Targeted Cytoplasmic Irradiation and its Biological Consequence

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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

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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
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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
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Cytoplasmic IrradiationBiological Consequence
Health Risk
Nucleus
a
a
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Charged Particle Microbeam of Columbia University
- Fast 1 sec to locate and irradiate each
cell - Beam spot of lt 1 µm (and soon much
smaller lt 100 nm) - Recovery of irradiated
cells from the dish is gt98
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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
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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
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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
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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)
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Mechanism Redox-modulated events
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Mechanism(s) of Genotoxicity Induced by
Cytoplasmic Irradiation
Zhou et al. J. Radiat. Res. 2009 Hong et al.
Brit. J. Cancer 2010
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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
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Immunostain of cytoplasm-irradiated AL cells
with anti-4-HNE
Control
8 alpha particles
8 alpha particles100µM 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
BHT8a
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Induction of peroxynitrite anions in targeted
cytoplasmic irradiation
Immunostaining for nitrotyrosine as a surrogate
marker for peroxynitrite anions
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Radiation-induced Non-targeted Responses
Endpoints Clonogenic survival
Mutation Neoplastic transformation ?H2AX 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
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Can Cytoplasmic Irradiation Induce Bystander
Mutagenesis in Neighboring, Non-irradiated Cells?
Alpha particles
8-OHdG
Damage signal
Mutation
Mutation
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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)
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Cytoplasm Irradiation Induces Bystander
Mutagenesis
Induced CD59 - Mutants per 105 Survivors
8a, 20 Cytoplasmic Irradiation
Control
8a, 100 Cytoplasmic Irradiation
8a, 20 Assuming No Bystander Effect
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Signal Transduction Pathways Modulating Bystander
Effect in vitro
Zhou et al., Proc. Natl. Acad. Sci.(USA) 102
14641-14646, 2005 Ivanov et al., Cell Signaling
22(7) 1076-1087, 2010.
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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
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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.

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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

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Heis 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