Cellulär och molekylär respons på låga doser av joniserande strålning
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1 Cellulär och molekylär respons på låga doser av joniserande strålning Bo Stenerlöw Biomedicinsk strålningsvetenskap Rudbecklaboratoriet Uppsala universitet Bo Stenerlöw Biomedical Radiation Sciences Uppsala University Rudbeck Laboratory S Uppsala Sweden
2 Ionising radiation and cytotoxic drugs: -breaks on DNA Ionising radiation DNA lesions senescence signalling DNA repair apoptosis mitotic cell death cell genomic instability protein activation gene activation cell cycle growth factor receptors
3 Cellulär och molekylär respons på låga doser av joniserande strålning Biomedicinsk strålningsvetenskap, Uppsala universitet Bakgrund och tidigare studier Molekylär och cellulär respons (SSM-projekt) Sammanfattning Pågående studier
4 Ionising radiation and cytotoxic drugs: -breaks on DNA Ionising radiation DNA lesions senescence signalling DNA repair apoptosis mitotic cell death cell genomic instability protein activation gene activation cell cycle growth factor receptors
5 Ionising radiation and cytotoxic drugs: -breaks on DNA Ionising radiation DNA lesions senescence signalling DNA repair apoptosis mitotic cell death cell genomic instability protein activation gene activation cell cycle growth factor receptors
6 DNA is the main target DNA damages induced by ionising radiation DNA Type of damage approx. number/cell after 1 Gy (γ- or X-ray) (SSB) base 1000 SSB 1000 (DSB) DSB 30 > simple DNA lesions(base damage, SSB etc.) are produced per day in every cell (without radiation exposure) metabolic processes -> free radicals replication errors heat instability etc. 2 nm These DNA lesions are repaired fast and efficiently!
7 DNA Damage & Repair Biomedical Radiation Sciences, Uppsala University Clustered DNA damaged sites: High LET DNA repair pathways ATM DSB DSB detection DNA-PKcs, KU80, KU70 Modifying DNA ends MRE11-RAD50-NBS1 complex DNA damage High LET (LET = Linear Energy Transfer) -Alpha-particles -Auger electron emitters -Heavy ions DNA ligation DNA ligase IV, XRCC4 SF 2Gy : 0.6 -> fractions ->25 X reduction in cell survival Growth factor receptors and DNA repair Affects the cellular sensitivity to ionising radiation
8 Measuring DNA double-strand breaks (DSB) in irradiated cells -Methodology is critical! High LET radiation: -clustered DNA damaged sites Non-DSB lesions
9 High relative biological effectiveness (RBE) 1 -x-rays -photons -electrons (high E) Cell Survival 0,1 0,01 -alpha particles -heavy ions 0, Radiation Dose (Gy) RBE S=0.2 = 4.3 (4.7 Gy/1.1 Gy)
10 Slow repair of high-let induced DSB Gamma 125 ev/nm ions 80 Initial damage Repair time (h)
11 Low-LET radiation: random distribution of ionisations and DSB Size standard 0 Gy irradiated DNA size Photons kbp t= DNA fragmentation analysed by pulsed-field gel electrophoresis (PFGE) 10 kbp Fraction of DNA DNA fragment size (kbp) Random (photons)
12 High excess of small DNA fragments after high-let irradiation Fraction of DNA (bp -1 ) t=0 Experimental (N ions, 125 ev/nm) Random distribution Size (kbp) small DNA fragments low mass, large numbers Höglund et al. RR 2000; Fakir et al. RR 2006
13 High excess of small DNA fragments after high-let irradiation Fraction of DNA (bp -1 ) t=0 Experimental (N ions, 125 ev/nm) Random distribution Size (kbp) small DNA fragments low mass, large numbers: increases the total DSB 2x Höglund et al. RR 2000; Fakir et al. RR 2006
14 High LET (125 ev/nm) Fraction of DNA (bp -1 ) Intact cells DNA Random distribution Size (kbp) Chromatin organization is responsible for non-random DNA fragmentation after high LET irradiation Radulescu et al., Radiat. Res. 161, 1 (2004)
15 125 I decays (Auger electrons): 1 decay = 1 DSB? -A single 125 I decay within DNA may induce multiple DSB 1 decay = 1 DSB? 125 I Clustered DSB may not be detected
16 Clustered DSB may not be detected -A single 125 I decay within DNA may induce multiple DSB 1 decay = 1 DSB? NO! 125 IdU incorporation DSB/cell/decay Asynchronous I Early S-phase 0.6 Late S-phase 2.3 Elmroth and Stenerlöw, Radiat. Res. 163, 369 (2005) Elmroth and Stenerlöw, Radiat. Res. 168, 175 (2007)
17 Conclusion 1: Clustering of DSB may lead to underestimation of the initial number of breaks (by a factor of 2 or more)
18 Conclusion 1: Clustering of DSB may lead to underestimation of the initial number of breaks (by a factor of 2 or more) This will also affect the estimates of repair kinetics! (Gustafsson, Hartman and Stenerlöw, submitted, 2013)
19 DNA double-strand breaks is critical for cell survival Non-homologous end joining (NHEJ): Broken DNA DSB DSB detection DNA-PKcs, KU80, KU70 Modifying DNA ends MRE11-RAD50-NBS1 complex Repair (h) Repair DNA ligation DNA ligase IV, XRCC4
20 DNA double-strand breaks is critical for cell survival Non-homologous end joining (NHEJ): Broken DNA DSB X DSB detection X Modifying DNA ends DNA-PKcs, KU80, KU70 MRE11-RAD50-NBS1 complex Little repair Little/No repair DNA ligation DNA ligase IV, XRCC4
21 DNA repair pathways Partial deficiency of DNA-PK Increase risk (deficiency) Lung cancer Uterine cervix cancer Breast cancer Colon cancer
22 Low levels of DNA-PKcs leads to radiosensitivity - Clonogenic survival assay Surviving fraction (SF) A Mock 0.01 sidna-pkcs Radiation dose (Gy) Surviving fraction (SF) HCT116 1 Mock sidna-pkcs Radiation Dose (Gy) SF (2 Gy) Cell line Mock sidna-pkcs A431 0,55 0,14 HCT116 0,26 0,09 H314 0,36 0,03 Surviving fraction (SF) H314 Mock sidna-pkcs Surviving fraction (SF) M059K/J M059K M059J M059K/J 0,44 (K) 0,04 (J) Radiation dose (Gy) Radiation Dose (Gy)
23 BUT: does not influence DNA repair - 53BP1 foci- single DSB - PFGE-DNA rejoining A431 2 Gy GM Gy 53BP1 foci/cell Ctr GM Gy Repair time (h) sidna-pkcs Mock 0 Gy 15 min sidna-pkcs % of intial damage % of intial damage HCT hours M059K/J hours Mock sidna-pkcs M059J M059K
24 Deficiency influences mitosis Syncronization with Nocodazole (G2)- release and IR Immuno co-localization of mitosis and p-dna-pkcs A431 % ph3 positive cells siprkdc Mock P-H2AX Ser2056 ph3 Thr CTR CTR Noc 2 Gy Noc 6h, 18 without Merged P-H2AX- Ser2056 Merged ph3- Thr2609 (Gustafsson, Abramenkovs and Stenerlöw, submitted, 2013)
25 Conclusion 2 Down-regulation of DNA-PKcs leads to radiosensitivity without affecting DSB repair Decrease survival No effect on DNA repair Stop in mitosis-indicating other roles of DNA-PKcs than in DSB repair
26 Summary: Large fraction of small DNA fragments after high-let Clustering of DSB may lead to underestimation of the initial number of breaks (by a factor of 2 or more) Rejoining of high-let induced DSB is not extremely slow Heat-labile sites: independent of LET DNA-PKcs is a critical protein for DSB repair, but it also regulates other important cellular stress response pathways
27 Pågående studier: DNA-PKcs reglering av mitos och check-points mekanismer DNA-skador och kromatinstruktur Klusterskador från hög-let-strålning
28 High LET induced clusters of DSB 10 µm 2 high LET particles 1 track 0.5 Gy
29 Clusters of DSB within γ-h2ax/53bp1 foci γ-h2ax 53BP1 Merged Ion DSB/focus LET (ev/nm) min post-irradiation
30 High LET induced clusters of DSB DSB DSB Complexity (<20-30 bp) Clusters of DSB within chromatin (<1 Mbp)
31 Tack! Uppsala University Biomedical Radiation Sciences Andris Abramenkovs Ann-Sofie Gustafsson Karin Karlsson (former PhD student) Irina Radulescu (former PhD student) Diana Spiegelberg
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