Article
CHMP4C Disruption Sensitizes the Human Lung
Cancer Cells to Irradiation
Kang Li, Jianxiang Liu, Mei Tian, Gang Gao, Xuesong Qi, Yan Pan, Jianlei Ruan, Chunxu Liu and
Xu Su *
Received: 17 November 2015; Accepted: 18 December 2015; Published: 24 December 2015
Academic Editor: William Chi-shing Cho
Key Laboratory of Radiological Protection and Nuclear Emergency, China CDC,
National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention,
2 Xinkang Street, Dewai, Beijing 10088, China; [email protected] (K.L.); [email protected] (J.L.);
[email protected] (M.T.); [email protected] (G.G.); [email protected] (X.Q.);
[email protected] (Y.P.); [email protected] (J.R.); [email protected] (C.L.)
* Correspondence: [email protected]; Tel.: +86-10-6238-9720
Abstract: Human lung cancer is highly invasive and the most malignant among human tumors.
Adenocarcinoma as a specific type of non-small cell lung cancer occurs with high frequency
and is also highly resistant to radiation therapy. Thus, how to avoid radiation resistance and
improve radiotherapy effectiveness is a crucial question. In the present study, human lung cancer
A549 and H1299 cells were irradiated using γ-rays from a Co60 irradiator. Protein expression
was detected by Western blotting. Cell cycle and apoptosis were measured by flow cytometry.
Surviving fraction was determined by colony formation assay. γH2AX and 53BP1 foci formation
were examined by fluorescence microscopy. In the results, we show that CHMP4C, a subunit
of Endosomal sorting complex-III (ESCRT-III), is involved in radiation-induced cellular response.
Radiation-induced Aurora B expression enhances CHMP4C phosphorylation in non-small cell lung
cancer (NSCLC) cells, maintaining cell cycle check-point and cellular viability as well as resisting
apoptosis. CHMP4C depletion enhances cellular sensitivity to radiation, delays S-phase of cell
cycle and reduces ionizing radiation (IR)-induced γH2AX foci formation. We found that Aurora
B targets CHMP4C and inhibition of Aurora B exhibits similar effects with silencing of CHMP4C
in radioresistance. We also confirm that CHMP4C phosphorylation is elevated after IR both in
p53-positive and-negative cells, indicating that the close correlation between CHMP4C and Aurora
B signaling pathway in mediating radiation resistance is not p53 dependent. Together, our work
establishes a new function of CHMP4C in radiation resistance, which will offer a potential strategy
for non-small cell lung cancer by disrupting CHMP4C.
Keywords: CHMP4C; Aurora B; human non-small lung cancer cells; radiation
1. Introduction
Human lung cancer is highly invasive and most malignant among human tumors, which is
classified into non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer
accounts for about 85% of lung cancers and the current therapeutic strategy includes surgical resection
together with radiation and/or chemo-treatment. Radiation resistance is the severe outcome of lung
cancer radiotherapy and therefore poses a critical barrier for the radio-therapeutic effect. Hence, how
to avoid the radiation resistance and to improve its treatment efficiency is a major challenge [1,2].
Ionizing radiation initiates DNA damage and arrests cell cycle progression, leading to cellular
genomic instability and loss of genetic information. In response to the damage stress, the ataxia
telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) are
Int. J. Mol. Sci. 2016, 17, 18; doi:10.3390/ijms17010018
www.mdpi.com/journal/ijms
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activated. ATM and ATR can also engage in cell division control via activating cyclins and
cyclin-dependent kinases (Cdks) [3,4]. A recent study found that in the cell division, there is a very
important cytokinetic abscission checkpoint regulated by CHMP4C, which controls abscission time
to coordinate mid-body resolution and prevents accumulation of DNA damage. CHMP4C can check
the abscission timing through Aurora B-directed phosphorylation [5].
Aurora B kinase is the key kinase of CPC (Chromosomal Passenger Complex) essential for the
mitotic processes. Aurora B expression and activity are altered during the cell cycle and peaks at
the G2-M transition. Aurora B is elevated in a variety of human tumors and its over-expression
is susceptible to tumor formation and poor outcomes of non-small cell lung cancer radiotherapy,
therefore indicating that Aurora B might be a drug target for lung cancer [6–8]. However, whether
Aurora B directly participates in lung cancer development is still unknown.
That ESCRT (Endosomal sorting complex) is required for the CPC (Chromosomal passenger
complex)-mediated cytokinetic abscission, during which CPC monitors the right abscission time of
two daughter cells. ESCRT has six distinct complexes (containing ESCRT-0, -I, -II, -III, ALIX and Vps4)
engaging in multi-vesicular body formation, cytokinesis and HBV (Hepatitis B virus) budding [9–11].
ESCRT-III (Endosome sorting complex-III) mediates membrane fission at the end of cytokinesis.
A recent study reported that CHMP4C, a subunit of ESCRT-III, retards abscission and inhibits DNA
damage accumulation in abscission checkpoint [12–15]. Meanwhile, CHMP4C enhances autophagy
and endosome production, during which p53 transcriptionally regulates CHMP4C via binding the
CHMP4C promoter from ´512~´450 DNA sequence in response to stress [16,17]. As the common
target of Aurora B and p53, CHMP4C has lower expression in normal tissues and high expression in
cancers [18]. Overall, despite CHMP4C is involved in abscission checkpoint and autophagy, how it
serves its function in DNA damage response as well as in lung cancer formation still remains unclear.
In the study, we first demonstrate that the subunit of ESCRT-III CHMP4C is involved in
cellular radiation reactions. Radiation enhances Aurora B expression and CHMP4C phosphorylation
in NSCLC cells, collectively directing cell cycle check-point and promoting cell survival.
CHMP4C silencing increases cellular sensitivity to radiation, hinders S-phase progression in cell cycle
and diminishes ionizing radiation (IR)-triggered γH2AX foci formation. We show that CHMP4C is
the major target of Aurora B and has a similar effect with Aurora B upon radiation. We further reveal
that the phosphorylation level of CHMP4C is rising both in p53-positive and-negative cells, showing
that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation
resistance is not p53 dependent. Overall, our work discovers a novel action of CHMP4C in radiation
resistance, suggesting CHMP4C as a new drug target for non-small cell lung cancer treatments.
2. Results
2.1. Radiation Induces CHMP4C Expression and Phosphorylation
To test if CHMP4C is involved in DNA damage response, cells were γ-irradiated with 2, 4, and
6 Gy and CHMP4C expression as well as phosphorylation was checked. We found that CHMP4C
protein increases after irradiation (IR) and peaks at 24 h in A549 but not in H1299 cells (Figure 1A,B).
However, the level of CHMP4C phosphorylation and Aurora B expression both increase with IR in
both A549 and H1299 (Figure 1C–E). Further, we detected p53 and CHMP4C expression after p53
silencing with or without IR (2 Gy). We found that CHMP4C protein did not increase after irradiation
in p53-cleaned A549 cells (Figure 1F). These data maybe indicate increased CHMP4C protein level
relies on p53 whereas its phosphorylation is just dependent of Aurora B.
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Figure 1.
1. Radiation induces CHMP4C expression
expression and
and phosphorylation.
phosphorylation. (A,B) The A549 or H1299 cells
cells
Figure
were irradiated
irradiated with
with 22 Gy,
Gy,and
andharvested
harvestedat
at4,
4,8,
8, 24
24 and
and 48
48 h.
h. CHMP4C expression or phosphorylation
phosphorylation
were
level was tested by Western
Western blot
blot using
using indicated
indicated antibodies;
antibodies; (C,D) Western
Western blot
blot analysis
analysis of
of CHMP4C
CHMP4C
level
expression or
or phosphorylation
phosphorylation level
level at
at 24
24 hh after
after treated
treated with
with 2,
2, 4,
4, and
and 66 Gy
Gy IR
IR in
in A549
A549 or
or H1299
H1299 cells;
cells;
expression
(E)
Aurora
B
expression
level
was
tested
by
Western
blot
at
24
h
after
cells
exposure
to
2,
4,
and
Gy
(E) Aurora B expression level was tested by Western blot at 24 h after cells exposure to 2, 4, and 66 Gy
IR; (F)
(F) Western
Westernblot
blotanalysis
analysisofofexpression
expressionofofCHMP4C
CHMP4C
and
P53
after
p53
silencing
with
or without
IR;
and
P53
after
p53
silencing
with
or without
IR
(2
in A549
cells.
IRGy)
(2 Gy)
in A549
cells.
2.2. CHMP4C Silencing Delayed S-Phase
S-Phase of
of the
the Cell
Cell Cycle
Cycle
2.2.
Next, we wondered
wondered ifif CHMP4C
CHMP4C exerts
exerts its
its regulatory
regulatory role
role in
in cell
cell cycle
cycle progression.
progression. A549 and
and
Next,
H1299 cells were
thethe
S phase
delay
waswas
checked,
indicating
that
H1299
were depleted
depleted ofofCHMP4C
CHMP4Cfor
for2424h and
h and
S phase
delay
checked,
indicating
CHMP4C
could
impact
S phase
exitexit
(Figure
CHMP4C
that
CHMP4C
could
impact
S phase
(Figure2A–D,G–J).
2A–D,G–J).Further,
Further,toto identify
identify whether CHMP4C
depletion could enhance the
thethe
cell
cycle
of
depletion
the radio-sensitivity
radio-sensitivity to
toA549
A549and
andH1299
H1299cells,
cells,we
weanalyzed
analyzed
cell
cycle
A549
andand
H1299
cellscells
cleared
of CHMP4C
afterafter
IR. When
A549 A549
and H1299
cells were
with 2
of
A549
H1299
cleared
of CHMP4C
IR. When
and H1299
cellstreated
were treated
Gy of2radiation,
G2/M transition
was severely
S-phase
retarded,
with
Gy of radiation,
G2/M transition
was blocked
severelyand
blocked
andwas
S-phase
was whereas
retarded,combined
whereas
radiation and
CHMP4C
knockdown
only results
S-phase
delay. Indelay.
the meantime,
similar similar
results
combined
radiation
and CHMP4C
knockdown
onlyinresults
in S-phase
In the meantime,
were found
in p21-cleaned
A549 A549
cells cells
(Figure
2A–D),
implying
thatthat
CHMP4C
might
overlay
or
results
were found
in p21-cleaned
(Figure
2A–D),
implying
CHMP4C
might
overlay
replenish
the
p21
function
in
cell
cycle
checkpoint
during
DNA
damage
response
in
A549
cells.
or replenish the p21 function in cell cycle checkpoint during DNA damage response in A549 cells.
In addition,
addition, CHMP4C
CHMP4C knockdown
knockdown has
has no
no influence
influence on
on p21
p21 expression
expression (Figure
(Figure 2E),
2E), and
and p21
p21
In
depletion cannot
which
reveals
thatthat
although
CHMP4C
and
depletion
cannot effect
effectCHMP4C
CHMP4Cexpression
expression(Figure
(Figure2F),
2F),
which
reveals
although
CHMP4C
P21 are
p53 target
genes,genes,
they may
in be
thein
different
signaling
pathway.
The double
siRNA
and
P21 both
are both
p53 target
they be
may
the different
signaling
pathway.
The double
experiments
show that
boththat
CHMP4C
and p21 depletion
exerts an additive
effect
in theeffect
S phase
siRNA
experiments
show
both CHMP4C
and p21 depletion
exerts an
additive
in exit
the
CHMP4C
overlay
or overlay
replenishorthe
p21 function
cell cycle
S(Figure
phase 3),
exitsuggesting
(Figure 3), that
suggesting
thatmight
CHMP4C
might
replenish
the p21infunction
in
checkpoint
during
DNA
damage
response
in
A549
cells,
by
participating
in
different
signaling
cell cycle checkpoint during DNA damage response in A549 cells, by participating in different
pathways.pathways.
signaling
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Mol. Sci.
Sci. 2016,
2016, 17,
17, 18
18
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Figure
2.
CHMP4C silencingdelayed
delayed S-phase of
of the cell
cycle.
transfected
with
Figure
2. 2.
CHMP4C
cycle.(A,B)
(A,B)A549
A549cells
cellswere
were
transfected
with
Figure
CHMP4Csilencing
silencing delayedS-phase
S-phase of the
the cell
cell cycle.
(A,B)
A549
cells
were
transfected
with
negative
control,siCHMP4C
siCHMP4Cororsip21
sip21for
for24
24 hh and/or
and/or exposed
toto22Gy
IR.
After
2424
h,h,
thethe
cells
were
negative
control,
exposed
Gy
IR.
After
cells
were
negative control, siCHMP4C or sip21 for 24 h and/or exposed to 2 Gy IR. After 24 h, the cells were
stained
with
Vibrant
Dyecycle greenstain
stain andtested
tested onaaflow
flow cytometer;(C,D)
(C,D) Cell cycle
cycle analyses of
stained
with
Vibrant
Dyecycle
stained
with
Vibrant
Dyecyclegreen
green stainand
and testedon
on a flowcytometer;
cytometer; (C,D)Cell
Cell cycleanalyses
analyses
of A549 cells. The data are presented as the mean ± S.E. of three independent experiments, ** p < 0.01;
A549
cells.cells.
TheThe
datadata
areare
presented
as as
thethe
mean
˘±S.E.
experiments,****p p< <0.01;
0.01;
of A549
presented
mean
S.E.of
ofthree
three independent
independent experiments,
(E,F) Western blot analysis of Expression of CHMP4C and P21 after CHMP4C or p21 silencing with
(E,F)
Western
blot
P21 after
after CHMP4C
CHMP4Cororp21
p21silencing
silencingwith
with
(E,F)
Western
blotanalysis
analysisofofExpression
Expressionof
of CHMP4C
CHMP4C and P21
or without IR (2 Gy); (G,H) H1299 cells were transfected with negative control or siCHMP4C for 24 h
oror
without
IRIR(2(2Gy);
with negative
negativecontrol
controlororsiCHMP4C
siCHMP4Cfor
for
without
Gy);(G,H)
(G,H)H1299
H1299cells
cellswere
were transfected
transfected with
2424
hh
and/or exposed to 2 Gy IR; After 24 h, the cells were stained with Vibrant Dyecycle green stain and
and/orexposed
exposedtoto22Gy
GyIR;
IR; After
After 24
24 h, the cells were
and/or
were stained
stainedwith
withVibrant
VibrantDyecycle
Dyecyclegreen
greenstain
stainand
and
analyzed on a flow cytometer; (I,J) Cell cycle analyses of H1299 cells. The data are presented as the
analyzed
flowcytometer;
cytometer;(I,J)
(I,J)Cell
Cellcycle
cycle analyses
analyses of H1299
analyzed
onona aflow
H1299 cells.
cells. The
Thedata
dataare
arepresented
presentedasasthe
the
mean ± S.E. of three independent experiments, ** p < 0.01.
mean
S.E.ofofthree
threeindependent
independent experiments,
experiments, **
mean
˘ ±S.E.
** pp <<0.01.
0.01.
Figure 3. CHMP4C and p21 double silencing exerts an additive effect in S-phase delay of the cell cycle
Figure
CHMP4C
and
p21cells
double
silencing
exerts
an
additive
ofofthe
cycle
Figure
3. 3.CHMP4C
and
p21
double
silencing
exerts
an negative
additiveeffect
effectin
inS-phase
S-phasedelay
delay
the
cell
cycle
in A549
cells.
(A,B)
A549
were
transfected
with
control,
siCHMP4C,
sip21,
orcell
double
A549
cells.
(A,B)
A549
cells
were exposed
transfected
with
negative
siCHMP4C,
sip21,
orVibrant
in in
A549
cells.
(A,B)
A549
cells
were
transfected
control,
siCHMP4C,
sip21,
ordouble
double
siCHMP4C
and
sip21
for 24
h and/or
to 2 with
Gy IR.negative
After 24control,
h, the cells
were stained
with
siCHMP4C
andsip21
sip21
for
hhand/or
to 2 to
Gy2IR.
24
h, the
cells
were
stained
with
Vibrant
siCHMP4C
and
for 24
24
and/or
exposed
GyAfter
IR.
After
24
h, the
cells
were
stained
with
Dyecycle green
stain and
tested
on a exposed
flow
cytometer;
(C,D)
Cell
cycle
analyses
of
A549
cells.
The
data
Dyecycle
green
stain
and
tested
on
a
flow
cytometer;
(C,D)
Cell
cycle
analyses
of
A549
cells.
The
data
are presented
asgreen
the mean
threeon
independent
experiments,
p < 0.05.
Vibrant
Dyecycle
stain± S.E.
and of
tested
a flow cytometer;
(C,D)**Cell
cycle analyses of A549 cells.
aredata
presented
as the mean
± S.E.
of ˘
three
experiments,
** p < 0.05. ** p < 0.05.
The
are presented
as the
mean
S.E.independent
of three independent
experiments,
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CHMP4C and
and Aurora
Aurora B Increase Radioresistance
2.3. CHMP4C
The above
above
data
indicate
that CHMP4C
can arrest
of cell
cycle.
The
data
indicate
that CHMP4C
inhibitioninhibition
can arrest S-phase
of cellS-phase
cycle. Colony
formation
Colony
formation
assays
were
next
employed
to
detect
if
CHMP4C
acts
in
cell
radioresistance.
assays were next employed to detect if CHMP4C acts in cell radioresistance. These assays showed
These
assays showed
that CHMP4C
celltosurvival
compared
to control
with
IR.
that
CHMP4C
knockdown
decreasesknockdown
cell survivaldecreases
compared
control with
IR. Similarly,
the
result
Similarly,
the result
was also
found in Aurora
B clearing.
Inversely,
CHMP4C
or Aurora
expression
was
also found
in Aurora
B clearing.
Inversely,
CHMP4C
or Aurora
B expression
can B
increase
cell
can increase
growth upon
irradiation
growth
uponcell
irradiation
(Figure
4A,B). (Figure 4A,B).
Figure
andand
Aurora
B increase
radioresistance.
(A) A549(A)
or (B)
H1299
cellsH1299
were transfected
Figure 4.4.CHMP4C
CHMP4C
Aurora
B increase
radioresistance.
A549
or (B)
cells were
with
siRNAs
or
plasmids
for
24
h
before
exposed
to
2,
4,
and
6
Gy-irradiation.
One
thousand
cells per
transfected with siRNAs or plasmids for 24 h before exposed to 2, 4, and 6 Gy-irradiation.
plate
seeded
immediately
and incubated
for 14after
days.IRColonies
were stained
with
1%
One were
thousand
cells
per plate after
wereIRseeded
immediately
and incubated
for 14
days.
crystal
violet.
The
number
of
colonies
was
counted
and
surviving
fraction
was
calculated
as
the
mean
Colonies were stained with 1% crystal violet. The number of colonies was counted and surviving
number
of colonies/(cells
× plating
efficiency).
The data are
expressed
as theefficiency).
mean ± S.E.The
of three
fraction was
calculated asseeded
the mean
number
of colonies/(cells
seeded
ˆ plating
data
independent
experiments.
are expressed as the mean ˘ S.E. of three independent experiments.
2.4.
2.4. CHMP4C
CHMP4C Acts
Acts Downstream
Downstream of
of Aurora
Aurora B
B
To
verify
whether
Aurora
B
regulates
CHMP4C,
wewe
silenced
or overexpressed
the Aurora
B with
To verify whether Aurora B regulates
CHMP4C,
silenced
or overexpressed
the Aurora
B
siRNA
or Aurora
B expression
plasmids
in A549
or H1299.
CHMP4C
knockdown
has has
no effect
on
with siRNA
or Aurora
B expression
plasmids
in A549
or H1299.
CHMP4C
knockdown
no effect
Aurora
B protein
level,
whereas
Aurora
B silencing
inhibits
CHMP4C
protein
levellevel
indicating
that
on Aurora
B protein
level,
whereas
Aurora
B silencing
inhibits
CHMP4C
protein
indicating
Aurora
B acts
upstream
of CHMP4C
(Figure
5A).
Moreover,
that Aurora
B acts
upstream
of CHMP4C
(Figure
5A).
Moreover,Aurora
AuroraBBoverexpression
overexpression leads
leads to
to
increased
CHMP4C
phosphorylation
and
inhibiting
Aurora
B
kinase
activity
using
phosphorylation
increased CHMP4C phosphorylation and inhibiting Aurora B kinase activity using phosphorylation
inhibitor
inhibitor AZD1152-HQPA
AZD1152-HQPA reduces
reduces CHMP4C
CHMP4C phosphorylation
phosphorylation (Figure
(Figure 5B–D).
5B–D). We
We then
then found
found the
the
CHMP4C
mRNA
level
was
unchanged
following
Aurora
B
downregulation
(Figure
5E),
further
CHMP4C mRNA level was unchanged following Aurora B downregulation (Figure 5E), further
making
surethat
thatAurora
Aurora
B regulates
CHMP4C
the protein
level. Collectively,
these
data
making sure
B regulates
the the
CHMP4C
at theatprotein
level. Collectively,
these data
suggest
suggest
that
Aurora
B
modulates
CHMP4C
expression
at
the
protein
level.
that Aurora B modulates CHMP4C expression at the protein level.
2.5.
2.5. CHMP4C
CHMP4C Silencing
Silencing Increases
Increases Cell
Cell Apoptosis
Apoptosis with
with Irradiation
Irradiation
The
The results
results of
of colony
colony formation
formation assay
assay indicate
indicate that
that the
the surviving
surviving fraction
fraction of
of NSCLC
NSCLC cells
cells was
was
significantly
attenuated
after
IR
treatment
of
4,
6
Gy.
We
next
want
to
ascertain
whether
CHMP4C
significantly attenuated after IR treatment of 4, 6 Gy. We next want to ascertain whether CHMP4C
knockdown
in A549
A549 cells:
cells: radiation
knockdown can
can enhance
enhance IR-induced
IR-induced apoptosis
apoptosis under
under three
three conditions
conditions in
radiation only
only
(6
knockdown,
combined
CHMP4C
knockdown
and IRand
(6 Gy).
WeGy).
foundWe
thatfound
combined
(6 Gy),
Gy),CHMP4C
CHMP4C
knockdown,
combined
CHMP4C
knockdown
IR (6
that
CHMP4C
silencing
and
irradiation
produce
more
evident
apoptosis
than
single
treatment
(Figure
6).
combined CHMP4C silencing and irradiation produce more evident apoptosis than single treatment
Hence,
assaysthese
reveal
that CHMP4C
facilitate
cellular
proliferation
and radiation
(Figure these
6). Hence,
assays
reveal thatcan
CHMP4C
can
facilitate
cellular proliferation
andresistance,
radiation
further
confirming
the
colony
formation
assays.
resistance, further confirming the colony formation assays.
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Figure 5. CHMP4C acts downstream of Aurora B. (A) Expression of Aurora B and CHMP4C after Aurora
Figure 5.5.CHMP4C
CHMP4C
acts
downstream
of Aurora
B. Expression
(A) Expression
of Aurora
B and CHMP4C
after
Figure
acts
downstream
of Aurora
B. (A)
of Aurora
B and CHMP4C
after Aurora
BAurora
or CHMP4C
in A549 andinH1299
The phosphorylation
level of CHMP4Clevel
in
B or knockdown
CHMP4C
knockdown
A549cells;
and (B)
H1299
(B) The phosphorylation
B or CHMP4C
knockdown
in A549 and H1299
cells;
(B) The cells;
phosphorylation
level of CHMP4C in
untreated
and
AZD1152
treated
(10
and
1000
nM)
A549
and
H1299
cells;
(C)
The
phosphorylation
of CHMP4C
untreated
and(10
AZD1152
(10 and
andH1299
1000 cells;
nM) (C)
A549
H1299 cells;
untreated
and in
AZD1152
treated
and 1000treated
nM) A549
Theand
phosphorylation
level
of CHMP4C
in untreated
and
AZD1152
treated
(100 nM)
A549
and H1299
cells;
(D)nM)
Western
(C)
The
phosphorylation
level
of
CHMP4C
in
untreated
and
AZD1152
treated
(100
A549blot
and
level of CHMP4C in untreated and AZD1152 treated (100 nM) A549 and H1299 cells; (D) Western
blot
analysis
of
CHMP4C
phosphorylation
level
in
A549
and
H1299
cells
transfected
with
pCDNA3.1H1299 cells;
(D) Western
blot analysis of
CHMP4C
in A549with
and pCDNA3.1H1299 cells
analysis
of CHMP4C
phosphorylation
level
in A549phosphorylation
and H1299 cellslevel
transfected
Aurora
B; (E)with
The mRNA expression level
of The
CHMP4C
Aurora B was
quantitated
with
transfected
(E)
mRNAoror
expression
of CHMP4C
or real-time
Aurora
B
Aurora
B; (E) ThepCDNA3.1-Aurora
mRNA expression B;
level
of CHMP4C
Aurora B level
was quantitated
with
real-time
PCR
± S.E., n = 3)real-time
after the A549(mean
and H1299
cells
of negative
or
was(mean
quantitated
˘ S.E.,
n =undergone
3) after thetransfection
A549 and H1299
cells control
undergone
PCR
(mean ± S.E.,with
n = 3) after thePCR
A549 and H1299
cells
undergone
transfection
of negative
control or
siAurora
B. of negative control or siAurora B.
transfection
siAurora
B.
Figure 6. CHMP4C silencing increases cell apoptosis with irradiation. (A) CHMP4C-defective cells
Figure
6. CHMP4C
with irradiation.
irradiation. (A)
Figure 6.
CHMP4C silencing
silencing increases
increases cell
cell apoptosis
apoptosis with
(A) CHMP4C-defective
CHMP4C-defective cells
cells
without IR; (B) siRNA transfected cells were exposed to 6 Gy of irradiation. (C) Quantification of cell
without
IR;
(B)
siRNA
transfected
cells
were
exposed
to
6
Gy
of
irradiation.
(C)
Quantification
of
without IR; (B) siRNA transfected cells were exposed to 6 Gy of irradiation. (C) Quantificationcell
of
apoptosis assays. The data are presented as the mean ± S.E. of three independent experiments.
apoptosis
assays.
TheThe
datadata
areare
presented
as as
the
cell apoptosis
assays.
presented
themean
mean± ˘S.E.
S.E.ofofthree
three independent
independent experiments.
experiments.
** p < 0.01
**
** pp << 0.01
0.01
2.6. CHMP4C Reduction Suppresses IR-Induced γH2AX and 53BP1foci Formation
2.6.
γH2AX and
Formation
2.6. CHMP4C
CHMP4C Reduction
Reduction Suppresses
Suppresses IR-Induced
IR-Induced γH2AX
and 53BP1foci
53BP1foci Formation
Next, to analyze if CHMP4C is immediately involved in DNA damage signaling, we examined
Next,
analyze
if CHMP4C
is
involved in
damage
we examined
Next, to
to
analyze
CHMP4C
is immediately
immediately
in DNA
DNACHMP4C
damage signaling,
signaling,
the γH2AX
and
53BP1 iffoci
assembly
in A549 cells involved
with or without
silencing. we
Theexamined
IR dose
the
γH2AX
and
53BP1
foci
assembly
in
A549
cells
with
or
without
CHMP4C
silencing.
The
dose
the
γH2AX
and
53BP1
foci
assembly
in
A549
cells
with
or
without
CHMP4C
silencing.
The IR
IR
dose
of 4 Gy was selected in this analysis according to the results of colony formation and apoptosis
assay.
of 4 Gy was selected in this analysis according to the results of colony formation and apoptosis assay.
As shown in Figure 6, the indicated dose (4 Gy) of γ-irradiation is sufficient to induce γH2AX and
As shown in Figure 6, the indicated dose (4 Gy) of γ-irradiation is sufficient to induce γH2AX and
53BP1foci formation in nuclear (Figures 7C,D and 8C,D). The results demonstrated that during 4 Gy
53BP1foci formation in nuclear (Figures 7C,D and 8C,D). The results demonstrated that during 4 Gy
Int. J. Mol. Sci. 2016, 17, 18
7 of 12
of 4 Gy was selected in this analysis according to the results of colony formation and apoptosis assay.
7 of 12
12
the indicated dose (4 Gy) of γ-irradiation is sufficient to induce γH2AX7 of
and
53BP1foci
formation
in nuclear
(Figures
7C,D and
Theand
results
demonstrated
that
during
4 Gy
of
irradiation,
CHMP4C
reduction
decreased
the 8C,D).
intensity
number
of γH2AX
and
53BP1foci
of
irradiation,
CHMP4C
reduction
decreased
the
intensity
and
number
of
γH2AX
and
53BP1foci
of
irradiation,
CHMP4C
reduction
decreased
the
intensity
and
number
of
γH2AX
and
53BP1foci
compared with the normal CHMP4C, proving that CHMP4C repression indeed represses recruitment
compared
with the
normal
CHMP4C, proving
CHMP4C repression
indeed represses
recruitment
compared
theand
normal
provingtothat
that
repression
recruitment
of
γH2AX,with
53BP1
otherCHMP4C,
related proteins
the CHMP4C
DNA lesions
(Figuresindeed
7 and represses
8).
of
γH2AX,
53BP1
and
other
related
proteins
to
the
DNA
lesions
(Figures
7
and
8).
of γH2AX, 53BP1 and other related proteins to the DNA lesions (Figures 7 and 8).
Int. J. Mol. Sci. 2016, 17, 18
Int.
Mol. Sci.in
2016,
17, 18 6,
As J.shown
Figure
Figure
γH2AX foci
foci formation.
formation. (A,B)
Figure 7.
7. CHMP4C
CHMP4C reduction
reduction suppresses
suppresses IR-induced
IR-induced γH2AX
(A,B) Negative
Negative control
control
Figure 7. CHMP4C reduction suppresses IR-induced γH2AX foci formation. (A,B) Negative control
and
siCHMP4C
transfected
cells
without
IR;
(C,D)
A549
cells
with
or
without
CHMP4C
and siCHMP4C transfected cells without IR; (C,D) A549 cells with or without CHMP4C knockdown
knockdown
and siCHMP4C transfected cells without IR; (C,D) A549 cells with or without CHMP4C knockdown
were
exposed to
to 44 Gy
Gy radiation;
radiation; and
and (E)
(E) image
image of
of γH2AX
γH2AX foci
foci formation.
formation. The
were exposed
The number
number of
of foci
foci per
per
were exposed to 4 Gy radiation; and (E) image of γH2AX foci formation. The number of foci per
nucleus was quantitated. The data are presented as the
the mean
mean ˘
± S.E. of three independent experiments,
nucleus was quantitated. The data are presented as the mean ± S.E. of three independent experiments,
* p << 0.05.
0.05.Scale
Scalebar,
bar,10
10µm.
µm.
* p < 0.05. Scale bar, 10 µm.
Figure
8.
CHMP4C
abrogation
represses
IR-induced
53BP1
foci
formation.
(A,B)
Negative
control
or
Figure 8.
8. CHMP4C
CHMP4Cabrogation
abrogationrepresses
repressesIR-induced
IR-induced53BP1
53BP1foci
fociformation.
formation.
(A,B)
Negative
control
Figure
(A,B)
Negative
control
or
siCHMP4C
transfected
cells
without
IR;
(C,D)
A549
infected
with
negative
control
or
siCHMP4C
or siCHMP4C
transfected
cells
without
(C,D)A549
A549infected
infectedwith
withnegative
negativecontrol
control or
or siCHMP4C
siCHMP4C
siCHMP4C
transfected
cells
without
IR;IR;
(C,D)
were
then
treated
with
4 Gy
Gy radiation;
radiation; and
and (E)
(E) image
image of
of 53BP1
53BP1 foci
foci formation.
formation. The
number
of
foci
per
were
then
treated
with
4
The
number
of foci
foci per
per
were then treated with 4 Gy radiation; and (E) image of 53BP1 foci formation. The number of
nucleus
was
quantitated.
The
data
are
presented
as
the
mean
±
S.E.
of
three
independent
experiments,
nucleus was
was quantitated.
quantitated. The
The data
data are
are presented
presented as
as the
S.E.of
ofthree
three independent
independent experiments,
experiments,
nucleus
the mean
mean ˘
± S.E.
** pp << 0.05.
Scale
bar,
10
µm.
0.05.
Scale
bar,
10
µm.
* p < 0.05. Scale bar, 10 µm.
Int. J. Mol. Sci. 2016, 17, 18
Int. J. Mol. Sci. 2016, 17, 18
8 of 12
8 of 12
3. Discussion
Discussion
Current investigations
investigations on CHMP4C mainly focus its function on endosome generation.
However,
serves its
itspart
partinincell
cellcycle,
cycle,viability
viabilityand
andapoptosis
apoptosis
under
irradiation
However, whether and how itit serves
under
irradiation
is
notnot
clear.
It has
been
reported
thatthat
p53 p53
regulates
transcription
of CHMP4C
in the
and
is
clear.
It has
been
reported
regulates
transcription
of CHMP4C
in autophagy
the autophagy
endosome
production
[16]. [16].
p53 plays
a critical
role in
regulating
DNA-damage-associated
cell cycle
and
endosome
production
p53 plays
a critical
role
in regulating
DNA-damage-associated
cell
progression,
DNA DNA
repair,repair,
or apoptosis
in the p53/p21
pathway.
As the classic
gene
of p53,
cycle
progression,
or apoptosis
in the p53/p21
pathway.
As thetarget
classic
target
genep21
of
regulates
the cyclin-Cdk
and PCNA
required required
for the Gfor
1 tothe
S transition
[19–21]. We
then
p53,
p21 regulates
the cyclin-Cdk
andcomplexes
PCNA complexes
G1 to S transition
[19–21].
supposed
that theythat
might
similar
in p53-directed
cell cycle
In support
of this
We
then supposed
theyhave
might
have function
similar function
in p53-directed
cellswitch.
cycle switch.
In support
hypothesis,
our results
indicated
that CHMP4C
knockdown
was engaged
in cellular
S-phase
delay,
of
this hypothesis,
our results
indicated
that CHMP4C
knockdown
was engaged
in cellular
S-phase
showing
the similar
regulation
withwith
p21 p21
in cell
cycle
delay,
showing
the similar
regulation
in cell
cycleprogression
progressionininA549.
A549. Moreover,
Moreover, double
effect in S-phase delay of the cell cycle.
knockdown of CHMP4C and p21 produced an additive effect
However, CHMP4C
CHMP4C knockdown
knockdown has
has no
no influence
influence on p21 expression and p21 depletion cannot effect
However,
expression, which
which reveals
reveals that
that the novel
novel role
role of
of CHMP4C
CHMP4C in cell cycle shows an alternative
CHMP4C expression,
paralleling the
the p21
p21 signal
signal pathway
pathway (Figure
(Figure 9).
9).
pathway paralleling
Figure 9.
9. The
CHMP4C signaling
signaling in
in response
response to
to stress.
stress. CHMP4C
CHMP4C is
is phosphorylated
phosphorylated by
by
Figure
The summary
summary of
of CHMP4C
Aurora
B
to
regulate
the
abscission
timing
checkpoint
and
cell
cycle
progression,
promote
cell
survival
Aurora B to regulate the abscission timing checkpoint and cell cycle progression, promote cell survival
and cell
cell viability
ATM)
and
viability and
and resist
resist apoptosis
apoptosis(Blue
(Bluearrows).
arrows). The
Theataxia
ataxiatelangiectasia
telangiectasiamutated
mutatedkinase
kinase ((ATM)
is activated
activated in
mitosis in
regulates
is
in mitosis
in Aurora
Aurora B-dependent
B-dependent manner
manner (Green
(Green arrows).
arrows). p53
p53 transcriptionally
transcriptionally regulates
autophagy and
and endosome
endosome production
production (Red
(Red arrows).
arrows). p21 is the target gene of
CHMP4C to enhance autophagy
p53 and functions
functions in
in the
the G1
G1 to
to SS checkpoint
checkpoint (Green
(Green arrows).
arrows).
Further, our results also showed that CHMP4C can promote cell survival and cell viability upon
Further, our results also showed that CHMP4C can promote cell survival and cell viability
IR, indicating that CHMP4C offer the radioresistance to the irradiated cells. We next studied if
upon IR, indicating that CHMP4C offer the radioresistance to the irradiated cells. We next studied
CHMP4C was connected with DNA repair, and found that CHMP4C inhibition repressed DNA
if CHMP4C was connected with DNA repair, and found that CHMP4C inhibition repressed DNA
repair by decreasing γH2AX and 53BP1 foci reacting to radiation stress. Hence, the novel functions
repair by decreasing γH2AX and 53BP1 foci reacting to radiation stress. Hence, the novel functions
of CHMP4C in cell cycle control and DNA repair not only enriches the content of the DNA damage
of CHMP4C in cell cycle control and DNA repair not only enriches the content of the DNA damage
pathway but also extends its specific role in cell regulation.
pathway but also extends its specific role in cell regulation.
p53 is often mutated in NSCLC and its mutation increases sensitivity to ionizing radiation in
p53 is often mutated in NSCLC and its mutation increases sensitivity to ionizing radiation
tumor cells [22]. CHMP4C transcription is controlled by p53 in endosome function [16]. CHMP4C
in tumor cells [22]. CHMP4C transcription is controlled by p53 in endosome function [16].
functions in the Aurora B-dependent abscission checkpoint and inhibits abscission upon phosphorylation
CHMP4C functions in the Aurora B-dependent abscission checkpoint and inhibits abscission
by Aurora B, eventually preventing premature resolution of intercellular chromosome bridges and
upon phosphorylation by Aurora B, eventually preventing premature resolution of intercellular
DNA damage accumulation. Our results reveal that increased Aurora B kinase enhances the
CHMP4C phosphorylation after IR, which is independent of p53. This is also supported by the facts
Int. J. Mol. Sci. 2016, 17, 18
9 of 12
chromosome bridges and DNA damage accumulation. Our results reveal that increased Aurora B
kinase enhances the CHMP4C phosphorylation after IR, which is independent of p53. This is also
supported by the facts that the cell lines displayed higher sensitivities to IR after CHMP4C inhibition
compared to IR alone, and CHMP4C can increase cell survival both in A549 and H1299 cells.
Several Aurora kinase inhibitors are tested in clinical trials [23]. Studies have shown that
inhibition of Aurora B radiosensitizes tumor cells [6,7]. In the study, we observed the reduction
of CHMP4C protein level but its mRNA expression remains unchanged following silencing of
Aurora B, revealing that Aurora B might regulate the CHMP4C protein stability. Inhibited Aurora
kinase B activity by AZD1152 suppresses CHMP4C phosphorylation. However, overexpression of
Aurora kinase B upregulates CHMP4C phosphorylation because of the large amount of Aurora B
proteins. Furthermore, CHMP4C suppression also radiosensitizes NSCLC cells similar to that during
Aurora B inhibition. We demonstrate that Aurora B kinase is essential to phosphorylate CHMP4C
and the role of CHMP4C in radioresistance is dependent on Aurora B, which proves that CHMP4C
is the major downstream target of Aurora B. Therefore, we identify the close relation between
CHMP4C and Aurora B signaling pathway in mediating radiation resistance in NSCLC cells, which
is independent on p53.
CHMP4C can check the cytokinetic abscission to prevent premature and accumulated DNA
damage as an abscission timer. CHMP4C deficiency generates the defective abscission check, which
makes injured cells quickly pass through M to S phase, leading to DNA repair disability and genomic
instability. These data suggests that CHMP4C deficiency disorganizes the cell cycle and enhances cell
sensitivity to radiation, thus providing a new combined strategy to diagnosis and treatment of the
lung cancer.
We propose that ATM is activated by Aurora B, followed by p53-mediated activation of
CHMP4C. In turn, CHMP4C promotes cell viability and proliferation. However, there is another
major tumor suppressor factor, the alternative reading frame (ARF), which is negatively regulated
by ATM in a transcription-independent manner. Contrary to the function of CHMP4C on cell fate,
inhibition of ATM enhances ARF levels and stimulates the tumor-suppressive effects of ARF in
human oncogene-transformed and cancer cells [24]. These findings provide insights into our study
and broaden the aspects of our further research.
4. Materials and Methods
4.1. Cell Culture and Irradiation
The human NSCLC cell lines A549 (p53 wild type) and H1299 (p53 deficient) (Cell resource
center, Peking union medical college, Beijing, China) were cultured in RPMI-1640 containing 10%
fetal bovine serum (Invitrogen, Carlsbad, CA, USA), incubated in 37 ˝ C humidified incubator with
5% CO2 and treated with different radiation of 2, 4, and 6 Gy using Co60 γ-rays with a dose rate of
1 Gy/min in the Irradiation (IR) Center (Beijing Radiation Center, Beijing Academy of Science and
Technology, Beijing, China).
4.2. siRNAs and Plasmids Transfection
A549 and H1299 cells were grown at 80% confluence and then introduced with siRNAs against
CHMP4C (Ambion, Austin, TX, USA), Aurora B (Ambion), p53 and p21 (GenePharma, Beijing,
China) as well as control siRNA (Ambion). CHMP4C, Aurora B, p53 and p21 siRNA sequence
is respectively: 51 -CCUGCGUCUCUACAACUAU-31 , 51 -CAUGGAUCUGAACAAAAUATT-31 ,
51 -CUACUUCCUGAAAACAACG-31
and
51 -CCUCUGGCAUUAGAAUUAUTT-31 .
siRNA tansfections were performed using Lipofectamine RNAi MAX reagent (Invitrogen).
Twenty-four hours after transfection, the cells were irradiated and harvested.
Int. J. Mol. Sci. 2016, 17, 18
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pcDNA3.1-CHMP4C or -Aurora B was contrasted and transfected into A549 or H1299 cells using
Lipofectamine 3000 reagent (Invitrogen). Twenty-four hours later, cells were collected and performed
for subsequent experiments as siRNAs treatment.
4.3. Kinase Inhibition Assays
Aurora B kinase inhibitor AZD1152-HQPA was dissolved in dimethyl sulphoxide (DMSO).
The A549 and H1299 cells were treated with AZD1152-HQPA at the concentration of 10, 100 and
1000 nM. After 24 h of treatment, CHMP4C phosphorylation was examined by Western blot.
4.4. Western Blot
The cells were harvested and lysed in RIPA lysis buffer (Thermo Scientific Pierce, Waltham, MA,
USA). The protein was collected at 12,000ˆ g for 15 min at 4 ˝ C and measured by BCA protein
assay kit (Thermo Scientific Pierce). Equal amounts of protein were separated on 10% sodium
dodecyl sulfate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes for Western blot
analysis. The membranes were blocked in 5% nonfat milk and then incubated with the following
primary antibodies: CHMP4C (Abcam, Cambridge, UK), phosphorylated (p)—CHMP4C (Abmart,
Arlington, MA, USA), Aurora B (Abcam), p53 (Cell Signaling Technology, Boston, MA, USA), p21
(Cell Signaling Technology) and β-actin (Cell Signaling Technology). The CHMP4C antibody is
diluted in 1:500, and the rest were used in 1:1000 dilutions. Membranes were washed in tris-buffered
saline containing 0.5% tween-20 and then incubated with goat anti-rabbit lgG (Abcam, 1:2000) or goat
anti-mouse lgG (Abcam, 1:3000) conjugated to horseradish peroxidase for 1 h at room temperature.
The membranes were detected using Chemiluminescence liquid (Thermo Scientific Pierce) according
to the manufacturer’s protocol and analyzed by the Image J software (Bio-Rad, Hercules, CA, USA).
4.5. Real-Time PCR
Total RNA was extracted using SV total RNA isolation system kit (Promega, Madison,
WI, USA) followed by reverse transcription using the GoScript reverse transcription system kit
(Promega). The subsequent cDNA products were used as templates to perform the real-time
PCR assays. The primers for the amplification of Aurora B or CHMP4C are as follows; Aurora
B forward: TTTGAGATTGGGCGTCCTCT and reverse: CGCCCTCCTTCTCTATCTGG; CHMP4C
forward: AGAAGCCCTGGAGAACTCAC and reverse: CTTGGGCAGTATCCTGTTGC. The β-actin
was used as the internal control using primers forward: TGCCAGAAAACAAGATGAG and reverse:
CACCTTCACCGTTCCAGTTT. PCR amplifications were performed in triplicate wells and each
experiment was repeated for three times. The relative expression levels of genes were analyzed
through the use of the 2´∆∆Ct method.
4.6. Cell Cycle Assay
Cells were harvested and treated with Vibrant Dyecycle green stain (Invitrogen) at 37 ˝ C for
30 min in a dark place. Then, cell cycle was analyzed on a flow cytometer using 488-nm excitation
and green emission.
4.7. Colony Formation Assay
To analyze colony formation, Single cell was plated in the 6 cm dish at a density of 1000 cells/mL
for 10 to 14 days to form spheres. Colonies were fixed with 4% paraformaldehyde for 20 min and
then stained with 1% crystal violet for 15 min. Colonies were counted and surviving fraction was
calculated as the mean number of colonies/(cells seeded ˆ plating efficiency).
Int. J. Mol. Sci. 2016, 17, 18
11 of 12
4.8. Cell Apoptosis Assay
Cells were harvested and washed twice with cold PBS. Cells were resuspended in 1ˆ binding
buffer (BD) at a concentration of 1 ˆ 106 cells/mL and stained with PE AnnexinV and 7-AAD (BD)
for 15 min at room temperature in the dark. Samples were analyzed by flow cytometry.
4.9. γH2AX and 53BP1 Foci Formation
Cells were seeded on the glass sheets into 6-well plates. Cells were transfected with siRNAs
and then exposed to 4 Gy of irradiation. 30 min later, cells were washed twice in PBS, and then
incubated with anti-γH2AX (Millipore, FITC conjugate, Billerica, MA, USA), anti-53BP1 (Abcam) or
anti-CHMP4C antibody at a dilution of 1:200 overnight at 4 ˝ C. After washing twice in washing
buffer (0.05% Tween-20 in PBS), the cells were incubated with goat anti-rabbit lgG (Abcam, Alexa
fluor conjugate) at 37 ˝ C for 1 h. Cells were washed twice with washing buffer and once with PBS.
The coverslips were then mounted with mounting medium for fluorescence with DAPI (Vectashield).
The image of γH2AX and 53BP1 foci was detected using a Leica DMRXA fluorescent microscope
(Leica, Wetzlar, Germany). γH2AX and 53BP1 foci were counted in cells with more than five foci.
4.10. Statistical Analysis
Data are presented as the mean ˘ S.E. of three independent experiments, and the statistics
were analyzed by students’ t test using Microsoft Excel (Microsoft Campus, Redmond, WA, USA).
p-value <0.05 indicates statistical significance.
5. Conclusions
In conclusion, we identify that CHMP4C is a new radiation-inducible protein engaged in cell
cycle and cell survival. We first confirm CHMP4C offers lung cancer cells radioresistance, which will
provide a potential radio-therapeutic strategy for non-small cell lung cancer by disrupting CHMP4C
expression. In future research, the role of CHMP4C combined with radiation in vivo, for example the
function of CHMP4C in xenografts [24], will be investigated, which would provide in vivo evidence
for the new action of CHMP4C.
Author Contributions: Xu Su, Jianxiang Liu, Mei Tian and Kang Li conceived and designed the experiments;
Kang Li performed the experiments; Xu Su, Jianxiang Liu, Mei Tian and Kang Li analyzed the data; Gang Gao,
Yan Pan and Xuesong Qi contributed analysis tools; Jianlei Ruan and Chunxu Liu contributed reagents and
materials; and Xu Su and Kang Li wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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