Article

Significance of p53-binding protein1 nuclear foci in uterine cervical lesions: Endogenous DNA double strand breaks and genomic instability during carcinogenesis

Department of Tumour and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Histopathology (Impact Factor: 3.45). 09/2011; 59(3):441-51. DOI: 10.1111/j.1365-2559.2011.03963.x
Source: PubMed
ABSTRACT
A defective DNA damage response can result in genomic instability (GIN) and lead to transformation to cancer. As p53-binding protein 1 (53BP1) localizes at the sites of DNA double strand breaks (DSBs) and rapidly forms nuclear foci (NF), the presence of 53BP1 NF can be considered to be an indicator of endogenous DSBs reflecting GIN. Our aim was to analyse the presence of DSBs by immunofluorescence for 53BP1 expression in a series of cervical lesions, to evaluate the significance of GIN during carcinogenesis.
A total of 80 archival cervical tissue samples, including 11 normal, 16 cervical intraepithelial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14 squamous cell carcinoma samples, were analysed for 53BP1 NF, human papillomavirus (HPV) infection, and p16(INK4a) overexpression. The number of 53BP1 NF in cervical cells appeared to increase with progression during carcinogenesis. The distribution of 53BP1 NF was similar to that of the punctate HPV signals as determined by in-situ hybridization and also to p16(INK4a) overexpression in CIN, suggesting an association with viral infection and replication stress.
Immunofluorescence analysis of 53BP1 expression can be a useful tool with which to estimate the level of GIN. During cervical carcinogenesis, GIN may allow further accumulation of genomic alterations, causing progression to invasive cancer.

Full-text

Available from: Kondo Hisayoshi, May 08, 2014
Significance of p53-binding protein 1 nuclear foci in uterine
cervical lesions: endogenous DNA double strand breaks and
genomic instability during carcinogenesis
Katsuya Matsuda,
1,2
Shiro Miura,
3
Tomomi Kurashige,
3
Keiji Suzuki,
4
Hisayoshi Kondo,
5
Makoto Ihara,
6
Hisayoshi Nakajima,
7
Hideaki Masuzaki
2
& Masahiro Nakashima
1,3
1
Department of Tumour and Diagnostic Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of
Biomedical Sciences,
2
Department of Obstetrics and Gynaecology, Nagasaki University Hospital,
3
Tissue and Histopathology
Section, Division of Scientific Data Registry, Atomic Bomb Disease Institute, Nagasaki University Graduate School of
Biomedical Sciences,
4
Department of Radiation Medical Sciences,
5
Biostatics Section, Division of Scientific Data Registry,
6
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical
Sciences, and
7
Nagasaki University School of Health Sciences, Nagasaki, Japan
Date of submission 27 July 2010
Accepted for publication 23 November 2010
Matsuda K, Miura S, Kurashige T, Suzuki K, Kondo H, Ihara M, Nakajima H, Masuzaki H & Nakashima M
(2011) Histopathology 59, 441–451
Significance of p53-binding protein 1 nuclear foci in uterine cervical lesions: endogenous
DNA double strand breaks and genomic instability during carcinogenesis
Aims: A defective DNA damage response can result in
genomic instability (GIN) and lead to transformation to
cancer. As p53-binding protein 1 (53BP1) localizes at
the sites of DNA double strand breaks (DSBs) and
rapidly forms nuclear foci (NF), the presence of 53BP1
NF can be considered to be an indicator of endogenous
DSBs reflecting GIN. Our aim was to analyse the
presence of DSBs by immunofluorescence for 53BP1
expression in a series of cervical lesions, to evaluate the
significance of GIN during carcinogenesis.
Methods and results: A total of 80 archival cervical tissue
samples, including 11 normal, 16 cervical intraepithe-
lial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14
squamous cell carcinoma samples, were analysed for
53BP1 NF, human papillomavirus (HPV) infection, and
p16
INK4a
overexpression. The number of 53BP1 NF in
cervical cells appeared to increase with progression
during carcinogenesis. The distribution of 53BP1 NF
was similar to that of the punctate HPV signals as
determined by in-situ hybridization and also to p16
INK4a
overexpression in CIN, suggesting an association with
viral infection and replication stress.
Conclusions: Immunofluorescence analysis of 53BP1
expression can be a useful tool with which to estimate
the level of GIN. During cervical carcinogenesis, GIN
may allow further accumulation of genomic altera-
tions, causing progression to invasive cancer.
Keywords: 53BP1, DNA damage response, genomic instability, immunofluorescence, uterine cervical cancer
Abbreviations: 53BP1, p53-binding protein 1; CIN, cervical intraepithelial neoplasia; DAB, diaminobenzidine;
DAPI-I, 4¢,6-diamidino-2-phenylindole dihydrochloride; DDR, DNA damage response; DSB, double strand break;
GIN, genomic instability; HPV, human papillomavirus; HR-HPV, high-risk human papillomavirus; IR, ionizing
radiation; ISH, in-situ hybridization; NF, nuclear foci; pRb, retinoblastoma protein; SCC, squamous cell carcinoma
Address for correspondence: M Nakashima, MD, PhD, Department of Tumour and Diagnostic Pathology, Atomic Bomb Disease Institute,
Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
e-mail: moemoe@nagasaki-u.ac.jp
Re-use of this article is permitted in accordance with the terms and conditions set out at http://www3.interscience.wiley.com/authorresources/
onlineopen.html
2011 Blackwell Publishing Limited.
Histopathology 2011, 59, 441–451. DOI: 10.1111/j.1365-2559.2011.03963.x
Page 1
Introduction
Any genotoxic agents can induce DNA damage and
consequently result in oncogenic alterations. The
importance of the DNA damage response (DDR) path-
way in tumour suppression is well recognized. A
defective DDR can result in genomic instability (GIN),
which is generally considered to be central to any
carcinogenic process.
1,2
Alternatively, the presence of
an activated DDR can be a hallmark of GIN, which may
subsequently enhance the carcinogenic process.
The p53-binding protein 1 (53BP1) belongs to a
family of evolutionarily conserved DDR proteins with C-
terminal BRCA1 C-terminus domains.
3,4
53BP1 is a
nuclear protein that rapidly localizes at the sites of DNA
double strand breaks (DSBs) and activates p53 along
with other kinases.
5–10
Activated p53 plays critical roles
in the DDR, such as cell cycle arrest, DNA repair, and
apoptosis.
11,12
It has been well documented in vitro with
immunofluorescence that 53BP1 exhibits diffuse nucle-
ar staining in untreated primary cells, whereas, after
exposure to radiation, 53BP1 localizes at the sites of DSBs
and forms discrete nuclear foci (NF).
5,6,13,14
We have
recently demonstrated that, with an immunofluores-
cence method, 53BP1 NF may serve as a valuable
molecular marker of GIN during carcinogenesis.
15,16
GIN seems to be induced at the precancerous stage
during thyroid and skin carcinogenesis, as follicular
adenoma and actinic keratosis show occasional 53BP1
NF.
15,16
Given that one manifestation of GIN is the
induction of endogenous DDR,
17
we propose that
immunofluorescence analysis of 53BP1 expression can
be a useful tool with which to estimate the level of GIN as
well as the malignant potential of human tumours.
Uterine cervical cancers are believed to develop
through a multistep process. Furthermore, it is well
established that persistent infections with high-risk
human papillomavirus (HR-HPV) represent a necessary
cause of high-grade premalignant lesions and subse-
quent invasive cancer of the uterine cervix. The HR-
HPV viral oncogenes, E6 and E7, have been shown to
be the main contributors to the development of human
papillomavirus (HPV)-induced cervical cancer, and
increased expression resulting from integration of the
viral DNA into the host genome has been detected in
invasive cancers and a subset of high-grade lesions.
18
It
has been shown that E6 and E7 together cause
polyploidy soon after they are introduced into cells, so
GIN is thought to play an essential role in the cellular
transformation of cervical epithelium during carcino-
genesis. The most manifest function of the E6 protein is
to promote the degradation of p53 through its inter-
action with a cellular protein, E6-associated protein, an
E3 ubiquitin ligase.
19
E7 is known to bind to the
retinoblastoma tumour suppressor gene product, reti-
noblastoma protein (pRb). Phosphorylation of pRb by
G
1
cyclin-dependent kinases releases E2F, leading to
cell cycle progression into the S phase. E7 is able to
bind unphosphorylated pRb, and this may induce cells
to prematurely enter the S phase by disrupting pRb–
E2F complexes. One cyclin-dependent kinase inhibitor,
p16
INK4a
, which prevents the phosphorylation of pRb
family members, is overexpressed when pRb is inacti-
vated by E7.
20
Normally, overexpression of p16
INK4a
results in cell cycle arrest, but with E7 expression, this
is overcome. Thus, overexpression of p16
INK4a
has been
suggested as a useful biomarker for evaluating HPV
pathogenic activity in cervical lesions.
The present study analysed the presence of endog-
enous DSBs by immunofluorescence for 53BP1 expres-
sion in a series of cervical tissues from patients to
evaluate the significance of GIN and its association
with HPV infection and p16
INK4a
overexpression dur-
ing cervical carcinogenesis. Like other tumours, GIN
was shown to be induced in cervical epithelium at a
precancerous stage, and increased significantly with
progression to cancer.
Materials and methods
cervical lesions
Eighty archival uterine cervical tissue samples were
selected for this study from the archives of the
Department of Obstetrics and Gynaecology, University
Hospital. Accuracy of diagnosis was confirmed by a
gynaecological pathologist (H.N.) and a general pathol-
ogist (M.N.). Histologically, the 80 primary cervical
tissue samples were as follows: 11 normal cervical
tissue samples from uteri that were surgically resected
because of leiomyoma; and 16 cervical intraepithelial
neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14 squamous
cell carcinoma (SCC) samples. All samples were forma-
lin-fixed and paraffin-embedded tissues, from which
sections were prepared for immunofluorescence and
in-situ hybridization (ISH) studies.
ish to detect hr-hpv
ISH was performed with the GenPoint Catalyzed Signal
Amplification System (Dako, North America, Inc.,
Carpinteria, CA, USA) for HR-HPV (types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59, and 68; code
Y1443), according to the manufacturer’s protocols.
After deparaffinization and rehydration, the sections
were incubated in 0.8% pepsin for 10 min at 37C and
442 K Matsuda et al.
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 2
treated with proteinase for 5 min at room temperature.
Sections were immersed in 0.3% H
2
O
2
in methanol
for 20 min, and denatured and hybridized in a
humidified chamber at 37C for 60 min. Detection of
hybridized probe was performed with the GenPoint
Tyramide Signal Amplification System for Biotiny-
lated Probes (Dako North America, Inc.), with applica-
tion of primary peroxidase-conjugated streptavidin,
biotinyl-tyramide secondary peroxidase-conjugated
streptavidin, and the chromogenic substrate diam-
inobenzidine (DAB). The slides were counterstained
with haematoxylin. Evaluation of the HPV ISH signals
was performed according to the criteria described
previously.
21–23
In brief, the patterns were as follows:
(i) a diffuse pattern representing episomal HPV that
correlates with viral replication; (ii) a punctate pattern
consisting of one or a few discrete signals in the
nucleus, indicating HPV integration into the cellular
genome; and (iii) a mixed pattern with separate areas
containing only integrated or episomal copies, and
areas where the integrated virus was hidden in
episomal HPV copies.
immunofluorescence for 53bp1 expression
After antigen retrieval with microwave treatment in
citrate buffer, deparaffinized sections were preincubated
with 10% normal goat serum. Tissue sections were
then reacted with anti-53BP1 rabbit polyclonal anti-
body (Bethyl Labs, Montgomery, TX, USA) at a 1:200
dilution. The slides were subsequently incubated with
Alexa Fluor 488-conjugated goat anti-rabbit antibody
(Invitrogen, Carlsbad, CA, USA). Specimens were
counterstained with 4¢,6-diamidino-2-phenylindole
dihydrochloride (DAPI-I; Vysis, Downers Grove, IL,
USA), analysed, and photographed with a High Stan-
dard All-in-One Fluorescence Microscope (Biorevo BZ-
9000; Keyence Japan, Osaka, Japan). Signals were
analysed at a ·1000 magnification.
evaluation of immunofluorescence results
A human cervical cancer cell line, HeLa, was also
analysed by immunofluorescence for 53BP1 expres-
sion. HeLa cells were cultured in RPMI-1640 medium
A B
Stable Low DDR High DDR Large foci
Figure 1. A, four types of p53-binding protein 1 (53BP1) expression found in HeLa cells: (1) stable type faint and diffuse nuclear staining;
(2) low DNA damage response (DDR) type one or two discrete nuclear foci (NF); (3) high DDR type three or more discrete NF; and (4) large
NF type discrete NF that are larger than 1.0 lm. B, measurements of the size of NF in a cell by use of a High Standard All-in-One
Fluorescence Microscope (Biorevo BZ-9000; Keyence Japan). 1, 1.01 lm; 2, 1.05 lm; 3, 0.53 lm.
53BP1 nuclear foci in cervical cancer 443
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 3
supplemented with 10% fetal bovine serum, and grown
at 37C in a 5% CO
2
95% air environment. After
fixation with methanol and preincubation with 10%
normal goat serum, cultured cells were treated accord-
ing to the same immunofluorescence method as men-
tioned above. The pattern of 53BP1 immunoreactivity
was classified into four types (Figure 1): (1) stable type
faint and diffuse nuclear staining; (2) low DDR type
one or two discrete NF; (3) high DDR type three or
more discrete NF; and (4) large NF type discrete NF
that are larger than 1.0 lm. The percentage of cervical
epithelial cells expressing each type of 53BP1 immu-
noreactivity was calculated in each cervical lesion.
dual immunofluorescence for 53bp1
expression and ish to detect hr-hpv
To assess the association between localization of 53BP1
NF and HR-HPV integration into the host genome in
neoplastic cells, dual immunofluorescence for 53BP1
expression and ISH to detect HR-HPV were performed
on the same tissue sections. Each section was first
labelled by immunofluorescence, subjected to ISH as
mentioned above, and photographed with a High
Standard All-in-One Fluorescence Microscope (Biorevo
BZ-9000; Keyence Japan). The images obtained were
merged and analysed by use of the accompanying
image analysis software with Biorevo BZ-9000.
immunohistochemistry for p16
ink4a
expression
After immersion in 0.3% H
2
O
2
methanol, sections
were preincubated with 10% normal goat serum. After
antigen retrieval, tissues were incubated overnight at
4C purified mouse anti-human p16 (BD Biosciences
Pharmingen, San Diego, CA, USA) at a 1:300 dilution.
The slides were subsequently incubated with biotiny-
lated goat anti-rabbit antibody for 1 h at room
temperature, and then avidin–peroxidase, and then
visualized with DAB.
double-label immunofluorescence
We also carried out double-label immunofluorescence
staining for 53BP1 and Ki67 expression to clarify the
association between type of 53BP1 expression and
cyclingecells. For double staining, tissues were incu-
bated with a mixture of rabbit anti-53BP1 and mono-
clonal mouse anti-Ki67 (MIB-1; DakoCytomation)
antibodies at 1:50 dilutions, and subsequently incu-
bated with a mixture of Alexa Fluor 488-conjugated
goat anti-rabbit and Alexa Fluor 546-conjugated goat
anti-mouse antibodies. Specimens were counterstained
with DAPI-I (Vysis), and visualized and photographed
with a High Standard All-in-One Fluorescence Micro-
scope (Biorevo BZ-9000; Keyence Japan). Signals were
analysed at a ·1000 magnification.
statistical analysis
The Jonckheere–Terpstra test was used to assess
associations between the type of HPV ISH signal
(negative, diffuse, mixed diffuse and punctate, and
punctate) or the type of 53BP1 expression (stable, low
DDR, high DDR, and large NF) and histological grade of
cervical neoplasia. Associations between the types of
53BP1 expression and HPV ISH signal were also
assessed with the Jonckheere–Terpstra test. The PHREG
procedure in the SAS 8.2 software (SAS Institute, Cary,
NC, USA) was utilized for calculation. All tests were
two-tailed, and a P-value of <0.05 was accepted as
statistically significant.
Table 1. Summary of high-risk human papillomavirus (HPV) in-situ hybridization (ISH) signal in the subjects used in this study
n
Mean age
[years (range)]
Type of HPV ISH signal, no. (%)
Negative Diffuse Mixed Punctate
Normal 11 42 (36–45) 11 (100) 0 0 0
CIN1 16 39 (18–68) 9 (56.3) 3 (18.8) 2 (12.5) 2 (12.5)
CIN2 15 36 (27–50) 3 (20) 3 (20) 3 (20) 6 (40)
CIN3 24 38 (28–62) 1 (4.2) 3 (12.5) 9 (37.5) 11 (45.8)
SCC 14 57 (31–76) 0 1 (7.1) 4 (28.6) 9 (64.3)
CIN, cervical intraepithelial neoplasia, SCC, squamous cell carcinoma.
P < 0.0001, Jonckheere–Terpstra test.
444 K Matsuda et al.
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 4
Results
hr-hpv ish signal in cervical lesions
The ISH results for each type of cervical lesions are
summarized in Table 1. Representative types of ISH
signals are illustrated in Figure 2. All normal cervical
epithelium cases were negative for HPV ISH signals. Of
the 16 CIN1 cases, nine (56.3%) expressed no signals,
three (18.8%) expressed the diffuse type, and two
(12.5%) were of the mixed and punctate type. Of the 15
CIN2 cases, three (20%) were of the negative and
diffuse type, three (20%) were of the mixed type, and
six (40%) were of the punctate type. Of the 24 CIN3
cases, only one (4.2%) was negative, three (12.5%)
were of the diffuse type, nine (37.5%) were of the mixed
type, and 11 (45.8%) were of the punctate type. All of
the 14 SCC cases were positive for HPV ISH signal: four
(28.6%) were of the mixed type and nine cases (64.3%)
were of the punctate type, whereas only one (7.1%)
expressed the diffuse type. Statistical analysis revealed
significant associations between type of HPV ISH signal
and type of cervical lesion (P < 0.0001). The incidence
of mixed and punctate types significantly increased in
the order of normal, CIN1, CIN2, CIN3, and SCC.
53bp1 expression in cervical lesions
The results of the immunofluorescence staining pat-
terns for 53BP1 in cervical lesions are presented in
Table 2, and typical examples are depicted in Figure 3.
Quantitation of the signals revealed immunopositivity
in 4877 nuclei (443.4 nuclei per case) in normal
cervical epithelium, 10 483 nuclei (655.2 nuclei per
case) in CIN1, 9976 nuclei (665.1 nuclei per case) in
CIN2, 14 953 nuclei (623.0 nuclei per case) in CIN3,
and 7979 nuclei (569.9 nuclei per case) in SCC. In the
normal cervical epithelium, 95.8% of nuclei showed
the stable type, and only 4.3% of nuclei showed the
DDR type. In CIN1, 73.4% of nuclei showed the stable
type, and 25.2% and 1.4% of nuclei showed the DDR
and large NF types, respectively. In CIN2, 58.4% of
nuclei showed the stable type, and 38.1% and 3.5% of
nuclei showed the DDR and large NF types, respec-
tively. In CIN3, 33.7% of nuclei showed the stable type,
and 58.5% of nuclei showed DDR types, including
37.5% with the high DDR type and 7.8% with the large
NF type. Finally, in SCC, only 18.3% of nuclei showed
the stable type, and 65.6% of nuclei showed DDR types,
including 49.8% with the high DDR type and 16.2% of
nuclei with the large NF type. The statistical analysis
ABC
Figure 2. In-situ hybridization signals of high-risk human papillomavirus in cervical lesions. Signal types were classified as diffuse [A,
cervical intraepithelial neoplasia (CIN)1], mixed (B, CIN2), or punctate (C, CIN3). Arrows in the inset of B indicate punctate signals.
Table 2. Results for type of p53-binding protein 1 (53BP1) expression in cervical lesions by immunofluorescence
n
Counted
nuclei
Type of 53BP1 expression (%)
Stable Low DDR High DDR Large foci
Normal 11 4877 95.8 3.1 1.2 0.0
CIN1 16 10 483 73.4 15.0 10.2 1.4
CIN2 15 9976 58.4 19.0 19.1 3.5
CIN3 24 14 953 33.7 21.0 37.5 7.8
SCC 14 7979 18.3 15.8 49.8 16.2
CIN, cervical intraepithelial neoplasia; DDR, DNA damage response; SCC, squamous cell carcinoma.
P < 0.0001, Jonckheere–Terpstra test.
53BP1 nuclear foci in cervical cancer 445
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 5
revealed that the histological type of cervical neoplasm
was significantly associated with type of 53BP1
expression (P < 0.0001).
association between 53bp1 nf and hr-hpv ish
signals in cervical epithelium
Associations between types of 53BP1 expression and
HR-HPV ISH signal in the cervical epthelium are
summarized in Table 3. In the cervical epithelium with
negative HR-HPV ISH signal, 78.5% of nuclei showed
the stable type, and 20.5% of nuclei showed the DDR
type. In the cervical epithelium with the diffuse type of
HR-HPV ISH signal, 55.6% of nuclei showed the stable
type, and 42.1% and 2.4% of nuclei showed the DDR
and large NF types, respectively. In the cervical
epithelium with the mixed type of HR-HPV ISH signal,
39.3% of nuclei showed the stable type, and 51.2% and
9.6% of nuclei showed the DDR and large NF types,
respectively. Finally, in the cervical epithelium with the
A
B
C
D
E
F
Figure 3. Immunofluorescence for p53-binding protein 1 (53BP1) expression in cervical lesions. Normal cervical epithelium (A) expressed
the stable type of staining, with rare, one or two nuclear foci (NF), whereas cervical intraepithelial neoplasia (CIN)1 (B) and CIN2 (C)
occasionally expressed three or more NF in dysplastic cells at the basal layer. CIN3 showed several discrete NF (D) with occasional large
foci (E) throughout the epithelium. Squamous cell carcinoma (F) showed several discrete NF including large foci.
Table 3. Association between types of p53-binding protein 1 (53BP1) expression and human papillomavirus (HPV) in-situ
hybridization (ISH) signal in cervical epithelium
Type of HPV ISH signal n
Type of 53BP1 expression (%)
Stable Low DDR High DDR Large foci
Negative 24 78.5 12.6 7.9 1.0
Diffuse 10 55.6 17.7 24.4 2.4
Mixed 18 39.3 20.4 30.8 9.6
Punctate 28 35.1 18.4 35.9 10.6
DDR, DNA damage response.
P < 0.0001, Jonckheere–Terpstra test.
446 K Matsuda et al.
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 6
punctate type of HR-HPV ISH signal, 35.1% of nuclei
showed the stable type, and 54.3% of nuclei showed
DDR types, including 35.9% with the high DDR type
and 10.6% with the large NF type. The statistical
analysis revealed that the type of HR-HPV ISH signal in
cervical neoplasms was significantly associated with
type of 53BP1 expression (P < 0.0001). The incidence
of the DDR and large NF types significantly increased in
the order of negative, diffuse, mixed and punctate types
of HR-HPV ISH signal.
The results of the dual immunofluorescence staining
for 53BP1 expression and ISH to detect HR-HPV are
shown in Figure 4. In CIN showing the mixed type of
HR-HPV ISH signal, 53BP1 NF and the punctate type
of HR-HPV ISH signal were similarly distributed in the
dysplastic cells at the basal portion in cervical epithe-
lium. Also in SCC showing the punctate type of HR-
HPV ISH signal, 53BP1 NF and the punctate type of
HR-HPV ISH signal were similarly distributed through-
out the cancer cells. However, co-localization of 53BP1
NF and the punctate type of HR-HPV signal was very
rare.
association between 53bp1 and p16
ink4a
expression in cervical lesions
The comparison of 53BP1 expression and p16
INK4a
expression in cervical lesions is shown in Figure 5. The
distribution of 53BP1 NF matched p16
INK4a
overex-
pression in each cervical lesion.
double-label immunofluorescence staining
for
53bp1 and ki67
CIN1 expressed the stable or low DDR type of 53BP1
immunoreactivity, and little Ki67 nuclear staining,
mainly at the basal layer (Figure 6). In contrast, a high
level of the DDR and large NF type of 53BP1 expression
and several nuclei showing Ki67 staining were
observed in CIN3 and SCC cells. Furthermore, double
staining for 53BP1 and Ki67 demonstrated that NF of
53BP1 immunostaining were not co-localized with
Ki67-positive dysplastic cells in CIN3, whereas 53BP1
NF-positive cells frequently expressed Ki67 nuclear
staining in SCC.
Discussion
Ionizing radiation (IR) effectively induces DSBs in
normal cells and activates DDR pathways to maintain
genomic integrity. Although non-irradiated cells typi-
cally show diffuse 53BP1 nuclear staining, many
53BP1 NF are immediately induced after exposure to
a low dose of IR.
13
Schultz et al.
6
showed that the
number of 53BP1 NF induced by IR increased linearly
with the dose of IR. On the other hand, we found that
HeLa cells express occasional discrete 53BP1 NF
without any genotoxic treatments, suggesting the
occurrence of endogenous DSBs in cancer cells. One
manifestation of GIN is induction of endogenous
DSBs;
17
thus, the level of 53BP1 NF formation can be
AB
Figure 4. Dual immunofluorescence for p53-binding protein 1 (53BP1) expression (green) and in-situ hybridization (ISH) signals of high-risk
human papillomavirus (red). The distribution of 53BP1 nuclear foci (NF) was the same as that of the punctate type of ISH signal in both cervical
intraepithelial neoplasia (CIN)1 with the mixed type (A) and squamous cell carcinoma with punctate type (B) of ISH signal. However,
co-localization of 53BP1 NF and the punctate type of ISH signal was very rare. Arrows indicate co-localization of 53BP1 NF and the punctate
type of ISH signal.
53BP1 nuclear foci in cervical cancer 447
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 7
A B
C D
E F
G H
Figure 5. Comparison of p53-binding protein 1 (53BP1) (A,C,E,G) and p16
INK4a
expression (B,D,F,H) in semi-serial sections of
cervical lesions. The distribution of 53BP1 nuclear foci was similar to that of p16
INK4a
overexpression in normal (A,B), cervical
intraepithelial neoplasia (CIN1) (C,D), CIN2 (E,F), and squamous cell carcinoma (G,H).
448 K Matsuda et al.
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 8
considered to be a cytological marker for GIN. The
present study has clearly demonstrated that the num-
ber of discrete NF (DDR type) of 53BP1 in cervical cells
increases with tumour progression, as seen by the
normal–CIN1–CIN2–CIN3–SCC sequence. The number
of discrete NF in the DDR type of 53BP1 NF in the
cervical epithelium seems to increase in precancerous
lesions, and the high DDR type and large NF type of
53BP1 immunoreactivity are predominantly observed
in SCC. Similar results, showing the differences in
53BP1 expression patterns during carcinogenesis, were
obtained in our previous studies on thyroid and skin
tumours resected from patients.
15,16
Therefore, we
propose that immunofluorescence analysis of 53BP1
expression can be a useful tool with which to estimate
the level of GIN as well as the malignant potential of
several human tumours.
The value of p16
INK4a
as a surrogate marker of HR-
HPV and CIN has been well established in recent years,
with studies showing both increased immunoexpres-
sion of p16
INK4a
in dysplastic cervical epithelium and a
positive correlation with HR-HPV infection and the
degree of CIN.
24–30
Cervical SCC develops through a
multistep process that involves replication stress. An
important occurrence in cervical carcinogenesis is
deregulated expression of the HR-HPV oncogenes E6
and E7. Several risk factors for cervical neoplastic
progression are likely to contribute to viral oncogene
deregulation, particularly integration of HR-HPV into
the host genome.
31,32
We found a significant associa-
A
B
C
D
E
F
G
H
I
Figure 6. Double-label immunofluorescence staining for p53-binding protein 1 (53BP1) (green) and Ki67 expression (red) in cervical lesions.
Cervical intraepithelial neoplasia (CIN)1 showed no or a few nuclear foci (NF) of 53BP1 immunoreactivity and little Ki67 nuclear staining,
mainly at the basal layer (AC). CIN3 showed several 53BP1 NF that were independent of Ki67 nuclear staining in cancer cells (DF). Squamous
cell carcinoma showed nuclei with both 53BP1 NF and Ki67 immunoreactivity (GI), suggesting disruption of the DNA damage response
pathway.
53BP1 nuclear foci in cervical cancer 449
2011 Blackwell Publishing Ltd, Histopathology, 59, 441–451.
Page 9
tion between the type of HR-HPV ISH signal and type of
53BP1 expression in the cervical epithelium. The
distribution of 53BP1 NF was similar to that of the
punctate type of ISH signals in both CIN and SCC,
suggesting that viral integration could induce endog-
enous DSBs through the induction of GIN in the host
genome. Furthermore, in cervical lesions, the distribu-
tion of 53BP1 NF was identical to that of p16
INK4a
overexpression; this represents a byproduct of viral
infection, and suggests an association between the
presence of GIN and replication stress that allows
enhancement of cell proliferation and avoidance of cell
death. As co-localization of 53BP1 NF and the punctate
type of HR-HPV ISH signal was rare, most viral
integrated sites might have been bypassed by the host’s
DDR machinery, and integrants may have a growth
advantage over others in a mixed population of cervical
cells.
This study also demonstrated little co-localization of
the DDR type of 53BP1 expression and Ki67 nuclear
staining as a marker for cycling cells in precancerous
lesions. An intact DDR pathway is activated, leading to
cell cycle arrest by p53 activity, so precancerous dys-
plastic cells may still preserve DDR function, at least
partially, and this may induce sporadic regression of
dysplasia. In contrast, cancer cells showing 53BP1 NF
were frequently co-localized with Ki67 staining, sug-
gesting a disruption of the DDR pathway that subse-
quently leads to an irreversible malignant
transformation. Taken together, these findings indicate
that GIN may have already occurred at the precancer-
ous stage during cervical carcinogenesis, and that
increasing GIN based on a disrupted DDR may allow
further accumulation of other genomic alterations,
causing progression to invasive cancer through accel-
eration of cell growth replication stress. Further
research is needed on the molecular mechanism of
the uncoupling of cell cycle progression in the face of
increasing severity of DNA damage at the advanced
phase of carcinogenesis.
In summary, this study has demonstrated a number
of 53BP1 NF in cervical lesions resected from patients
that were similar to those found in irradiated cells with
a DDR pathway activated to eliminate DSBs, suggesting
the occurrence of endogenous DSBs. GIN seems to be
induced at the precancerous stage through integration
of the HR-HPV genome into the host cell genome.
Furthermore, invasive cancers exhibited 53BP1 NF in
cycling cells, which suggested that the disrupted DDR
subsequently led to further amplification of the geno-
mic injury. A recent study on immunohistochemical
detection of DDR-associated molecules, including
53BP1, suggests that GIN is an early event that occurs
in pulmonary hyperplasia prior to changes in the p53
tumour suppressor gene during lung carcinogenesis in
patients, suggesting that GIN may serve as a causative
link between precancer and cancer.
33
Thus, measure-
ment of GIN, a hallmark feature of solid tumours that is
implicated in both the initiation and progression of
cancers, may serve as a valuable molecular marker of
malignant potential. Our recent study also demon-
strated that the detection of 53BP1 NF by immunoflu-
orescence can be a useful histological marker with
which to estimate the malignant potential of thyroid
and skin tumours.
15,16
Thus, we propose that immu-
nofluorescence analysis of 53BP1 expression can be a
useful tool with which to estimate the level of GIN and,
simultaneously, the stage of cancerous progression of
uterine cervical lesions.
Acknowledgements
This work was supported in part through the Nagasaki
University Global Centre of Excellence (COE) pro-
gramme ‘Global Strategic Center for Radiation Health
Risk Control’ and by a Grant-in-Aid for Scientific
Research from the Japanese Ministry of Education,
Science, Sports and Culture (No. 18590334). We also
thank Ms Oozono for her secretarial assistance in
preparing this manuscript.
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    • "We note that patients with oncocytic FA were older than those with conventional FA, consistent with previous findings regarding the general population of patients with oncocytic FA [34] . Although we cannot completely exclude the possibility that age difference contributed to the differential expression of 53BP1, our previous studies indicated that 53BP1 expression pattern is largely influenced by pathological grade and the pattern of neoplasms [22, 23, 27, 35]. Oncocytic cells contain large numbers of mitochondria harboring morphologic, functional, and genetic abnormalities . "
    [Show abstract] [Hide abstract] ABSTRACT: Oncocytic follicular adenomas (FAs) of the thyroid are neoplasms of follicular cell origin that are predominantly composed of large polygonal cells with eosinophilic and granular cytoplasm. However, the pathological characteristics of these tumors are largely unexplored. Both the initiation and progression of cancer can be caused by an accumulation of genetic mutations that can induce genomic instability. Thus, the aim of this study was to evaluate the extent of genomic instability in oncocytic FA. As the presence of p53-binding protein 1 (53BP1) in nuclear foci has been found to reflect DNA double-strand breaks that are triggered by various stresses, the immunofluorescence expression pattern of 53BP-1 was assessed in oncocytic and conventional FA. The association with the degree of DNA copy number aberration (CNA) was also evaluated using array-based comparative genomic hybridization. Data from this study demonstrated increased 53BP1 expression (i.e., “unstable” expression) in nuclear foci of oncocytic FA and a higher incidence of CNAs compared with conventional FA. There was also a particular focus on the amplification of chromosome 1p36 in oncocytic FA, which includes the locus for Tumor protein 73, a member of the p53 family implicated as a factor in the development of malignancies. Further evaluations revealed that unstable 53BP1 expression had a significant positive correlation with the levels of expression of Tumor protein 73. These data suggest a higher level of genomic instability in oncocytic FA compared with conventional FA, and a possible relationship between oncocytic FA and abnormal amplification of Tumor protein 73.
    No preview · Article · Mar 2016 · Endocrine Journal
  • Source
    • "The tumor suppressor gene pRb prevents the cell from replicating damaged DNA, and previous studies have shown that analyzing pRb can be helpful in prediction of CIN2-3 regression [6] [17]. p53 also has some value, but the staining is less reproducible and cannot always distinguish between wild type and mutated p53 [18] [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: Cervical intraepithelial neoplasia grades 2-3 (CIN2-3) are usually treated by cone excision, although only 30% progress to cancer and 6-50% regress spontaneously. Biomarkers predicting CIN2-3 regression would be of great clinical value and could reduce unnecessary cone excision and associated complications. The aim of this study was to investigate whether punch-biopsy derived immunohistochemical biomarkers, local immune response, CIN lesion size and condom use are independently correlated to regression of CIN2-3. Methods: A prospective population-based cohort study of 162 women aged 25-40, with first-time onset diagnosis of CIN2-3 in colposcopy-directed biopsies was carried out. The median biopsy-cone interval was 16 weeks. Regression was defined as CIN1 or less in the cone biopsy. Results: The regression rate was 21% (34/162). pRb>30% in the lower epithelial half was the strongest predictor for regression (30% regression, p<0.0001). If additionally a CIN-lesion was smaller than 2.5mm and CD4+ lymphoid cells in the subepithelial stroma ≤ 195 per 1.04 mm basal membrane, the regression rate was 53%. In CIN-lesions>2.5mm and CD4+-stroma ≤195, consistent condom use increased the regression rate from 13% to 67% (p=0.003). If pRb was ≤30%, the regression rate was low (6%). Conclusion: Biomarkers and CIN lesion length can predict CIN2-3 regression, and might be helpful to identify patients who can increase the regression rate of CIN lesions by consistent condom use.
    Full-text · Article · Sep 2012 · Gynecologic Oncology
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    [Show abstract] [Hide abstract] ABSTRACT: Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.
    Full-text · Article · Aug 2012 · Cell
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