The human body is constantly exposed to ionizing radiation of different qualities. Especially the exposure to high-LET (linear energy transfer) particles increases due to new tumor therapy methods using e.g. carbon ions. Furthermore, upon radiation accidents, a mixture of radiation of different quality is adding up to human radiation exposure. Finally, long-term space missions such as the mission to mars pose great challenges to the dose assessment an astronaut was exposed to. Currently, DSB counting using γH2AX foci is used as an exact dosimetric measure for individuals. Due to the size of the γH2AX IRIF of ~ 0.6 μm, it is only possible to count DSB when they are separated by this distance. For high-LET particle exposure, the distance of the DSB is too small to be separated and the dose will be underestimated. In this study, we developed a method where it is possible to count DSB which are separated by a distance of ~ 140 nm. We counted the number of ionizing radiation-induced pDNA-PKcs (DNA-PKcs phosphorylated at T2609) foci (size = 140 nm ± 20 nm) in human HeLa cells using STED super-resolution microscopy that has an intrinsic resolution of 100 nm. Irradiation was performed at the ion microprobe SNAKE using high-LET 20 MeV lithium (LET = 116 keV/μm) and 27 MeV carbon ions (LET = 500 keV/μm). pDNA-PKcs foci label all DSB as proven by counterstaining with 53BP1 after low-LET γ-irradiation where separation of individual DSB is in most cases larger than the 53BP1 gross size of about 0.6 μm. Lithium ions produce (1.5 ± 0.1) IRIF/μm track length, for carbon ions (2.2 ± 0.2) IRIF/μm are counted. These values are enhanced by a factor of 2-3 compared to conventional foci counting of high-LET tracks. Comparison of the measurements to PARTRAC simulation data proof the consistency of results. We used these data to develop a measure for dosimetry of high-LET or mixed particle radiation exposure directly in the biological sample. We show that proper dosimetry for radiation up to a LET of 240 keV/μm is possible.
- MeSH
- biologické markery MeSH
- dávka záření MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- fluorescenční mikroskopie metody MeSH
- fosforylace účinky záření MeSH
- HeLa buňky MeSH
- lidé MeSH
- lineární přenos energie MeSH
- lithium škodlivé účinky MeSH
- oprava DNA účinky záření MeSH
- proteinkinasy účinky záření MeSH
- radiační expozice MeSH
- radiometrie metody MeSH
- těžké ionty škodlivé účinky MeSH
- uhlík škodlivé účinky MeSH
- záření gama škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.
- MeSH
- ATM protein MeSH
- DNA vazebné proteiny MeSH
- fosforylace účinky záření MeSH
- G2 fáze účinky záření MeSH
- ionizující záření MeSH
- jaderné proteiny * fyziologie MeSH
- lidé MeSH
- nádorové supresorové proteiny MeSH
- protein-serin-threoninkinasy * fyziologie MeSH
- proteinkinasy * fyziologie metabolismus účinky záření MeSH
- proteiny buněčného cyklu * fyziologie MeSH
- replikace DNA účinky záření MeSH
- S fáze účinky záření MeSH
- serin metabolismus MeSH
- Check Tag
- lidé MeSH
- MeSH
- buněčné dělení fyziologie genetika MeSH
- CDC geny fyziologie MeSH
- Chlamydomonas reinhardtii fyziologie genetika účinky záření MeSH
- finanční podpora výzkumu jako téma MeSH
- mitóza fyziologie MeSH
- proteinkinasy účinky záření MeSH
- rostlinné proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
