Radiation Enhanced Cancer Progression
in Human Colonic Epithelial Cells
Andres I. Roig
U.T. Southwestern Medical Center
UT Southwestern Project Studying Effects of
Space Radiation on Human Cells and Tissues
• Several NASA funded projects studying the
effects of space radiation on different tissues:
–
–
NSCOR (NASA Specialized Center of Research)
• Lung cancer risk assessment
Individual grants:
• Space radiation effects on central nervous system
• Space radiation shielding approaches
• Space radiation effects on DNA damage sensing and repair
• Space radiation and risk for cancer progression in human
colonic epithelial cells
** Large uncertainty exists in risk estimates for cancer
development after a 3 year mission to Mars or extended
stays in the Moon
Rems per year (log scale)
Radiation Levels Experienced by Space Travelers
Distance from Earth’s Surface (not to scale)
Scientific American
Space Radiation and DNA Damage
•
On travelling to Mars
– Every cell nucleus will hit by a proton or electron every few days
– By a high mass and charge ion (HZE particle) once a month
Cucinotta, et al. Lancet Onclogy. 2006
HZE particle
Cell nucleus
srag.jsc.nasa.gov/SpaceRadiation/Why/DNA1.gif
¾ Susceptible tissues: Lung, gastrointestinal, blood, breast, CNS,
Tissue damage, concern for cancer
eye lens
HZE Particles Have Complex Track Structures
High Linear Energy Transfer (LET) Core
and Low LET Penumbra
PENUMBRA
(LOW-LET)
PENUMBRA
Track End (LOW-LET)
PENUMBRA
(LOW-LET)
CORE
(High-LET)
PENUMBRA
(LOW-LET)
CORE
PENUMBRA
(LOW-LET)
Track End
Track End
PENUMBRA
(LOW-LET)
Cross Section
Track End
Modified from Aloke Chatterjee
HZE Particles Cause Complex Damage
to Tissues
• Cells directly traversed by
HZE particle may not survive
• Cells irradiated with
secondary particles or delta
rays may be more likely to
survive and progress to cancer
¾ It is generally accepted that
Stem cells
accumulated DNA damage
increases the risk of cancer
progression
Colon Cancer
• Third most common type of cancer and second
most frequent cause of cancer-related death in
the united states. It is estimated that for 2008
approximately 108,000 new cases occurred with
50,000 deaths (www.cancer.org)
• Usually begins as a noncancerous polyp that
can, over time, become a cancerous tumor
Prevalence of Colonic Polyps Increases with Age
• 40 to 49 age group: 22% to 40%
• 50 to 60 age group: 50% to 60%
¾ With increasing age more time to accumulate mutations
Genomic
Instability, 18q
Trisomy 7, 5q
NORMAL
EPITHELIUM
ABERRANT
CRYPTS
APC
EARLY
ADENOMA
INTERMEDIATE
ADENOMA
K-ras
Smad4
Adapted from Fearon and Vogelstein
17p, Trisomy 20
LATE
ADENOMA
CARCINOMA
p53
Telomerase
METASTASIS
Other
alterations
¾ Is there a risk of enhanced colonic tumorigenesis in middle
age astronauts after long-term missions and exposure to
chronic low doses of space radiation?
Current Estimates for Risk of Cancer Death After
Extended Extra-orbital Space Missions
Reason for uncertainties
• Correctness of scaling
data from atomic bomb
survivors/patients receiving
radiation to astronauts
exposed to chronic low
doses of space radiation
• Differences in radiation
quality
• Dose rate
• Inter-individual variability
Durante, et al. Nature Reviews Cancer. 2008
Need Models to Determine the Risk for Space
Radiation-Enhanced Colon Cancer Progression
•
Human models
– Tissue culture: two and three dimensional (2-D, 3-D models)
– Intact tissues (colonic biopsies)
•
Research focus:
– Creating new cell lines of adult human colonic
epithelial cells
• Non-transformed
• Able to sustain long-term growth
– Characterize the acute effects of DNA damage at the
cellular and tissue level
– Determine if cells have progressed to cancer after
irradiation
– Test medical and dietary countermeasures with well
known mechanisms of action that can reduce cancer
progression
Creating Adult Human Colonic Epithelial Cell
Lines
•
•
Prior human colon cell lines are derived from cancerous
tissues.
– Disadvantage: already contain cancer-causing
mutations
Extract and immortalize human colonic epithelial cells
(HCEC) from colonic biopsies of normal tissues
Reya et al. Nature. 434: 843-850, 2005
Crypt Extraction Procedure
Human colon biopsies
Antibiotic washes
24 hrs after
plating
Cut
Antibiotic washes
Liberated crypt
Collagenase
and dispase
Crypts
Early Growth of Human Colonic Epithelial Cells
24 hrs
12 days
E-cadherin
ZO-1
Serum free
Merge
Dividing cell
¾ Growth on supplemented basal media, 2% oxygen, Primaria® flasks, colon
fibroblast feeder layers; serum gradually reduced to zero percent within first ten days
Infection with Retroviral Vectors Containing
Cdk4 and hTERT for Long-Term Growth
First passage
Post-infection
• Retroviral infections with Cdk4 (to bypass culture stress) and
hTERT (to immortalize)
• Antibiotic selection
Long-Term Growth of Immortalized HCECs
Data 1
Population Doublings, PD
Patient 1
150
HCEC 1 CT
(Cdk4 and hTERT)
HCEC Cdk4 + hTERT
100
PD 15
Days in Culture
HCEC 1
50
0
0
50
100
150
Days in Culture
200
HCEC non-infected control
Patient 2
Population Doublings, PD
PD 125
150
HCEC 2 CT
(Cdk4 and hTERT)
100
50
HCEC 2
0
0
50
100
Days in Culture
150
PD 15
PD 24
2D Models of Radiation-Enhanced Colonic
Tumorigenesis
or
Irradiate cells at
UTSW with
gamma rays
Irradiate cells at NSRL
with Fe or Protons
Colonic cells
Evaluate for:
Soft Agar
Ionizing radiation
Top Layer
Cells
Bottom Layer
-PK
DNA
γH2AX
ATM
53BP1
DNA damage/repair response
Top Layer
Colony
Bottom Layer
Anchorage independent growth to
assess for cancer progression
Persistence of DNA Damage in 2D Colonic
Epithelial Cells After HZE Irradiation
1 Gy (1 GeV/n) 56Fe Irradiated Normal Colonic Epithelial Cells
2hr
24hr
2hr
1 Gy (1 GeV/n) Proton Irradiated Normal Colonic Epithelial Cells
Control
30min
2hr
24hr
100
Percent foci remaining
Control
30min
1 Gy (1GeV/n)
56
Fe
80
1 Gy (1GeV/n)
Protons
60
40
20
0
DAPI
Gamma H2AX
DNA- PKcs
pT2609
Merge
10
20
Time (hrs)
30
Persistence of DNA Damage in 3D Cultures of
Colonic Epithelial Cells after HZE Irradiation
Layer human colonic cells
on top of human colonic
fibroblasts in collagen I gels
Remove medium to form a
medium/air interface
Control
4hrs
30min
24 hrs
24hr
1 Gy (1GeV/n) 56Fe
Control
4hrs
30min
24 hrs
Percent foci remaining
1 Gy Gamma Rays
DNA-PKC pT2609
DAPI
DNA-PKC pT2609
Low LET 1
Gy Gamma
100
80
High LET 1Gy
(1GeV) Fe56
60
40
20
0
DAPI
Human colonic biopsy
Epithelial Cells on
collagen/fibroblast gel
0
10
20
Time
30
What is the Pattern of DNA Damage in Human
Colonic Tissues After Low or High LET Radiation?
Colonic Biopsy
Crypt openings
H&E demonstrating normal tissue
microenvironment in biopsies
Stereoscopic image
More Pronounced DNA Damage Response in the
Epithelial Compared to Stromal Compartment after 1Gy
Gamma Rays
DAPI
DNA-PKcs pT2609
control
30min
2hr
24hrs
Colonic
epithelial
cells
Stromal
cells
Average percent remaining foci
DAPI
DNA-PKcs pT2609
100
Colonic epithelial cells
Stromal cells
80
60
40
20
0
0
5
10
15
Time (hours)
20
25
Similar Pattern for Other DNA Damage-Repair
Proteins and in HZE Irradiated Biopsies
DAPI
30 minutes: 1 Gy, 1GeV
53BP1
56Fe
particles
Colonic
epithelial
crypt
Stromal
cells
DAPI
DNA-PKC pT2609
Stromal
cells
Colonic
epithelial
cells
Stromal
cells
Colonic
epithelial
crypt
DAPI
53BP1
Conclusions from DNA Damage and Repair Studies
•
•
•
High LET HZE particles cause more complex and
difficult to repair DNA damage
Cells of various types existing in organized systems
(tissues) may respond differently to radiation
Implications to cancer progression:
– Complex, more difficult to repair DNA damage may accumulate
–
¾
in cells over time, especially quiescent cells
Example of quiescent cell: colonic stem cells
Colonic stem and stem-like progenitor cells are
important targets of radiation-induced DNA damage.
¾ Do our immortalized HCECs display stem cell markers?
Location of Stem Cells in Human Colonic Crypts
Colonic Crypt
Differentiated
Cells
Proliferative
Cells
Mesenchymal
cells
PCNA
Modified from Clevers
Stem cells
Development of Adenomas
Colonic Crypt
Differentiated
Cells
Adenoma
Proliferative
Cells
Mesenchymal
cells
PCNA
Stem cell
Mutated stem cell
Radiation Risk Assessment Using
Cancer-Initiated Cells
ABERRANT
CRYPTS
EARLY
ADENOMA
APC
INTERMEDIATE
ADENOMA
K-ras
LATE
ADENOMA
Smad4
Trisomy 7 (HCEC CT+7 [PD 106])
METASTASIS
CARCINOMA
Other
alterations
p53
Telomerase
APC down-regulation
HC
EC
Diploid HCEC (PD 26)
17p, Trisomy 20
(P
HC
D
26
EC
)
CT
HC
(P
D
EC
54
CT
)
A
HC
(P
T1
D
16
15
0)
DL
D1
Trisomy 7, 5q
NORMAL
EPITHELIUM
Adapted from Fearon and Vogelstein
Genomic
Instability, 18q
METASTASIS
250KD
In situ
Chromosome 7
150KD
100KD
Dypti Lulla
Ugur Eskiocak
Mutant186 bp
Wild 156 bp
β-actin
80% p53 knockdown
HCEC
CTAR+7
KRASV12
HCEC
CTAP+7
p53 knock down
CT
R
Mo
lec
ula
rM
ar
ke
r
p53
HC
EC
CT
HC
EC
HCEC CTA +7
HC
EC
CT
P
HC
EC
CT
Establishment of Cellular Reagents with Additional
Mutations in the Colon Cancer Pathway
200 bp
100 bp
Mutant Kras expression
Subpopulation:
Trisomy 20 (HCEC CTARP+7,+20)
Growth in Soft Agar
CT
CTAP+7
CTR+7
HELA
Ugur Eskiocak
Conclusions From Cancer Progression Studies
• Cellular reagents established representing distinct
stages in the in vivo colon cancer progression model
– Normal diploid (express stem cell markers, non-tumorigenic)
– Cancer-initiated cells (APC down-regulated, trisomy 7)
– Cancer-progressed cells (mutant Kras, p53 knockdown, trisomy
20)
• CDDO-Me exerts a radio-protective effect via induction
of antioxidant enzymes
• Preliminary results from experiments on HCEC CTs
show:
– CDDO-Me decreases DNA double-strand breaks in the acute
setting
– Diminishes long-term tumorigenic transformation after irradiation
Future Directions
• Establishment of additional colonocytes from patients
undergoing routine colonoscopy of a range of donor
ages
• Experimenting with other HZE particles: oxygen, silicon,
titanium
• Administration of CDDO-Me after IR. Is CDDO-Me also a
mitigator of cancer progression?
• Try other cancer mitigating agents: Vitamin D3, NSAIDs,
calcium
• Extend experiments to mouse models
– APC min/+ , CDX2P Apc flox/+
Acknowledgements
•
NASA grant NNX08BA54G
Dr. Jerry Shay
Ugur Eskiocak
Dr. Woody Wright
Suzie Hight
Kim Batten