of 11
RESEA
RCH
ARTICL
E
Interference
with
DNA
repair
after
ionizing
radiation
by
a pyrrole-imidazole
polyamide
Silvia
Diaz-Perez
1
, Nathanael
Kane
1
, Alexis
A.
Kurmis
2
, Fei
Yang
2
, Nicolas
T. Kummer
1
,
Peter
B.
Dervan
2
, Nicholas
G.
Nickols
1,3
*
1
Department
of Radiation
Oncology,
Universit
y of Californi
a, Los
Angeles
, California,
United
States
of
America,
2
Division
of Chemis
try
and
Chemic
al Engineering,
California
Institute
of Technolo
gy,
Pasadena,
Californi
a, United
States
of America,
3
Department
of Radiation
Oncolo
gy,
VA
Greater
Los
Angeles
Healthcare
System
, Los
Angeles
, California,
United
States
of America
*
nnickols@
mednet.ucla
.edu
Abstract
Pyrrole-imidazole
(Py–Im)
polyamides
are
synthetic
non-genotoxic
minor
groove-bind
ing
small
molecules.
We
hypothesized
that
Py–Im
polyamides
can
modulate
the
cellular
response
to ionizing
radiation.
Pre-treatment
of cells
with
a Py-Im
polyamide
prior
to expo-
sure
to ionizing
radiation
resulted
in a delay
in resolution
of phosphoryla
ted
γ
-H2AX
foci,
increase
in XRCC1
foci,
and
reduced
cellular
replication
potential.
RNA-seque
ncing
of cell
lines
exposed
to the
polyamide
showed
induction
of genes
related
to the
ultraviolet
radiation
response.
We
observed
that
the
polyamide
is almost
10-fold
more
toxic
to a cell
line
deficient
in DNA
ligase
3 as
compared
to the
parental
cell
line.
Alkaline
single
cell
gel
electrophoresis
reveals
that
the
polyamide
induces
genomic
fragmentation
in the
ligase
3 deficient
cell
line
but
not
the
corresponding
parental
line.
The
polyamide
interferes
directly
with
DNA
ligation
in vitro
.
We
conclude
that
Py-Im
polyamides
may
be
further
explored
as
sensitizers
to geno-
toxic
therapies.
Introduction
Half
of
all
cancer
patients
undergo
radiotherapy
[1].
A primary
mechanism
of
action
of
radio-
therapy
is induction
of
DNA
damage.
Combinations
of
radiation
with
radiosensitizing
drugs
is a fundamental
treatment
paradigm
in
clinical
oncology
[2].
Most
systemic
therapies
cur-
rently
used
as
radiosensitizers
(e.g.,
flurouracil,
cisplatin,
mitomycin,
gemcitabine,
topoisom-
erase
poisons)
interfere
with
DNA
repair
as
part
of
their
mechanism
of
radiosensitization
[3].
However,
all
of
these
drugs
are
genotoxic.
Non-genotoxic
small
molecules
that
potentiate
the
effects
of
ionizing
radiation
in
malignant
cells
could
be
used
to
augment
radiotherapy
for
solid
tumors.
Pyrrole-imidazole
(Py–Im)
polyamides
comprise
a class
of
small
molecule
minor
groove-
binders
that
are
non-genotoxic
[4].
Py-Im
polyamides,
oligomers
of
aromatic
amino
acids
linked
in
series,
fold
into
an
antiparallel
hairpin
structure
upon
binding
DNA
[4].
The
side-by-
side
pairings
of
the
Py
and
Im
subunits
determine
DNA
target
sequence
specificity.
The
ring-
pairs
conform
to
steric
and
hydrogen
bonding
pattern
differences
in
the
floor
of
the
minor
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OPEN
ACCESS
Citation:
Diaz-Per
ez S, Kane
N, Kurmis
AA, Yang
F,
Kummer
NT, Dervan
PB, et al. (2018)
Interference
with DNA repair
after ionizing
radiation
by a
pyrrole-im
idazole
polyamide.
PLoS
ONE 13(5):
e0196803.
https://d
oi.org/10.1371/j
ournal.
pone.019680
3
Editor:
Robert
W. Sobol,
University
of South
Alabama
Mitchell
Cancer
Institute,
UNITED
STATES
Received:
January
22, 2018
Accepted:
April 19, 2018
Published:
May 1, 2018
Copyright:
This is an open
access
article,
free of all
copyright,
and may be freely
reproduced,
distributed,
transmi
tted, modifie
d, built upon,
or
otherwise
used
by anyone
for any lawful
purpose.
The work
is made
available
under
the Creative
Commons
CC0 public
domain
dedication.
Data
Availabilit
y Statement:
All relevant
data are
within
the paper
and its Support
ing Information
files. The sequencing
data is uploaded
to https://
www.ncb
i.nlm.nih.go
v/bioproject.
BioProjec
t ID:
PRJNA445611
.
Funding:
This study
was supported
by Prostate
Cancer
Foundation
Young
Investigator
Award
to Dr
Nicholas
George
Nickols,
Stop Cancer
Career
Developmen
t Award
to Dr Nicholas
George
Nickols,
National
Institute
of General
Medical
Sciences
grant
R01GM276
81 to Peter
B
Dervan,
groove
presented
by
the
edges
of
the
base
pairs
[5].
DNAse
I footprinting
titrations
and
other
methods
have
established
the
binding
affinity
and
specificity
of
these
molecules
[6].
Protein-
DNA
interactions
can
be
inhibited
by
allosteric
changes
in
the
DNA
minor
and
major
grooves
induced
by
Py-Im
polyamide-DNA
binding
[7].
Py-Im
polyamides
composed
of
eight
aro-
matic
rings
localize
to
the
nucleus
in
live
cells
[8].
Py-Im
polyamides
fail
to
activate
a canonical
DNA
damage
response
[9],
are
not
genotoxic
on
their
own
[10,11],
and
do
not
significantly
alter
cell
cycle
distribution
at
concentrations
used
for
gene
expression
studies
[9].
Py-Im
poly-
amides
have
been
used
as
molecular
probes
in
cell
culture
to
modulate
gene-expression
path-
ways
[12,13]
and
interfere
with
RNA
Polymerase
II elongation
[10,14].
We
hypothesized
that
a Py–Im
polyamide
could
modulate
the
cellular
response
to
ionizing
radiation.
Pre-treatment
of
LNCaP
and
VCaP
cells
with
a Py-Im
polyamide
prior
to
exposure
to
ionizing
radiation
resulted
in
a delay
in
resolution
of
phosphorylated
γ
-H2AX
foci
indica-
tive
of
delayed
repair
of
double
strand
breaks,
and
increased
induction
of
XRCC1
foci
consis-
tent
with
a higher
frequency
of
single
strand
breaks.
RNA-sequencing
of
cell
lines
treated
with
the
polyamide
showed
induction
of
genes
related
to
the
ultraviolet
radiation
response.
We
observed
the
polyamide
is almost
10-fold
more
toxic
to
a LN-428
cell
line
deficient
in
DNA
ligase
3 as
compared
to
its
parental
cell
line.
Alkaline
comet
assay
reveals
that
the
polyamide
induces
genomic
fragmentation
in
the
Ligase
3 deficient
but
not
the
parental
line.
The
polyam-
ide
interferes
directly
with
DNA
ligation
in vitro
.
We
conclude
that
Py-Im
polyamides
may
be
further
explored
as
sensitizers
to
genotoxic
therapies.
Materials
and
methods
Cell
culture
Early
passage
LNCaP
(ATCC,
CRL-1740)
and
VCaP
(ATTCC
CRL-2876)
were
cultured
in
RPMI
1640
with
10%
FBS
and
DMEM
with
10%
FBS,
respectively.
LN428
glioma
cell
lines
KD-BER-LN428-control
and
KD-BER-LN428-LIG3
(Trevigen
54999-001-01
and
5504-001-
01)
were
cultured
in
alpha
MEM
medium
supplemented
with
10%
Heat
Inactivated
FBS,
10
mg/ml
Gentamycin,
1
μ
g/ml
Puromycin.
Immunofluorescence
Irradiation
was
performed
using
a laboratory
irradiator
(Gulmay
Medical).
Immunofluores-
cence
was
performed
on
cells
grown
on
cover
slips
coated
with
0.1
mg/ml
of
Poly-D-lysine,
fixed
with
4%
paraformaldehyde
(electron
microscopy
science)
in
PBS
for
15
min
at
room
temperature.
Cell
permeabilization
was
performed
with
0.5%
ice-cold
Triton
X-100
for
15
min
at
room
temperature.
Cells
were
incubated
with
blocking
solution
(10%
FBS,
heat
inactivated
on
0.05%
Tween
on
PBS)
for
1 hour
at
room
temperature.
Cover
slip
were
incubated
with
1:30
dilution
with
primary
antibodies
anti
γ
-H2AX-FITC
(Millipore,
# 16-202A),
Anti-XRCC1
(Novus,
# NB100-532),
4 ̊C
overnight.
Anti-XRCC1
was
detected
with
1:200
dilution
of
don-
key
anti-Rabbit
DyLight
594
(Novus,
# NB1P1-75642).
Nuclei
were
counterstained
with
DAPI,
viewed
with
Leica
DMR
fluorescent
microscope,
images
captured
with
Quips
mFISH
software
(Vysis).
Three
fields
were
selected
at
random
and
10
nuclei
per
field
were
counted.
Cell
viability
measured
using
xCELLigence
The
xCELLigence
system
noninvasively
monitors
viability
of
cultured
cells
by
impedance,
quantified
as
cell
index
(CI),
representing
cell
number,
viability,
morphology.
Assays
were
in
96
well
plates
with
KD-BER-LN428-control,
KD-BER-LN-428-LIG3
cells
at
6000
cells/well.
Interferenc
e with
DNA
repair
after
ionizing
radiation
by
a pyrrole-imid
azole
polyamide
PLOS
ONE
| https://doi.or
g/10.137
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ne.01968
03
May
1, 2018
2 / 11
Jonsson
Cancer
Center
Foundation
Seed
Grant
to
Dr Nicholas
George
Nickols.
The funders
had no
role in study
design,
data collection
and analysis,
decision
to publish,
or preparation
of the
manuscript.
Competing
interests
:
F. Yang
is the vice president
(research)
at and has ownership
interest
(including
patents)
in Gene
Sciences,
Inc. N.G. Nickols
has
ownership
interest
(including
patents)
in Gene
Sciences,
Inc. P.B. Dervan
is the founder
of, has
ownership
interest
(including
patents)
in, and is a
consultant/a
dvisory
board
member
for Gene
Sciences,
Inc. This does
not alter our adherence
to
PLOS
ONE policies
on sharing
data and
materials.
Varying
concentrations
of
1
(0.3.
0.1,
0.3,
1.0,
3.0
10,
30
μ
M)
were
added,
incubated
at
37 ̊C
with
readout
every
5 minutes
for
72
hours.
The
experiment
was
run
in
biological
triplicate.
Cell
viability
assay
with
PrestoBlue
Exponentially
growing
LNCaP
cells
on
6 well
plates
were
treated
with
10
μ
M
1
for
24
hours
and
then
irradiated.
The
cells
were
pelleted
and
resuspended
twice
to
remove
polyamide
from
the
media,
plated
at
4000
and
8000
cells/mL
in
96
well
plates.
The
plates
were
incubated
at
37 ̊C
for
2 weeks,
PrestoBlue
(Invitrogen)
was
added
and
incubated
30
min
at
room
tempera-
ture,
fluorescence
read
at
560
nm
by
spectrophotometry
(SPECTRMax).
Alkaline
comet
assay
Alkaline
comet
assay
was
perform
as
described
[15]
using
a Trevigen
kit
(4250-050-K).
After
treatment
with
1
or
vehicle,
cells
were
centrifuged.
Cells
suspension
of
1x10
5
/ml
in
PBS
(Ca
+
+
and
Mg
++
free)
were
embedded
in
300
μ
l
of
1%
low-melt
agarose
at
37 ̊C
and
50
μ
L
were
mounted
on
CometSlides
(Trevigen)
pre-incubated
at
37 ̊C.
Embedded
cells
were
lysed
at
4 ̊C
for
60
minutes
in
the
dark,
treated
20
min
in
alkaline
unwinding
solution
(200mM
NaOH,
1
nM
EDTA
pH
>
13.3)
at
room
temperature
in
the
dark,
and
electrophoresed
(21V
for
30
min-
utes)
in
a pre-chilled
apparatus
with
fresh
un-winding
buffer
as
previously
described.
Slides
were
fixed
in
70%
ethanol
for
5 minutes,
dried
at
37 ̊C,
stained
with
100
l of
2X
diluted
SYBR
Gold
(Invitrogen)
for
30
minutes.
Slides
were
imaged
with
a Leica
DMR
fluorescent
micro-
scope
and
quantified
using
OpenComet
in
ImageJ.
DNA
T4
Ligase
experiments
Sequences
of
oligonucleotides
used
in
ligation
assay
are:
1a
FAM,
FAM-GACGCAAGTTC
AGCT
CGA;
1b CAAGTTCAGACGC
;
2a
CTGCGTTCAAGTCGAGCTG
TTCAAGTCTGCG
(Integrated
DNA
Technology).
Ligation
was
performed
in
presence
of
varying
concentrations
of
1
and
2
,
100
nM
of
the
annealing
oligos
and
4U
of
T4
ligase
for
1 hour
at
room
temperature.
The
liga-
tion
was
stop
by
adding
5
μ
l
of
TBE-Urea
sample
buffer
and
incubation
at
70 ̊C
for
3 minutes.
The
ligation
products
were
analyzed
by
acrylamide
electrophoresis
on
a 15%
TBE-Urea
gel
[16]
at
180
V for
1 hour.
Image
acquisitions
of
the
gels
were
done
by
Typhoon
Imaging
System
and
image
quantification
by
Quant
Software.
RNA
sequencing
and
analysis
LNCaP
and
VCaP
cells
were
plated
at
5
×
10
4
cells/mL
in
10-cm
2
dishes,
treated
with
or
with-
out
10
μ
M
1
in
RPMI
1640
and
DMEM
supplemented
with
10%
FBS,
respectively,
for
24
hours.
Total
RNA
was
TRIzol
extracted,
sequenced
(Illumina
HiSeq2000),
and
mapped
against
the
human
genome
(hg19)
with
Tophat2
using
Ensembl
GRCh37
gene
annotations.
Htseq-
count
was
used
for
exon
alignment
and
DESeq2
for
differential
expression.
Pathway
analysis
was
performed
with
the
gene
set
enrichment
analysis
(GSEA)
software
on
genes
with
padj
<
0.05
and
p
<
0.05
for
LNCaP
and
VCaP,
respectively.
Cell
cycle
Cells
cultured
at
70%
confluence
were
treated
with
10
μ
M
1
for
48
hours
and
cell
cycle
distri-
bution
assessed
by
monoparametric
propidium
iodide
flow
cytometry,
analyzed
by
FacScan
I
(Becton
Dickinson)
and
ModFit
software.
Interferenc
e with
DNA
repair
after
ionizing
radiation
by
a pyrrole-imid
azole
polyamide
PLOS
ONE
| https://doi.or
g/10.137
1/journal.po
ne.01968
03
May
1, 2018
3 /
11