Apoptoseinduktion und Zellzyklusarretierung nach Bestrahlung mit Photonen oder Kohlenstoffionen

In this study the biological impact of carbon ions in comparison to photons was analyzed in the human lung cancer cell line A549. Apoptosis induction and cell cycle arrest were examined after exposure to the two radiation types. In order to detect the relative biological effectiveness (RBE) of carbon ions compared to photons, a colony forming assay was employed by our research group. The relative expression of genes involved in cell cycle progression and apoptosis was measured by quantitative Real-Time-PCR in a monolayer cell culture (photons max. 7.5 Gy, 12C max. 4.5 Gy) as well as in a xenograft model (photons 6 Gy, 12C 2 Gy). Furthermore, cell cycle distribution after radiation was detected by flow cytometric measurement of DNA content. Additionally, radiation-induced apoptosis was monitored using an Annexin V/ PI assay. The following results were obtained: · The RBE of carbon ions (9.8 MeV/u, LET 170 keV/μm) compared to photons (6 MV) for 10% survival was 2.88. · After exposure to carbon ions expression of the pro-apoptotic gene BAX lasted longer and was up-regulated at lower doses in the monolayer cell culture as compared to photon irradiated cells. · The anti-apoptotic gene BIRC5 was down-regulated to a greater extent after carbon ion exposure (0.18-fold, photons: below threshold) in the monolayer cell culture. · There was a higher percentage (max. 12%) of radiation-induced cell death in the cancer cell line A549 after exposure to carbon ions (up to 4 Gy) than after exposure to photon beams up to 10 Gy (max. 6.7%). · The gene CDKN1A, which is involved in the G1/S checkpoint, was up-regulated up to 4-fold and up to 18-fold following photon and carbon ion irradiation in the monolayer cell culture, respectively. · GADD45A, which is involved in G2/M arrest, was barely up-regulated after exposure to photons, whereas it was up-regulated 12-fold after carbon ion exposure in the monolayer cell culture. · Exponentially growing cells were arrested in G2/M and in G1 phases after exposure to photons. After carbon ion irradiation the arrest was more pronounced and longer-lasting, even in response to smaller physical doses. · Gene expression data in the xenograft model differed from that of the monolayer cell culture model. After equivalent physical doses of photons, gene expression in the xenografts was partly higher than in the monolayer cells. Contrarily, gene expression was, to some extent, lower after carbon ion exposure in the xenografts compared to in monolayer cells. This study demonstrates that three times more cells lost their ability to divide after exposure to carbon ions than after an equivalent physical dose of photons. The considerabe respective induction and suppression of apoptosis-related genes emphasizes the more distinct activation of the apoptosis pathway after carbon ion irradiation. Additionally, apoptosis induction detected by the Annexin V assay was higher in carbon ion-exposed cells, despite being low for both treatments. The higher RBE for cell survival following carbon ion irradiation can be partly explained by differences in apoptosis induction. The marked and longer lasting cell cycle arrest and the more distinct induction of cell cycle inhibitors after exposure to carbon ions are most likely due to the more complex damage after High-LET radiation, as the cells need more time for repair. Apoptosis detection revealed that the repair capacity of the cells was exceeded more often after carbon ion irradiation. The different gene expression results in the xenograft model support the important role of the tumor microenvironment for the response to radiation in vivo. Further studies are needed to explore the types of influences that operate during the response to High-LET radiation in vivo. For future studies, it would be interesting to analyze other genes that are involved in apoptosis and cell cycle arrest pathways after carbon ion irradiation and, accordingly protein expression changes.