Targeted Cytoplasmic Irradiation and its Biological Consequence

1. Targeted Cytoplasmic Irradiation and its Biological Consequence Tom K. Hei, Ph.D. Professor and Vice-Chairman of Radiation Oncology Professor of Environmental Health Sciences Columbia University, New York, NY. Presented by: Edouard Azzam Department of Radiology New Jersey Medical School, Newark, NJ

2. Radiation Carcinogenesis • A stochastic & late effect • No (or very low) dose threshold • Severity is not dose related • Probability of occurrence increases with dose • In radiation protection: Linear No-Threshold model is accepted practice • For years, it was taught that nucleus is a prerequisite to produce genetic damage or an important biological response * Polonium needle experiment Munro TR, Radiat. Res.42: 451, 1970

3. Effect (Cancer Incidence) ? Radiation Dose Non-nuclear targeted effects and possible shapes of dose response curve for carcinogenesis at low doses Gold standard: A-bomb survivors Supra-linear Cancer Risk Linear quadratic Linear Threshold Radiation Dose

4. Cytoplasmic Irradiation:Biological Consequence & Health Risk Nucleus a a

5. Charged Particle Microbeam of Columbia University - Fast: 1 sec to locate and irradiate each cell - Beam spot of < 1 μm (and soon much smaller: < 100 nm) - Recovery of irradiated cells from the dish is >98%

6. Paradigm shift: Extranuclear irradiation induces genomic mutations Use of Hoechst and Nile Red to distinguish nucleus from cytoplasm … Advances in microscopy Wu, L.J. et al., Proc. Natl. Acad. Sci. (USA) 96: 4959, 1999

7. The human hamster hybrid AL cells contain a single human chromosome 11 and a gene at 11p13.1 encodes the cell surface CD59 antigen that forms the basis of the mutagenic assay 7

8. Cytoplasmic irradiation (with 8  particles) induced mutations similar to those that arise spontaneously (i.e. small deletions in CD59 locus) Nuclear targeting resulted in multi-locus deletionsWu et al., PNAS 1999 8  particles through nucleus Spontaneous mutants 8  particles through cytoplasm Human chromosome 11 markers used in analysis

9. Involvement of Reactive Oxygen Species Use of fluorogenic probes [e.g.  2’,7’ –dichlorofluorescein diacetate (DCFDA)] to detect and quantify ROS in cytoplasmic irradiated (left panel) and control AL cells (right panel)

10. Mechanism: Redox-modulated events

11. Mechanism(s) of Genotoxicity Induced by Cytoplasmic Irradiation Zhou et al.J. Radiat. Res. 2009 Hong et al. Brit. J. Cancer 2010

12. Genotoxicity Induced by Cytoplasmic Irradiation  Immunoperoxidase staining for 8-OHdG in AL cells Zhou et al.J. Radiat. Res. 2009 Hong et al. Brit. J. Cancer 2010

13. Immunostain of cytoplasm-irradiated AL cells with anti-4-HNE Control 8 alpha particles 8 alpha particles+100µM BHT * 40.0 35.0 30.0 25.0 Fluorescence Intensity (arbitrary unit) 20.0 15.0 10.0  5.0 0.0 0 8a BHT+8a

14. Induction of peroxynitrite anions in targeted cytoplasmic irradiation Immunostaining for nitrotyrosine as a surrogate marker for peroxynitrite anions

15. Bystander Mutagenesis Incubation Expression Period Absence of bystander yieldsbackground mutation frequency Radiation-induced Non-targeted Responses Endpoints: Clonogenic survival Mutation Neoplastic transformation γH2AX foci induction Apoptosis Chromosomal aberrations Micronucleus Intracellular oxidant levels DNA damage signaling Zhou et al., Proc. Natl. Acad. Sci. (USA) 97: 2000 Zhou et al., Proc. Natl. Acad. Sci. (USA) 98: 2001 5 Gy

16. Can Cytoplasmic Irradiation Induce Bystander Mutagenesis in Neighboring, Non-irradiated Cells? Alpha particles 8-OHdG Damage signal Mutation Mutation

17. Targeted cytoplasmic irradiation induces micronucleus formation in bystander cells in vicinity Targeted cytoplasmic traversal of glioblastoma T98G cells induces micronuclei in co-cultured, bystander human fibroblasts. The bystander induction was suppressed in the presence of the nitric oxide scavenger c-PTIO (Shao et al. PNAS, 2004)

18. Cytoplasm Irradiation Induces Bystander Mutagenesis Induced CD59 - Mutants per 105 Survivors 8a, 20% Cytoplasmic Irradiation 8a, 100% Cytoplasmic Irradiation 8a, 20% Assuming No Bystander Effect Control

19. Signal Transduction Pathways Modulating Bystander Effect in vitro Zhou et al., Proc. Natl. Acad. Sci.(USA)102: 14641-14646, 2005 Ivanov et al.,Cell Signaling22(7): 1076-1087, 2010.

20. Do progeny of bystander cells demonstrate genomic instability ? Bystander cells Human hamster hybrid AL cells Irradiated Cells Overnight incubation Colony pickup, Culture, Cell killing, Mutation, and Chromosome assay Died

21. Targeted cytoplasmic irradiation induces chromosomally unstable clones Dr. Burong Hu • Chromosomal instability clone: Clone derived from a single cell that shows at least three distinct metaphase subpopulations involving rearrangements of the human chromosome, • Rearrangements account for a minimum of 5% of the total metaphases scored.

22. S U M M A R Y • Development of microbeam technology has empowered a paradigm shift in our understanding of basic radiobiological concepts • From cytoplasmic damage, to non-targeted effects, and to genomic instability among progeny of non-hit cells, the availability of microbeam technology has offered unparallel research opportunities • These studies have moved beyond the traditional 2D cell cultures to include 3D human tissues, plants and whole organisms • Mechanistic insight on non-targeted effects and targeted cytoplasmic irradiation have been made possible • Together, these studies are basic to understanding the stress response, intercellular communication and late health effects of radiation

23. Hei’s Research Team http://www.crr-cu.org/hei.htm Vladimir Ivanov Yunfei Chai Hong N. Zhou Yong L. Zhao Gloria Calaf Geng Wan Winston Liao Amish Shah Brian Ponnaiya NIH grants: P01-CA 49062-19; Superfund ES 10349-10; R01-ES11804-S1 R01-ES11804-05; NIEHS Center grant ES 09089-12