Cellulär och molekylär respons på låga doser av joniserande strålning - PDF

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Cellulär och molekylär respons på låga doser av joniserande strålning Bo Stenerlöw Biomedicinsk strålningsvetenskap Rudbecklaboratoriet Uppsala universitet Bo Stenerlöw Biomedical Radiation Sciences Uppsala

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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 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 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 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 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 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! 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 Measuring DNA double-strand breaks (DSB) in irradiated cells -Methodology is critical! High LET radiation: -clustered DNA damaged sites Non-DSB lesions 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) Slow repair of high-let induced DSB Gamma 125 ev/nm ions 80 Initial damage Repair time (h) 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) 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 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 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) 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 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) Conclusion 1: Clustering of DSB may lead to underestimation of the initial number of breaks (by a factor of 2 or more) 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) 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 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 DNA repair pathways Partial deficiency of DNA-PK Increase risk (deficiency) Lung cancer Uterine cervix cancer Breast cancer Colon cancer 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) 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 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) 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 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 Pågående studier: DNA-PKcs reglering av mitos och check-points mekanismer DNA-skador och kromatinstruktur Klusterskador från hög-let-strålning High LET induced clusters of DSB 10 µm 2 high LET particles 1 track 0.5 Gy Clusters of DSB within γ-h2ax/53bp1 foci γ-h2ax 53BP1 Merged Ion DSB/focus LET (ev/nm) min post-irradiation High LET induced clusters of DSB DSB DSB Complexity ( 20-30 bp) Clusters of DSB within chromatin ( 1 Mbp) 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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