International Journal of
Molecular Sciences
Technical Note
Impact of Premature Senescence on Radiosensitivity
Measured by High Throughput Cell-Based Assays
Razmik Mirzayans *, Bonnie Andrais and David Murray
Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, AB T6G 1Z2, Canada;
[email protected] (B.A.); [email protected] (D.M.)
* Correspondence: [email protected]; Tel.: +1-780-432-8897
Received: 11 June 2017; Accepted: 1 July 2017; Published: 6 July 2017
Abstract: In most p53 wild-type human cell types, radiosensitivity evaluated by the colony
formation assay predominantly reflects stress-induced premature senescence (SIPS) and not cell
death (Int. J. Mol. Sci. 2017, 18, 928). SIPS is a growth-arrested state in which the cells acquire
flattened and enlarged morphology, remain viable, secrete growth-promoting factors, and can give
rise to tumor-repopulating progeny. The impact of SIPS on radiosensitivity measured by short-term
assays remains largely unknown. We report that in four p53 wild-type human solid tumor-derived
cell lines (HCT116, SKNSH, MCF7 and A172): (i) the conventional short-term growth inhibition
assay (3 days post-irradiation) generates radiosensitivity data comparable to that measured by the
laborious and time-consuming colony formation assay; (ii) radiation dose-response curves obtained
by multiwell plate colorimetric/fluorimetric assays are markedly skewed towards radioresistance,
presumably reflecting the emergence of highly enlarged, growth-arrested and viable cells; and (iii)
radiation exposure (e.g., 8 Gy) does not trigger apoptosis or loss of viability over a period of 3 days
post-irradiation. Irrespective of the cell-based assay employed, caution should be exercised to avoid
misinterpreting radiosensitivity data in terms of loss of viability and, hence, cell death.
Keywords: ionizing radiation; p53 signaling; premature senescence; viability; proliferation; apoptosis;
colony forming ability; MTT; XTT; CellTiter-Blue
1. Introduction
Activation of the p53 signaling pathway by ionizing radiation was originally proposed to result
either in cell cycle checkpoint activation to facilitate DNA repair and promote survival, or in apoptotic
cell death (e.g., [1]). Based on this two-armed model, radiosensitivity assessed by multiwell plate
colorimetric/fluorimetric assays (e.g., MTT, XTT, CellTiter-Blue) would be expected to reflect cell death.
Accordingly, the outcomes of such radiosensitivity (and indeed chemosensitivity) assays are often
interpreted to reflect loss of viability and hence cell death. However, such an assumption may be
misleading. As extensively reviewed recently [2–4], a large body of evidence has established that the
primary response triggered by moderate doses of ionizing radiation (between 2 and 8 Gy) in solid
tumor-derived cell lines is in fact a sustained proliferation block, and not apoptosis. The proliferation
block predominantly reflects stress-induced premature senescence (SIPS) in cells that express wild-type
p53 [5–9].
SIPS is a sustained growth arrested state characterized by the acquisition of flattened and enlarged
cell morphology in cells that retain viability for a long time (months) post-treatment and acquire the
ability to secrete factors that can promote proliferation and invasiveness in cell culture models and
tumor development in vivo [10,11]. In addition, under some conditions, cancer cells undergoing SIPS
can give rise to stem cell-like progeny, thereby contributing to cancer relapse following therapy [3,12,13].
Thus, although cancer cells undergoing SIPS might be scored as “dead” in the short-term (colorimetric)
Int. J. Mol. Sci. 2017, 18, 1460; doi:10.3390/ijms18071460
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Int. J. Mol. Sci. 2017, 18, 1460
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and long-term (colony formation) assays in vitro, and can potentially contribute to tumor-growth
delay in animal model studies, the senescence-like growth-arrested response (tumor dormancy) may
in fact represent an important cell-survival mechanism, ultimately resulting in tumor regrowth and
disease recurrence [2,3,14,15].
The p21WAF1 (p21; also called CDKN1A) protein is the founding member of the CIP/KIP family of
cyclin-dependent kinase (CDK) inhibitors [16,17]. It is a p53 transcriptional target that plays a pivotal
role in the DNA damage surveillance network, not only by activating early cell cycle checkpoints,
but also through suppressing apoptosis (e.g., by inhibiting caspases), switching on the SIPS program,
and contributing to the maintenance of the sustained growth arrested response, a hallmark of
SIPS [18,19]. Consistent with these multifunctional properties of p21, radiosensitivity as measured by
the colony formation assay in most cell types that express wild-type p53 (e.g., skin fibroblast strains;
solid tumor-derived cell lines) is shown to be largely, if not totally, associated with growth arrest
through p21-mediated SIPS [2,3,6,7,20].
The purpose of the present study was to determine the impact of SIPS on radiosensitivity
when evaluated by short-term tests in the commonly-used p53 wild-type cancer cell lines HCT116
(colon carcinoma), MCF7 (breast carcinoma), SKNSH (neuroblastoma), and A172 (malignant glioma).
These cell lines are known to respond to moderate doses of ionizing radiation (e.g., 8 Gy) by exhibiting
sustained nuclear accumulation of p21, coupled with growth arrest through SIPS. We demonstrate that
radiosensitivity as measured by multiwell plate colorimetric/fluorimetric assays is markedly skewed
towards radioresistance when compared to that measured by growth inhibition (3 days post-irradiation)
and colony formation (10 days post-irradiation) assays, and that the response measured by these
cell-based assays reflects growth inhibition (SIPS) and not apoptosis or loss of viability.
2. Results and Discussion
2.1. Colony Formation and Growth Inhibition Assays Indicate a Comparable Degree of Radiosensitivity in p53
Wild-Type Cancer Cells
The colony formation assay (also called clonogenic survival) for assessment of radiosensitivity in
cultured human cells was developed by Puck and Marcus over half a century ago [21]. It has since
been used for studying the effectiveness of different agents (radiation, chemicals, or a combination
of the two) on the survival and proliferation of various mammalian cell types. The typical assay for
evaluating radiosensitivity involves preparing a single-cell suspension from a monolayer culture,
plating out cells at very low densities (e.g., 300 cells per 60-mm tissue culture dish), exposing them
to graded doses of ionizing radiation, and incubating them for 10–14 days to allow the formation of
macroscopic colonies, each comprised of aggregates of at least 50 cells. The reduction in macroscopic
colony number in irradiated compared to sham-treated samples reflects the degree of radiosensitivity,
i.e., loss of reproductive potential. We have performed pilot experiments with skin fibroblast strains
and cancer cell lines and found that irradiating the cells either prior to seeding them at cloning
densities or shortly afterwards (within ~4 h post-plating for cancer cell lines [22]) does not impact on
the outcome of the assay (unpublished observations).
As alluded to earlier, in most cell types, activation of the p53 pathway in response to moderate
doses of ionizing radiation (e.g., 8 Gy) results in activation of early cell cycle checkpoints, suppression
of apoptosis and sustained growth arrest through SIPS [2–4]. Consistent with these observations,
radiosensitivity as measured by the colony formation assay in normal fibroblast strains and p53
wild-type solid tumor-derived cell lines has been shown to primarily reflect p21-dependent growth
arrest through SIPS, and not cell death [2,3,6,7,20]. We predicted that the long-term clonogenic assay
and the short-term (3 days post-irradiation) growth inhibition assay involving cell counting would
indicate a comparable degree of radiosensitivity in p53 wild-type cells, given that the same p53
downstream effector (p21) is responsible for activating both early growth arrest (checkpoints) and
sustained growth arrest (SIPS). The results presented in Figure 1 support this interpretation for the p53
wild-type lines MCF7, HCT116, SKNSH and A172. For each cell line, radiation doses resulting in 50%
Int. J. Mol. Sci. 2017, 18, 1460
Int. J. Mol. Sci. 2017, 18, 1460
3 of 12
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effectcell
(Inhibiting
Dose
50%; resulting
ID50 ) were
comparable
when measured
formation and
Int.
J.line,
Mol. Sci.
2017,
18, doses
1460
3 ofgrowth
11
radiation
in 50%
effect (Inhibiting
Dose 50%;by
IDcolony
50) were comparable when
inhibition
assays.
measured by colony formation and growth inhibition assays.
cell line, radiation doses resulting in 50% effect (Inhibiting Dose 50%; ID50) were comparable when
measured by colony formation and growth inhibition assays.
Figure 1. Radiosensitivity of the indicated cell lines evaluated by growth inhibition and colony
Figure 1. Radiosensitivity of the indicated cell lines evaluated by growth inhibition and colony
formation assays. Growth inhibition and colony forming ability measurements were performed 3
formation
assays.
Growth inhibition
colonycell
forming
ability measurements
were performed
3 days
Figureand
1. 10
Radiosensitivity
of the and
indicated
lines
growth
andforming
colony
days
days post-irradiation,
respectively.
Bars,evaluated
standard by
error
(SE). inhibition
CFA, colony
and 10
days post-irradiation,
standard
(SE).
CFA, colonywere
forming
ability.
formation
assays. Growth respectively.
inhibition andBars,
colony
formingerror
ability
measurements
performed
3
ability.
days and 10 days post-irradiation, respectively. Bars, standard error (SE). CFA, colony forming
ability.
Interestingly,
we have
recently
reported
these
assays
indicate
a comparable
Interestingly,
we have
recently
reported
thatthat
these
twotwo
assays
alsoalso
indicate
a comparable
degree
degree of radiosensitivity for solid tumor-derived cell lines that lack p21 or wild-type p53 activity
of radiosensitivity
for
solid
tumor-derived
cell
lines
that
lack
p21
or
wild-type
p53
activity
[23].
weinhave
that
thesethe
two
assaysofalso
indicate a comparable
[23]. Interestingly,
Radiosensitivity
suchrecently
cells wasreported
shown to
reflect
creation
multinucleated
giant cells,
Radiosensitivity
in such cells
shown
to reflect
the creation
of multinucleated
giant cells,
degreeremain
of radiosensitivity
for was
solid
tumor-derived
cellextended
lines
thatperiods
lack p21
or wild-type
p53 activity
which
viable and exhibit
metabolic
activity for
(weeks)
post-irradiation.
which[23].
remain
viable
and
exhibit
metabolic
activity
for
extended
periods
(weeks)
post-irradiation.
Radiosensitivity
in
such
cells
was
shown
to
reflect
the
creation
of
multinucleated
giant
cells,
Although the colony formation assay is considered to be the “gold standard” for genotoxicity
which
remain
viable
and
exhibit
metabolic
activity
for
extended
periods
(weeks)
post-irradiation.
Although
the
colony
formation
assay
is
considered
to
be
the
“gold
standard”
for
genotoxicity
assessment, it has several drawbacks. Aside from being time consuming and labor intensive,
Although
colony
formation
assay
is considered
to be
the “gold
for macroscopic
genotoxicity
assessment,
it has
several
drawbacks.
Aside
from being
time
consuming
and
labor intensive,
preparation
of the
good
quality
single-cell
suspensions
and
evaluation
ofstandard”
borderline
assessment,
it
has
several
drawbacks.
Aside
from
being
time
consuming
and
labor
intensive,
preparation
good
quality
suspensions
and
evaluationexpertise.
of borderline
macroscopic
colonies
coloniesof
are
not trivial
forsingle-cell
some cell types,
requiring
considerable
In addition,
some
solid
preparation
of
good
quality
single-cell
suspensions
and
evaluation
of
borderline
macroscopic
tumor-derived
cell
lines
(e.g.,
SUM52PE
breast
carcinoma)
cannot
be
reliably
evaluated
by
this
assay
are not trivial for some cell types, requiring considerable expertise. In addition, some solid
colonies of
aretheir
not poor
trivialcloning
for some
cell types,and
requiring
considerable
In addition,
some
solid
because
efficiencies,
some cell
lines
(e.g.,expertise.
SKBR3
breast
carcinoma)
notassay
tumor-derived
cell lines
(e.g., SUM52PE
breast carcinoma)
cannot
be reliably
evaluated
bydothis
tumor-derived
cell linessuspension
(e.g., SUM52PE
breast carcinoma)
cannot
be reliably
evaluated
by this assay
yield
a
good
single-cell
by
conventional
approaches
(e.g.,
after
exposure
to
trypsin)
[22].
because of their poor cloning efficiencies, and some cell lines (e.g., SKBR3 breast carcinoma) do not
because
their poor
cloning
efficiencies,
and some
lines (e.g.,
breast
carcinoma)[23],
do not
The of
growth
inhibition
assay
circumvents
these cell
limitations.
AsSKBR3
pointed
out previously
in
yield yield
a good
single-cell
suspension
bybyconventional
approaches
(e.g.,after
after
exposure
to trypsin)
[22].
a good
single-cell
suspension
conventional
approaches
(e.g.,
exposure
to
trypsin)
[22].
addition
to reproducibility
and relative
ease of performance,
the
growth inhibition
assay,
which
is
The growth
inhibition
assay
circumvents
these
limitations.
As
pointed
out
previously
The growth
circumvents
limitations.
As pointed
out previously
[23], to
in [23],
completed
withininhibition
5 days ofassay
seeding
the cells,these
enables
one to utilize
microscopic
examination
in addition
to
reproducibility
and
relative
ease
of
performance,
the
growth
inhibition
assay,
which
is
addition
reproducibility
and relative
ease
performance,
growth
which is
track
the to
long-term
fate of adherent
cells.
Toofthis
end, cells inthe
one
set of inhibition
dishes canassay,
be evaluated,
completed
5ofdays
of seeding
the 3cells,
enables
one
to utilize
microscopic
examinationfor
completed
within
5 days
of seeding
the
cells,
enables
to utilize
microscopic
tototrack
either
at thewithin
time
cell
counting
(i.e.,
days
afterone
irradiation)
or
after
furtherexamination
incubation,
track
the
long-term
fate
of
adherent
cells.
To
this
end,
cells
in
one
set
of
dishes
can
be
evaluated,
the long-term
fate
of
adherent
cells.
To
this
end,
cells
in
one
set
of
dishes
can
be
evaluated,
either
different responses using a battery of single-cell methods. We routinely use one set of dishes for cellat the
either
the time(i.e.,
ofbycell
counting
(i.e., 3 days assay
after
irradiation)
or after
incubation,
for
time viability
of
cell at
counting
3the
days
after
irradiation)
or
after
further
incubation,
for different
responses
assessment
trypan
blue-exclusion
under
conditions
thatfurther
minimize
the creation
different
responses
using
a
battery
of
single-cell
methods.
We
routinely
use
one
set
of
dishes
for
cell
positives
(e.g., cellmethods.
detachment
exposureuse
to trypsin)
Basedfor
on cell
this viability
assay, weassessment
found
usingofa false
battery
of single-cell
Webyroutinely
one set [23].
of dishes
viability
assessment
by the
trypan
blue-exclusion
assay under
conditions
that minimize
the of
creation
that
radiation
exposure
does
not induce
of viability
(trypan
blue staining)
in cultures
these
by the
trypan
blue-exclusion
assay
underloss
conditions
that
minimize
the creation
of false
positives
of false
positives
(e.g., cell detachment
bytimes
exposure
to trypsin)
[23].
Based
on
this
assay, we
found
cell
lines
when
determined
at
different
between
3
days
(Figure
2)
and
3
weeks
(data
not
(e.g., that
cell radiation
detachment
by exposure to trypsin) [23]. Based on this assay, we found that radiation
exposure does not induce loss of viability (trypan blue staining) in cultures of these
shown)
post-irradiation.
exposure
doeswhen
not induce
loss of
blue staining)
in cultures
lines
cell lines
determined
at viability
different (trypan
times between
3 days (Figure
2) andof3these
weekscell
(data
notwhen
determined
at
different
times
between
3
days
(Figure
2)
and
3
weeks
(data
not
shown)
post-irradiation.
shown) post-irradiation.
Figure 2. Viability of the indicated cell lines before (0 Gy) and 3 days after radiation exposure,
evaluated by the trypan blue-exclusion assay. Bars, SE.
Figure
2. Viability
the indicated
cell before
lines before
Gy)3 and
daysradiation
after radiation
exposure,
Figure
2. Viability
of theofindicated
cell lines
(0 Gy)(0and
days3after
exposure,
evaluated
by the trypan blue-exclusion
by theevaluated
trypan blue-exclusion
assay. Bars, assay.
SE. Bars, SE.
Int. J. Mol. Sci. 2017, 18, 1460
Int. J. Mol. Sci. 2017, 18, 1460
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2.2. Multiwell
Plate
Assays
Markedly
Radiosensitivity
2.2. Multiwell
Plate
Assays
MarkedlyUnderestimate
Underestimate Radiosensitivity
2.2.1.2.2.1.
Multiwell
Plate
Colorimetric/Fluorimetric
Assays:Description
Description
Multiwell
Plate
Colorimetric/Fluorimetric Assays:
Short-term
cellcell
metabolic
are widely
widelyused
usedto toassess
assess
radiosensitivity
Short-term
metabolicactivity
activity assays
assays are
radiosensitivity
and and
chemosensitivity
withwith
solidsolid
tumor-derived
cell cell
lineslines
[24–27].
SuchSuch
assays
are performed
in a multiwell
chemosensitivity
tumor-derived
[24–27].
assays
are performed
in a
multiwell
format and
abilitycells
of living
cells toa convert
a detection
to its
format
and measure
the measure
ability oftheliving
to convert
detection
reagent reagent
to its metabolite,
metabolite,
often
mediated
by
the
mitochondrial
oxidoreductases.
The
tetrazolium
salts
often mediated by the mitochondrial oxidoreductases. The tetrazolium salts 3-(4,5-dimethylthiazol3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide
(MTT)
and
2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid (XTT) are the most
tetrazolium-5-carboxanilid (XTT) are the most commonly-used detection reagents in multiwell plate
commonly-used detection reagents in multiwell plate assays. Figure 3 summarizes the general steps
assays. Figure 3 summarizes the general steps for genotoxicity assessment by the XTT colorimetric assay.
for genotoxicity assessment by the XTT colorimetric assay.
Figure 3. Depiction of anticipated outcome of multiwell plate colorimetric (XTT) assays after
Figure 3. Depiction of anticipated outcome of multiwell plate colorimetric (XTT) assays after treatments
treatments that result in cytostatic (growth inhibition) effect only (e.g., 5 Gy of ionizing radiation), or
that result in cytostatic (growth inhibition) effect only (e.g., 5 Gy of ionizing radiation), or cytostatic
cytostatic plus cytotoxic effects (e.g., >40 μM cisplatin). The example is given for a culture with a
plus cytotoxic effects (e.g., >40 µM cisplatin). The example is given for a culture with a control
control population doubling time of 24 h. In this case, for “control” wells (upper left well), seeding
population
doubling
h.incubating
In this case,
forwith
“control”
(upper
well),
seeding
2000 cells
2000 cells
per welltime
(dayof
0) 24
and
them
growthwells
medium
for 3left
days
(upper
right well)
per well
(day
0)
and
incubating
them
with
growth
medium
for
3
days
(upper
right
well)
will
result in
will result in 16,000 cells/well at the time of optical density measurement. For “radiation” wells
16,000
cells/well
at
the
time
of
optical
density
measurement.
For
“radiation”
wells
(middle
left
(middle left well) and “cisplatin” wells (lower left well), the number of cells per well is expected not well)
and “cisplatin”
wellsday
(lower
left 3,
well),
theinduction
numberof
ofgrowth
cells per
wellinisvirtually
expected
increase from
to increase from
0 to day
due to
arrest
allnot
cellsto(irradiation)
or day
induction
arrestof
plus
loss ofarrest
viability
(cisplatin treatment).
It is important
note that of
most
0 to day
3, dueoftogrowth
induction
growth
in virtually
all cells (irradiation)
or to
induction
growth
cell (cisplatin
lines (e.g., treatment).
HCT116) have
population
doubling
timessolid
than tumor-derived
24 h [22].
arrestsolid
plustumor-derived
loss of viability
It shorter
is important
to note
that most
cell lines (e.g., HCT116) have shorter population doubling times than 24 h [22].
In a typical assay, the cells are plated at “optimal” densities in the wells of a microtiter plate,
exposed to a genotoxic agent, and incubated for 3 days. XTT (or MTT) is added to each well, and
In
a typical
assay, the
are
plated
at “optimal”
in the
a microtiter
cultures
are incubated
forcells
a few
hours
to allow
the viable densities
cells to covert
thewells
yellowoftetrazolium
saltplate,
exposed
genotoxic
agent, formazan
and incubated
for XTT
3 days.
XTTis(or
MTT)
is added
to each
(XTTto
or aMTT)
to its colored
derivative;
formazan
orange
in color
whereas
MTT well,
and cultures
incubated
for a few
hours
to allowper
thewell
viable
to covert
yellow tetrazolium
formazanare
is purple.
The number
of cells
inoculated
andcells
the duration
of the
cell incubation
with
XTT or
need
to be
optimized
for each
cell line. XTT formazan is orange in color whereas MTT
salt (XTT
orMTT
MTT)
to its
colored
formazan
derivative;
is readily
up byofthe
cells,
but its formazan
metabolite
water insoluble,
requiring a with
formazanMTT
is purple.
Thetaken
number
cells
inoculated
per well
and theisduration
of cell incubation
solubilization
step
before
data
evaluation.
XTT,
on
the
other
hand,
requires
an
electron
coupling
step
XTT or MTT need to be optimized for each cell line.
to facilitate
its uptake
by theup
cells,
thecells,
XTT metabolite
water soluble.
MTT
is readily
taken
bybut
the
but its is
formazan
metabolite is water insoluble,
After these treatments, either with (MTT) or without (XTT) the metabolite solubilization step,
requiring a solubilization step before data evaluation. XTT, on the other hand, requires an electron
the medium overlaying the control (sham-treated) cultures turns purple (MTT) or orange (XTT), as a
coupling step to facilitate its uptake by the cells, but the XTT metabolite is water soluble.
result of conversion of the yellow tetrazolium salt to a colored metabolite by viable cells. Genotoxic
After these treatments, either with (MTT) or without (XTT) the metabolite solubilization step,
the medium overlaying the control (sham-treated) cultures turns purple (MTT) or orange (XTT),
as a result of conversion of the yellow tetrazolium salt to a colored metabolite by viable cells.
Genotoxic treatment results in a decrease in color intensity of the medium, which is proportional to
Int. J. Mol. Sci. 2017, 18, 1460
Int. J. Mol. Sci. 2017, 18, 1460
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the number of viable cells/well at the time of incubation with XTT or MTT (Figure 3). The results are
treatment
results
in a decrease
in color intensity
of the medium,
which
proportional
to the
number
recorded
in a plate
reader,
using absorbance
measurements
at 475
nmisfor
XTT and 570
nm
for MTT.
of
viable
cells/well
at
the
time
of
incubation
with
XTT
or
MTT
(Figure
3).
The
results
are
recorded
The CellTiter-Blue assay is also widely used for genotoxicity assessment. This assay isinbased
a plate reader, using absorbance measurements at 475 nm for XTT and 570 nm for MTT.
on the
ability of living cells to convert the redox dye resazurin (blue) into the resorufin product
The CellTiter-Blue assay is also widely used for genotoxicity assessment. This assay is based on
(pink). Resazurin has little intrinsic fluorescence until it is converted to its metabolite, which is highly
the ability of living cells to convert the redox dye resazurin (blue) into the resorufin product (pink).
fluorescent
(579 nm excitation/584 nm emission). Thus, the CellTiter-Blue assay not only eliminates
Resazurin has little intrinsic fluorescence until it is converted to its metabolite, which is highly
the need
for
electron
coupling
or metabolite
solubilization
steps
required for
tetrazolium-based
assays,
fluorescent
(579 nm
excitation/584
nm emission).
Thus, the
CellTiter-Blue
assay
not only eliminates
but itthe
also
enables
fluorimetric
evaluation
of thesolubilization
data, which steps
is considered
more sensitive than
need
for electron
coupling
or metabolite
required to
forbe
tetrazolium-based
absorbance
assays, measurements.
but it also enables fluorimetric evaluation of the data, which is considered to be more
sensitive than absorbance measurements.
2.2.2. Enlarged Cells Created Post-Irradiation Metabolize MTT
2.2.2. Enlarged Cells Created Post-Irradiation Metabolize MTT
The properties of MTT make it particularly useful for microscopic assessment of individual cells
The properties
oftreatment.
MTT make it
useful forare
microscopic
of individual
cells MTT
in the absence
of solvent
Toparticularly
this end, cultures
incubatedassessment
with medium
containing
in
the
absence
of
solvent
treatment.
To
this
end,
cultures
are
incubated
with
medium
containing
to allow viable cells to covert MTT to its purple, water-insoluble formazan. Under these conditions,
MTT to allow viable cells to covert MTT to its purple, water-insoluble formazan. Under these
MTT metabolites appear as intercellular formazan granules as well as needle-like formazan crystals [23].
conditions, MTT metabolites appear as intercellular formazan granules as well as needle-like
We used this single-cell observation method to determine the metabolic activity of individual cells
formazan crystals [23]. We used this single-cell observation method to determine the metabolic
within
cultures
of the MCF7
A172
cell lines
andA172
at two
and
6 days)
activity
of individual
cells and
within
cultures
of thebefore
MCF7 and
celltime
linespoints
before (3
and
at two
time after
exposure
to ionizing
(8 Gy).
Representative
are (8
presented
in Figures 4 and
5A,are
and the
points
(3 and 6 radiation
days) after
exposure
to ionizingimages
radiation
Gy). Representative
images
results
of
image
analysis
are
shown
in
Figure
5B.
In
these
experiments,
cells
were
incubated
presented in Figures 4 and 5A, and the results of image analysis are shown in Figure 5B. In these with
experiments,
cellsMTT
werefor
incubated
with
medium
MTT for
~1 h and
their images
were 5A).
medium
containing
~1 h and
their
imagescontaining
were acquired
(Figure
4, upper
row; Figure
(Figure
4, upper
row;and
Figure
medium
removedtoand
the cells
Next,acquired
the medium
was
removed
the 5A).
cellsNext,
werethe
treated
withwas
methanol
dissolve
andwere
remove
treated with
methanol
to dissolve
remove
intercellular
tetrazolium
granules
and crystals;
their row)
intercellular
tetrazolium
granules
andand
crystals;
their
images were
acquired
before (Figure
4, middle
images were acquired before (Figure 4, middle row) and after (Figure 4, lower row) mild staining
and after
(Figure 4, lower row) mild staining with trypan blue. Two observations should be noted.
with trypan blue. Two observations should be noted. First, enlarged cells that emerged in response
First, enlarged cells that emerged in response to radiation exposure (i.e., cells undergoing or destined
to radiation exposure (i.e., cells undergoing or destined to undergo SIPS) showed a high degree of
to undergo
SIPS) showed a high degree of metabolic activity (Figure 4, upper row; Figure 5A). Second,
metabolic activity (Figure 4, upper row; Figure 5A). Second, the level of metabolic activity per cell
the level
of
metabolic
activity
per cell
signal intensity
in the greater
“regionfor
of irradiated
interest”) was
(estimated
from signal
intensity
in (estimated
the “regionfrom
of interest”)
was markedly
markedly
greater
for
irradiated
cultures
than
for
sham-irradiated
controls
(Figure
5B).
cultures than for sham-irradiated controls (Figure 5B).
Figure 4. Representative bright-field microscopy images depicting the metabolic activity of MCF7
Figure
4. Representative bright-field microscopy images depicting the metabolic activity of MCF7
cultures before (control) and at indicated times after 8-Gy irradiation. Metabolic activity was
cultures before (control) and at indicated times after 8-Gy irradiation. Metabolic activity was measured
measured by the ability of the cells to convert the yellow MTT to its purple formazan metabolite,
by the ability of the cells to convert the yellow MTT to its purple formazan metabolite, appearing
appearing as dark granules and crystals. Upper row: images were acquired after incubation of cells
as dark
granules and crystals. Upper row: images were acquired after incubation of cells with MTT
with MTT for ~1 h. Middle row: following incubation with MTT, cells were fixed in methanol for 0.5
for ~1min
h. to
Middle
row:
incubation
with MTT,
cells
were was
fixed
in methanol
0.5 min to
dissolve
the following
MTT metabolite.
The resulting
purple
medium
removed
before for
acquiring
dissolve
the of
MTT
resulting
purple
medium
before
acquiring
images of
images
cells.metabolite.
Lower row:The
MTT
treated and
methanol
fixedwas
cellsremoved
were mildly
stained
with trypan
cells. blue
Lower
MTT treated
and methanol
cells
were
mildlyatstained
with
trypan blueThe
(TB) to
(TB)row:
to visualize
their morphology.
Allfixed
images
were
acquired
the same
magnification.
border
of some
cells is marked
for clarity.
visualize
their
morphology.
All images
were acquired at the same magnification. The border of some
cells is marked for clarity.
Int. J. Mol. Sci. 2017, 18, 1460
Int. J. Mol. Sci. 2017, 18, 1460
6 of 12
6 of 11
Figure 5. (A) Representative images of A172 cells used for image analysis. The images of MTT
Figure 5.
(A) Representative images of A172 cells used for image analysis. The images of MTT
metabolites were acquired as described in Figure 4 legend (upper row). The images were then
metabolites were acquired as described in Figure 4 legend (upper row). The images were then converted
converted to grayscale and inverted. Blue and red ovals mark some regions of interest (reflecting
to grayscale
inverted.
Blue
and red ovals
mark some
regions
of interest
(reflecting
MTT metabolites)
MTT and
metabolites)
and
corresponding
background
regions
used for
image analysis,
respectively.
(B)
and corresponding
background
regions
usedactivity
for image
(B) as
Densitometric
Densitometric evaluation
of MTT
metabolic
for theanalysis,
indicated respectively.
cultures, expressed
signal
evaluation
of MTT
activity
for the
indicated
cultures,
expressed
as signal
intensity
intensity
for metabolic
regions of interest
(cells)
after
corresponding
background
corrections.
Mean
valuesfor
for regions
at least
30 cells
arecorresponding
presented for each
sample. Bars,corrections.
SE. ROI, regionMean
of interest;
BG,for
background.
of interest
(cells)
after
background
values
at least 30 cells are
presented for each sample. Bars, SE. ROI, region of interest; BG, background.
2.2.3. Multiwell Plate Assays Lack Specificity
Several
pitfalls
of Lack
multiwell
plate colorimetric assays have been extensively reviewed,
2.2.3. Multiwell
Plate
Assays
Specificity
including the impact of cell density and possible interference of test compounds with optical density
measurements
However,
a fundamentalassays
caveathave
related
to extensively
cell fate hasreviewed,
been largely
Several
pitfalls of[28–33].
multiwell
plate colorimetric
been
including
overlooked.
the MTT/XTT
for example,
the change
in optical
density for
control
wellsdensity
the impact
of cellIn density
and assays,
possible
interference
of test
compounds
with
optical
(purple or
orange)However,
versus treated
wells is often
assumed
to reflect
loss
of has
viability
to
measurements
[28–33].
a fundamental
caveat
related
to cell
fate
beenconsequent
largely overlooked.
genotoxic treatment. This interpretation would be tenable only if the number of cells at the time of
In the MTT/XTT assays, for example, the change in optical density for control wells (purple or orange)
assay evaluation (e.g., 3 days following genotoxic treatment) would be identical in control and
versus treated
wellshowever,
is oftenthis
assumed
to the
reflect
loss of genotoxic
viability stress
consequent
genotoxic
treatment.
treated wells;
is often not
case because
activates to
transient
cell cycle
This interpretation
tenableproliferation
only if theblock
number
of cells
theSIPS
time
of assay
evaluation
checkpoints aswould
well as abesustained
associated
with,at
e.g.,
and/or
creation
of
polyploid/multinucleated
giant
cells, which
are characterized
maintenance
cell membrane
(e.g., 3 days
following genotoxic
treatment)
would
be identical by
in control
and of
treated
wells; however,
integrity
to secretegenotoxic
growth-promoting
factors (reviewed
in cell
[2–4]).
Suchcheckpoints
growth-arrested
this is often
notand
the ability
case because
stress activates
transient
cycle
as well as
cancer cells exhibit metabolic activity, including the ability to convert MTT to its purple formazan
a sustained proliferation block associated with, e.g., SIPS and/or creation of polyploid/multinucleated
metabolite ([23]; Figures 4 and 5A, and unpublished observations).
giant cells, which
are characterized by maintenance of cell membrane integrity and ability to secrete
Thus, although many suppliers refer to these colorimetric assays in terms of “Cell Viability” or
growth-promoting
in [2–4]).
Such
cancer
cells exhibit
metabolic
“Cytotoxicity,”factors
all such(reviewed
assays that are
performed
in a growth-arrested
multiwell plate format
lack specificity
and can
activity,lead
including
the ability
to convert
to itsinpurple
([23]; Figures
4 and 5A,
to incorrect
interpretations.
As MTT
illustrated
Figure formazan
3, multiwellmetabolite
plate colorimetric
assays are
expected to observations).
indicate comparable levels of genotoxicity for treatments that cause growth inhibition in
and unpublished
the although
absence of many
cell killing
(e.g., after
moderate doses
of ionizing
radiation)
for
Thus,
suppliers
referexposure
to thesetocolorimetric
assays
in terms
of “Celland
Viability”
or
treatments that induce loss of viability (e.g., after treatment with high concentrations of cisplatin).
“Cytotoxicity,” all such assays that are performed in a multiwell plate format lack specificity and can
lead to 2.2.4.
incorrect
interpretations.
Asbyillustrated
in Figure
multiwell
plate
assays are
Radiosensitivity
Measured
Multiwell Plate
Assays 3,
in p53
Wild-Type
Cellcolorimetric
Lines
expected to indicate comparable levels of genotoxicity for treatments that cause growth inhibition
Assuming that radiation exposure would minimally alter the size of cancer cells,
in the absence
of cellaskilling
(e.g.,
exposure
to moderate
oftoionizing
radiation)
radiosensitivity
measured
by after
multiwell
plate assays
would be doses
expected
be comparable
to thatand for
treatments
that induce
lossinhibition
of viability
treatment
of cisplatin).
measured
by growth
and (e.g.,
colonyafter
formation
assays.with
This high
is notconcentrations
always the case, however;
cancer cells undergoing SIPS following radiation exposure exhibit a markedly enlarged
2.2.4. Radiosensitivity
Measured
by example,
Multiwell
Plate that
Assays
in p53
Celllocalization
Lines
morphology. Sohn
et al. [34], for
reported
HCT116
cellsWild-Type
exhibit nuclear
of
p21 and a dramatic increase in size at 48 h after irradiation. We made a similar observation with our
Assuming
that radiation exposure would minimally alter the size of cancer cells, radiosensitivity
panel of cell lines (data not shown). Accordingly, colorimetric assays are likely to underestimate
as measured
by multiwell
plate assays
would
bethat
expected
to be comparable
to that measured by
radiosensitivity
in p53 wild-type
cell lines,
given
highly enlarged
cells created post-irradiation
growth remain
inhibition
colony
assays.
This is
not always
the case,
cancer cells
viableand
(e.g.,
Figureformation
2), and exhibit
metabolic
activity
(e.g., Figures
4 andhowever;
5). The results
presented
Figure 6 are
consistent
with thisexhibit
notion.aInmarkedly
MCF7 cultures,
for example,
the ID50Sohn
values
undergoing
SIPS in
following
radiation
exposure
enlarged
morphology.
et al. [34],
measured
by
growth
inhibition
and
multiwell
plate
assays
(both
measured
3
days
post-irradiation)
for example, reported that HCT116 cells exhibit nuclear localization of p21 and a dramatic increase in
amounted to ~3 Gy and >8 Gy, respectively.
size at 48 h after irradiation. We made a similar observation with our panel of cell lines (data not shown).
Accordingly, colorimetric assays are likely to underestimate radiosensitivity in p53 wild-type cell lines,
given that highly enlarged cells created post-irradiation remain viable (e.g., Figure 2), and exhibit
metabolic activity (e.g., Figures 4 and 5). The results presented in Figure 6 are consistent with this
notion. In MCF7 cultures, for example, the ID50 values measured by growth inhibition and multiwell
plate assays (both measured 3 days post-irradiation) amounted to ~3 Gy and >8 Gy, respectively.
Int. J. Mol. Sci. 2017, 18, 1460
7 of 12
Int. J. Mol. Sci. 2017, 18, 1460
7 of 11
Int. J. Mol. Sci. 2017, 18, 1460
7 of 11
6. Radiosensitivity
of indicated
the indicated
evaluated
by the
96-well
plate
XTT
(solid
FigureFigure
6. Radiosensitivity
of the
cell cell
lineslines
evaluated
by the
96-well
plate
XTT
(solid
squares)
squares) and CellTiter-Blue
(openassays.
squares)Bars,
assays.
ID50 values
for colorimetric
(this
and CellTiter-Blue
(open squares)
SE.Bars,
TheSE.
IDThe
values
for
colorimetric
(this
Figure),
50
Figure),6.growth
inhibition and
colony
formation
(Figure
1) assaysbyarethe
shown.
CTB,
CellTiter-Blue;
Figure
Radiosensitivity
of the
indicated
cell lines
evaluated
96-well
plate
XTT (solid
growth inhibition and colony formation (Figure 1) assays are shown. CTB, CellTiter-Blue; CFA,
50%.
CFA, colony
ability;(open
ID50, inhibiting
squares)
and forming
CellTiter-Blue
squares) dose
assays.
Bars, SE. The ID50 values for colorimetric (this
colony forming ability; ID50 , inhibiting dose 50%.
Figure), growth inhibition and colony formation (Figure 1) assays are shown. CTB, CellTiter-Blue;
2.3. Cancer
Cells Expressing
Wild-Type
p53 Do Not
doseReadily
50%. Undergo Apoptosis within 3 Days
CFA, colony
forming ability;
ID50, inhibiting
2.3. Cancer
Cells Expressing Wild-Type p53 Do Not Readily Undergo Apoptosis within 3 Days Post-Irradiation
Post-Irradiation
2.3. Cancer Cells Expressing Wild-Type p53 Do Not Readily Undergo Apoptosis within 3 Days
In most
human
types,
activation of
of p53
p53 signaling
to genotoxic
stressstress
has been
In
most
human
cellcell
types,
activation
signalingininresponse
response
to genotoxic
has been
Post-Irradiation
shown
to
supress
(rather
than
promote)
apoptosis
[2,19].
Suppression
of
apoptosis
by
p53
signaling
shown to supress (rather than promote) apoptosis [2,19]. Suppression of apoptosis by p53 signaling
In most
human not
cell only
types,
activation
ofacts
p53 at
signaling inlevels
response
to death
genotoxic
stressbut
has been
has been
attributed
p21, which
of the
cascade,
has been
attributed
not only
to to
p21,
which acts
at different
different levels
of the
death
cascade,also
buttoalso to
shown
to supress
(rather than
promote) apoptosis
[2,19].proteins
Suppression
of apoptosis
p53(wild-type
signaling
p53-dependent
expression
of numerous
anti-apoptotic
including
14-3-3δ, by
WIP1
p53-dependent expression of numerous anti-apoptotic proteins including 14-3-3δ, WIP1 (wild-type
has
been attributed
not only
p21,
which(DNAJ
acts at homolog
different subfamily
levels of the
death cascade,
also to
p53-induced
phosphatase
1), to
and
DNAJB9
B member
9) [19]. but
Consistent
p53-induced
phosphatase
1), and
DNAJB9 (DNAJ
homolog
subfamily
B member
[19].(wild-type
Consistent
p53-dependent
expression
of numerous
anti-apoptotic
proteins
14-3-3δ,
WIP1
with these
observations,
p53
wild-type
cancer
cell
lines
such
asincluding
HCT116
[34,35],9)MCF7
[6,35]
and with
these p53-induced
observations,
p53
wild-type
cancer
cell
lines
such
as
HCT116
[34,35],
MCF7
[6,35]
and
A172 [36]
1), and DNAJB9
(DNAJapoptosis
homologin
subfamily
[19]. Consistent
A172 [36] arephosphatase
markedly refractory
to undergoing
responseBtomember
ionizing9)radiation.
are markedly
refractory
to undergoing
apoptosis
in lines
response
to
radiation.
with Apoptosis
these
observations,
p53 wild-type
cancer
cell
such
as ionizing
HCT116 [34,35],
MCF7of[6,35]
and
in our
previous
work
was evaluated
by microscopic
examination
Hoechst
A172
[36] areinmarkedly
refractorywork
to undergoing
apoptosis
in response
tofragmentation).
ionizing
radiation.
33342-stained
cells
morphological
criteria
condensation
and
We sought
Apoptosis
ourforprevious
was (nuclear
evaluated
by
microscopic
examination
of Hoechst
Apoptosis
in morphological
our
previous by
work
was(nuclear
evaluated
by
microscopic
examination
Hoechst
to verify
these
observations
flow
cytometric
determination
of Annexin ofV-positive
33342-stained
cells
for
criteria
condensation
and fragmentation).
We sought to
33342-stained
cells for morphological
criteria
(nuclear
condensation
and
fragmentation).
We sought
(phosphatidylserine-externalized)
cells.
The
results
are
presented
in
Figure
7.
In
these
experiments,
verify these observations by flow cytometric determination of Annexin V-positive (phosphatidylserineto
verifyof the
these
by used
flowin the
cytometric
determination
of Annexin
cultures
fourobservations
cancer cell lines
radiosensitivity
studies were
exposed toV-positive
radiation
externalized) cells. The results are presented in Figure 7. In these experiments, cultures of the four
(phosphatidylserine-externalized)
cells. The results
are presented
in Figure
7. In
thesecombined
experiments,
and incubated for 3 days. The corresponding
floating
and adherent
cells
were
and
cancercultures
cell lines
used
the radiosensitivity
studies
were
exposed
to in
radiation
incubated
for
of by
the
fourin
cancer
cell As
linesexpected,
used in the
radiosensitivity
exposedand
to
radiation
evaluated
flow
cytometry.
radiation
exposure studies
resultedwere
apoptosis
(Annexin
V
3 days.
The
corresponding
floating
and
adherent
cells
were
combined
and
evaluated
by
flow
cytometry.
and
incubated
3 days.
The of
corresponding
floating
and adherent cells were combined and
staining)
in onlyfor
a small
fraction
these p53 wild-type
cells.
As expected,
radiation
in apoptosis
V staining)
in only a(Annexin
small fraction
of
evaluated
by flow exposure
cytometry.resulted
As expected,
radiation(Annexin
exposure resulted
in apoptosis
V
in only cells.
a small fraction of these p53 wild-type cells.
these staining)
p53 wild-type
Figure 7. (A) Representative flow cytometry profiles of SKNSH cells stained with Annexin V (FITC)
before and 3 days after irradiation. (B) The percentages of Annexin V-positive cells in the indicated
cell
before
and 3 daysflow
after
irradiation.
SE.ofofSKNSH
Figure
(A) Representative
flowcytometry
cytometry Bars,
profiles
cells
stained
withwith
Annexin
V (FITC)
Figure
7. lines
(A)7.Representative
profiles
SKNSH
cells
stained
Annexin
V (FITC)
before
and
3
days
after
irradiation.
(B)
The
percentages
of
Annexin
V-positive
cells
in
the
indicated
before and 3 days after irradiation. (B) The percentages of Annexin V-positive cells in the indicated cell
cell lines before and 3 days after irradiation. Bars, SE.
lines before and 3 days after irradiation. Bars, SE.
Int. J. Mol. Sci. 2017, 18, 1460
Int. J. Mol. Sci. 2017, 18, 1460
8 of 12
8 of 11
As aAs
positive
control, we also determined the apoptotic response of selected cancer cell lines
a positive control, we also determined the apoptotic response of selected cancer cell lines
(e.g., (e.g.,
HCT116)
following
incubation
with with
high high
concentrations
of theofchemotherapeutic
drugdrug
cisplatin
HCT116) following
incubation
concentrations
the chemotherapeutic
or thecisplatin
nuclear or
export
leptomcyin
B. Treatment
cisplatin (60
µM)
or leptomycin
(2 µM)
the inhibitor
nuclear export
inhibitor
leptomcyinwith
B. Treatment
with
cisplatin
(60 μM)B or
for 3 leptomycin
days resulted
apoptosis
in resulted
a high proportion
cells when
evaluated
bywhen
Annexin
B (2in
μM)
for 3 days
in apoptosisof
inHCT116
a high proportion
of HCT116
cells
evaluated
by Annexin
(Figurecriteria
8) and morphological
criteria (data not shown).
V/flow
cytometry
(FigureV/flow
8) andcytometry
morphological
(data not shown).
Figure 8. (A) Representative flow cytometry profiles of HCT116 cells assessed for Annexin V
Figure
8. (A) Representative flow cytometry profiles of HCT116 cells assessed for Annexin V positivity
positivity after incubation in the absence (control) or presence of leptomycin B (2 μM) for 3 days. (B)
after incubation in the absence (control) or presence of leptomycin B (2 µM) for 3 days. (B) The
The percentages of Annexin V-positive cells in HCT116 cultures incubated in the absence or presence
percentages of Annexin V-positive cells in HCT116 cultures incubated in the absence or presence of
of leptomycin B (2 μM) or cisplatin (60 μM) for 3 days. Bars, SE.
leptomycin B (2 µM) or cisplatin (60 µM) for 3 days. Bars, SE.
3. Materials and Methods
3. Materials and Methods
3.1. Cells and Culture Conditions
3.1. Cells and Culture Conditions
The four cancer cell lines used in the present study were purchased from the American Type
Culture
(Rockville,
MD, in
USA).
werestudy
cultured
as monolayers
DMEM/F12
nutrientType
The
fourCollection
cancer cell
lines used
the Cells
present
were
purchased in
from
the American
medium
supplemented
with
5% USA).
(v/v) fetal
bovine
as described
in [7]. All
were free
Culture
Collection
(Rockville,
MD,
Cells
wereserum
cultured
as monolayers
in cultures
DMEM/F12
nutrient
of
Mycoplasma
contamination.
medium supplemented with 5% (v/v) fetal bovine serum as described in [7]. All cultures were free of
Mycoplasma contamination.
3.2. Reagents
The vital dye trypan blue (Sigma, St. Louis, MO, USA), the CellTiter-Blue reagent (Promega,
3.2. Reagents
Madison, WI, USA) and the tetrazolium dyes MTT and XTT (Roche Diagnostics, Penzberg,
The
vital dye trypan blue (Sigma, St. Louis, MO, USA), the CellTiter-Blue reagent (Promega,
Germany) were used as recommended by the manufacturers.
Madison, WI, USA) and the tetrazolium dyes MTT and XTT (Roche Diagnostics, Penzberg, Germany)
were 3.3.
used
as recommended
by the manufacturers.
Radiation
Exposure
Exposure to Co γ-rays was performed in a Gammacell 220 unit as described [37].
3.3. Radiation
Exposure
60
3.4. Radiosensitivity
Assays was performed in a Gammacell 220 unit as described [37].
Exposure
to 60 Co γ-rays
Radiosensitivity evaluation by growth inhibition, colony formation and 96-well plate assays
3.4. Radiosensitivity
Assays
was performed as described [23]. Flow cytometric assessment of Annexin V-positive cells was
performed as described
[38]. by growth inhibition, colony formation and 96-well plate assays was
Radiosensitivity
evaluation
performed as described [23]. Flow cytometric assessment of Annexin V-positive cells was performed
4. Conclusions
as described [38].
In the current study, we have demonstrated that the conventional growth inhibition assay
4. Conclusions
generates radiosensitivity data comparable to that obtained by the slower and more technically
challenging colony formation assay for p53 wild-type cancer cell lines. On the other hand, the
In
the current study, we have demonstrated that the conventional growth inhibition assay
response measured by multiwell plate colorimetric/fluorimetric assays is markedly skewed towards
generates
radiosensitivity
data
comparable
to that
obtained
by the
slowergrowth-arrested
and more technically
radioresistance,
which we
assume
to reflect the
emergence
of highly
enlarged,
and
challenging
colony
formation
assay
for
p53
wild-type
cancer
cell
lines.
On
the
other
viable cells post-irradiation (i.e., cells undergoing SIPS). In addition, we have confirmed thathand,
the response
multiwell plate
colorimetric/fluorimetric
assaysradiation
is markedly
skewed
exposure measured
to moderateby(“clonogenic
survival-curve-range”)
doses of ionizing
does not
induce
apoptosis (as which
judgedwe
by assume
the Annexin
V/flow
approach)
orenlarged,
loss of viability
in the
towards
radioresistance,
to reflect
thecytometry
emergence
of highly
growth-arrested
and viable cells post-irradiation (i.e., cells undergoing SIPS). In addition, we have confirmed that
exposure to moderate (“clonogenic survival-curve-range”) doses of ionizing radiation does not induce
apoptosis (as judged by the Annexin V/flow cytometry approach) or loss of viability in the p53
Int. J. Mol. Sci. 2017, 18, 1460
9 of 12
wild-type cancer cell lines that we examined. These observations, in concert with those recently
reported by us for p53 null or mutant p53-expressing cancer cell lines [23], give credence to the caution
advised by the Nomenclature Committee on Cell Death [39] and others [40] with regard to the potential
for misinterpreting the outcome of cell-based genotoxicity data in terms of loss of viability and hence
cell death.
Short-term multiwell plate assays are indispensable for high throughput studies, e.g., screening
compound libraries for genotoxicity (proliferation block and/or cell death) towards the US NCI panel
of cancer cell lines. However, it is becoming increasingly evident that the effect measured by such
assays primarily reflects growth inhibition and not loss of viability [3,19,23]. Our current studies
with p53 wild-type cancer cell lines exposed to moderate doses of ionizing radiation provide further
support for this conclusion.
Acknowledgments: The authors wish to thank April Scott and Ying W. Wang for technical support.
This work was supported by the Canadian Breast Cancer Foundation—Prairies/North West Territories region,
Alberta Innovates-Health Solutions (grant 101201164) and the Alberta Cancer Foundation—Transformative
Program (file 26603).
Author Contributions: Razmik Mirzayans conceived and designed the experiments, interpreted data and wrote
the manuscript. Bonnie Andrais performed most of the experiments. David Murray interpreted data and edited
the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
SIPS
CFA
DNAJB9
WIP1
CDK
MTT
XTT
CTB
ID50
SE
TB
ROI
BG
Stress-induced premature senescence
Colony forming ability
DNAJ homolog subfamily B member 9
Wild-type p53-induced phosphatase 1
Cyclin dependent kinase
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid
CellTiter-Blue
Inhibiting dose, 50%
Standard error
Trypan blue
Region of interest
Background
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