Transcriptional tales of heavy ion-hit cells – consequences for space exploration missions

Kurzfassung

INTRODUCTION Space radiation exposure is a major limiting factor in human spaceflight. Radiation-induced cellular DNA damage, if unrepaired or mis-repaired during the cellular radiation response, increases the risk of cancer and degenerative diseases. Transcriptional modulation is a major component of this response and regulates many downstream outcomes such as cell cycle arrest, apoptosis, senescence, and differentiation. Characterizing transcriptional responses to space radiation may provide mechanistic insight beyond DNA damage induction, and help identify targets for countermeasures. To determine qualitative and quantitative differences in the cellular radiation response, we compared the transcriptional responses of human and murine cells to heavy ions and X-rays.

METHODS Murine pre-osteoblasts (OCT-1), human embryonic kidney (HEK) cells and A549 (p53 wt) were exposed to X-rays or heavy ions (C, O, Ne, Ar, Ti, Ni, Pb). At different time points after irradiation, ribonucleic acid (RNA) was extracted and gene expression was analysed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) or RNA sequencing (RNA-Seq). In HEK cells, NF-B activation was monitored using a stably transfected d2EGFP reporter plasmid. Based on RNA-Seq data, bioinformatics tools (pre-ranked gene set enrichment analysis – GSEA, over-representation analysis - ORA) were employed to identify a gene expression signature of high-LET C ions in A549 cells.

RESULTS In OCT-1 cells, CDKN1A mRNA levels did not correlate with the extent of G2/M arrest, but with physical dose of X-rays and C, Ne and Fe ions. In HEK cells, NF-B activation was strongest after exposure to heavy ions with a linear energy transfer (LET) of 50 - 300 keV/µm, accompanied by increased expression of NF-B target genes that overlapped with the expression profile induced by treatment with tumor necrosis factor α (TNF-α). Also, in A549 cells, the increase in expression of NF-B target genes was greater after exposure to C ions in comparison to X-rays. A549 cells demonstrated stronger enrichment of p53 pathway, G2M checkpoint, E2F targets and mitotic spindle response after exposure to C ions compared to X-rays. Furthermore, C ions activated inflammatory and mesenchymal stress programs not observed after X-rays irradiation. While many histones were upregulated in response to X-rays, they were downregulated after irradiation with C ions, representing a distinct transcriptional signature specific to high-LET ion exposure.

CONCLUSION High-LET ion irradiation induces a transcriptional response that is quantitatively and qualitatively distinct from that elicited by low-LET X-rays at equal physical doses. Typical pathways involved in the cellular radiation response are more strongly enriched after high-LET radiation exposure (p53 pathway, G2M checkpoint, E2F targets and mitotic spindle response). Striking qualitative differences were observed in inflammatory and mesenchymal stress programs and chromatin architecture; the latter might be caused by the dense ionization pattern and complex DNA damage induced by high LET ions, resulting in the need for chromatin remodelling for efficient DNA repair.

ACKNOWLEDGEMENT We thank DLR, the Higher Education Commission of Pakistan (HEC) - HRDI-UESTP’s/UET’s-Faculty Training in cooperation with the “Deutscher Akademischer Austauschdienst” - German Academic Exchange Service (DAAD) and the European Union for funding (ENSAR no. 262010 & 654002). We acknowledge the GANIL dosimetry team and beam operators, and the DLR Biodiagnostics Group for their help during the heavy ion beam-times.