Cell and molecular biological analyses of the induction of fibrotic changes in human skin and lung fibroblasts exposed to heavy ion irradiation

As compared to conventional photon radiation heavy ions may offer specific therapeutic advantages in radiation oncology with respect to the higher relative biological effectiveness (RBE) at beam terminus. It has been demonstrated for heavy ion irradiation that the RBE in irradiated normal tissue, especially for acute effects is only slightly increased. However, since normal tissue reactivity, i.e. acute and late effects, is dose limiting in radiation therapy the proposed research project is concerned with the underlying cellular and molecular processes of normal tissue reactivity induced by heavy ions in comparison to photon irradiation. A prominent and clinically highly relevant radiation therapy-induced late reaction of normal tissue is fibrosis [1,2]. Fibrosis can occur especially in skin and lung and is characterized by remodelling of connective tissue resulting in enhanced collagen production and deposition. Although fibrosis is the result of a multicellular process involving endothelial cells smooth muscle cells, immunocompetent cells and fibroblasts within the irradiated tissue, the fibroblast cell system is the cell type responsible for the expression of the fibrotic tissue phenotype. Over the recent years our laboratory could demonstrate that the radiation-induced terminal differentiation of the fibroblast cell system, i.e. the induced differentiation of progenitor fibroblasts to postmitotic highly collagen synthesizing fibrocytes, is the key event in the induction and manifestation of the fibrotic tissue remodelling [3-8]. Molecular biological studies into the basic mechanisms of radiation-induced terminal fibroblast differentiation revealed that the cytokine TGF-β1 is the key factor which mediates the radiation-induced differentiation of progenitor fibroblasts to fibrocytes at the level of signal transduction in an autocrine as well as paracrine fashion. This has been demonstrated in a number of experiments analysing the cellular responses of both normal human or rat skin and lung fibroblasts to ionizing radiation (photons and heavy ions) with and without concomitant treatment with TGFß1neutralizing antibodies [4-6,9]. Finally, by the use of lung fibroblast cultures from homozygous TGFß1-knock out mice, it could be demonstrated that TGFß1 is the main determinator of fibroblast radiation sensitivity [10]. On the basis of these molecular studies into the cellular mechanism of radiation-induced fibrosis it can be concluded and hypothesized that fibroblasts are stimulated to produce enhanced levels of activated TGFß1 in response to radiation exposure. Activated TGFß1 then binds to the TGFß1 receptor II. In a cascade of phosphorylation reactions several members, i.e. Smad proteins (esp. Smad 4), of the TGFß1-receptor-dependent signal transduction are activated and the TGFß1-signal is transduced to the nucleus. Consequently, the expression of TGFß1target genes is upregulated resulting in an enhanced expression and activity of the cyclin-dependent kinase inhibitors (cdk-inh.) p21, p27, p15, and p16. Most likely these inhibitors are responsible for the induction of radiation-induced terminal fibroblast differentiation (Rodemann et al. 2001, in prep.). This assumption is based on the observation that TGFß1-neutralizing antibodies can block the long lasting or permanent (>6-8 hrs, TP53 independent) upregulation of p21 expression in response to both radiation and TGFß1-treatment, but not the transient, TP53-dependent p21 expression (< 6 hrs). The results of the ongoing project will give detailed insights into the regulatory mechanisms of radiation-induced terminal fibroblast differentiation by heavy ions. Together with the results to be established by the Biophysics group at the GSI [11], which investigates the molecular mechanisms and processes of the radiation-dependent activation of the latent inactive form of newly synthesized and secreted LTGFß1, the data will help to describe the specific molecular and cellular pathways, which lead the development of fibrosis in response to radiation exposure. Consequently, these data will not only allow a specific estimation of the risk of fibrosis in radiation therapy applying heavy ions, but also lead to the development of new strategies to prevent or interfere with late normal tissue complications, like fibrosis in radiation oncology.