if confirmed by other studies including prospective clinical trials, as future biomarkers for therapy stratification according to the genetic make-up. Molecularly, clarification of the mechanisms behind the identified associations may assist in the development of new intervention strategies to further optimize individual radiotherapy.