Molecular and cellular response pattern of human fibroblasts exposed to heavy ion ir- radiation

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]. 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 [2-6]. 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 [4,7,8]. 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 [9]. As a consequence of both photon and heavy ion irradiation, it could be demonstrated in the present study that human skin fibroblasts respond similar in terms of TGF-β1 mRNA gene expression, which was stimulated slightly (1.2x – 1.4x) by radiation exposure. However, analyzed at the active level of the cytokine TGF-β1 in the culture medium, radiation exposure led to a app. 2.8-3-fold increase within 60 min after radiation. Consequently, it can be postulated that radiation (photon and heavy ion radiation) rather leads to the proteolytic activation of latent TGF-β1 (LTGF-β1) to the active form of this cytokine than to a stimulation of gene expression [10]. For practical reasons, this hypothesis was tested first in photon-irradiated cells by the use of a specific inhibitor of the protease furin, which is known to be a major component of the proteolytic activation process of LTGF-β1 to TGF-β1. Preincubation with the furin inhibitor CMK resulted in a significant decrease of the phosphorylation of Smad-2 and –3 proteins involved in the intracellular TGF-β1 receptor-dependent signal transduction. Although not yet analyzed, but based on the original data of significantly elevated and presumably TGF-β1-dependent terminal differentiation of fibroblasts exposed to heavy ion as compared to photons, it can be assumed that heavy ion irradiation will lead to a similar if not more pronounced activation of LTGF-β1 [10].