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Esophageal squamous cell carcinoma (ESCC) is one
of the main types of esophageal carcinoma, arising from
the epithelial cells that line the esophagus. It is common
in developing countries and has a relatively high frequen-
cy in China [1], accounting for 90% of all esophageal car-
cinoma cases [2]. Risk factors for ESCC include tobacco,
alcohol, hot drinks, and poor diets. The current general
therapeutic methods rely on surgery, like endoscopic
mucosal resection for early stages of ESCC, and require
the assistance of chemotherapy with or without radiation
therapy for some more severe cases [3]. Adjuvant
chemotherapy with cisplatin, vindesine, and bleomycin
has been applied on esophageal carcinoma patients [4],
which exploits the effects of inducing DNA damage to
accelerate cancer cell death [5].
Many proteins respond to DNA damage and partic-
ipate in the DNA damage repair process, among which
mediator of DNA damage checkpoint 1 (MDC1) plays
important roles in early stages. Upon DNA damage,
MDC1 not only itself is recruited to the double-strand
break site, but also helps to recruit other proteins related
to DNA damage repair [6]. It was reported that the down-
regulation of MDC1 accelerates DNA damage-induced
cell apoptosis [7]. Minichromosome maintenance
(MCM) complex, a heterohexamer composed of
MCM2-7, is recruited to DNA replication sites after the
anchor of origin recognition complex (ORC) [8], thus it
is also required for DNA damage repair [9]. These DNA
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM16-122, September 12, 2016.
1221
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
Knockdown of Minichromosome Maintenance Proteins
Inhibits Foci Forming of Mediator of DNA-Damage Checkpoint 1
in Response to DNA Damage in Human Esophageal
Squamous Cell Carcinoma TE-1 Cells
Jinzhong Yu1#, Ruijie Wang2#*, Jinfeng Wu3, Zhongqin Dang1, Qinsheng Zhang1, and Bo Li1
1Henan Province Hospital of TCM, Department of Gastroenterology, 450002 Zhengzhou, Henan, China2Henan University of Chinese Medicine, 450008 Zhengzhou, Henan, China; E-mail: [email protected]
3Shenzhen Luohu People’s Hospital, Department of Gastroenterology, Shenzhen, 518001 Guangdong, China
Received May 11, 2016
Revision received July 1, 2016
Abstract—Esophageal squamous cell carcinoma (ESCC) has a high morbidity in China and its treatment depends greatly on
adjuvant chemotherapy. However, DNA damage repair in cancer cells severely affects the outcome of treatment. This study
investigated the potential mechanism regarding mediator of DNA-damage checkpoint 1 (MDC1) and minichromosome
maintenance proteins (MCMs) during DNA damage in ESCC. Recombinant vectors of MDC1 and MCMs with tags were
constructed and transfected into human ESCC cell line TE-1. Immunoprecipitation and mass spectrometry were performed
to screen the MCMs interacting with MDC1, and direct interaction was confirmed by glutathione S-transferase (GST) pull-
down assay in vitro. MCM2 and MCM6 were knocked down by shRNAs, after which chromatin fraction and foci forming
of MDC1 upon bleomycin-induced DNA damage were examined. The results showed that MCM2/3/5/6 were immuno-
precipitated by antibodies against the tag of MDC1 in TE-1 nuclei, and the GST pull-down assay indicated the direct inter-
action. Knockdown of MCM2 or MCM6 reduced the chromatin fraction of MDC1 according to Western blot results.
Moreover, knockdown of MCM2 or MCM6 could significantly inhibit foci forming of MDC1 in TE-1 nuclei in response
to bleomycin-induced DNA damage (p < 0.001). This study indicates the direct interaction between MDC1 and MCMs in
TE-1 nuclei. Downregulation of MCMs can inhibit chromatin fraction and foci forming of MDC1 in TE-1 cells upon DNA
damage, which suggests MCMs and MDC1 as potential targets to improve the outcome of chemotherapy in ESCC.
DOI: 10.1134/S0006297916100205
Key words: esophageal squamous cell carcinoma (ESCC), DNA damage repair, mediator of DNA-damage checkpoint 1
(MDC1), minichromosome maintenance protein (MCM)
1222 JINZHONG YU et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 10 2016
repair molecules respond to DNA damage and constitute
a network to enable cells to survive.
Like normal cells, cancer cells exhibit ability for
DNA damage repair and have developed their specific
DNA damage repair mechanisms [10]. However, DNA
damage repair in cancer cells severely affected the out-
come of chemotherapy in various diseases including
ESCC [11, 12]. Thus, it is imperative to find means for
effective modulation of DNA damage repair to suppress
the survival of ESCC cells during chemotherapy.
This study investigated the potential mechanism
regarding MDC1 and MCMs during DNA damage in
ESCC. Immunoprecipitation and mass spectrometry in
human ESCC cell line TE-1 and glutathione S-trans-
ferase (GST) pull-down assay in vitro were performed to
reveal the interaction between MDC1 and MCMs in cell
nuclei. We further knocked down MCMs by shRNAs to
analyze the involvement of MCMs in the modulation of
MDC1 upon DNA damage. This study adds to under-
standing of the functions of MDC1 and MCMs and pro-
vides potential strategies to improve the outcome of
chemoradiotherapy in ESCC.
MATERIALS AND METHODS
Cells. Human esophageal squamous carcinoma cell
line TE-1 (Institute of Biochemistry and Cell Biology,
China) was used in this study. The cells were cultured and
passaged in Dulbecco’s modified Eagle Medium
(DMEM; Gibco, USA) supplemented with 10% fetal
bovine serum (FBS; Gibco) and incubated in a humidi-
fied atmosphere with 5% CO2 at 37°C. This study was
performed according to the instructions of our institute.
Plasmid construction. The cDNA sequences encod-
ing the complete open reading frames of human MDC1
(BC152556), MCM2 (GenBank Accession No.
BC017258), MCM3 (BC003509), MCM5 (BC003656),
and MCM6 (BC032374) were amplified by polymerase
chain reaction (PCR) and verified by sequencing. The
HA and FLAG tag sequences were added to the 3′ end of
MDC1 and MCMs by PCR. Then the fusion sequences
were inserted into pcDNA3.1 overexpression vectors
(Thermo Scientific, USA), and the resulting plasmids
were named pcDNA3.1-MDC1-HA, pcDNA3.1-
MCM2-FLAG, pcDNA3.1-MCM3-FLAG, pcDNA3.1-
MCM5-FLAG, and pcDNA3.1-MCM6-FLAG. The
plasmids were purified with Plasmid Purification Kit
(Qiagen, China) according to the manufacturer’s instruc-
tions.
Cell transfection and treatment. The plasmids were
transfected or co-transfected into TE-1 cells in serum-
free DMEM using Lipofectamine 2000 (Invitrogen,
USA) according to the supplier’s instructions. Before
transfection, the cells were adjusted to confluency of
about 80%. Plasmids of 1 µg were added to each well of
24-well plates. The cells were collected for further analy-
sis at 48 h post-transfection. The TE-1 cells stably
expressing HA-tagged human MDC1 were screened by
neomycin (300 µg/ml).
Similar procedures were conducted on TE-1 cells for
the transfection of the specific shRNAs for MCM2 and
MCM6, sh-MCM2 and sh-MCM6 (Genechem, China),
or the negative control (NC) for shRNAs. Bleomycin
(2 µM) was added to the medium for a treatment of 1 h to
induce DNA damage in TE-1 cells at 48 h post-transfec-
tion. Thus, the TE-1 cells were divided into four groups:
NC, bleomycin ± NC, bleomycin ± sh-MCM2, and
bleomycin ± sh-MCM6.
Chromatin fraction. Chromatin fraction was pre-
pared according to a previous study [8]. Briefly, TE-1
cells stably expressing HA-tagged MDC1 were lysed in
buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl,
10 mM MgCl2, 0.34 mM sucrose, 10% glycerol, 0.2%
Triton X-100, 1 mM dithiothreitol (DTT), and Protease
Inhibitor Cocktail (Roche, Switzerland) for 10 min at
4°C. After centrifugation at 1000 rpm for 5 min, the
supernatants were collected, which contained the cyto-
plasmic fraction. The pellet was resuspended in buffer
containing 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT,
and Protease Inhibitor Cocktail and lysed for 30 min at
4°C. After centrifugation at 1500 rpm for 5 min, the
supernatant was mixed with the cytoplasmic fraction as
the non-chromatin fraction.
The remaining pellet was resuspended in buffer con-
taining 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1%
Nonidet P-40, 1 mM DTT, and Protease Inhibitor
Cocktail for 10 min at 4°C, after which it was sonicated
and centrifuged at 13,000 rpm for 10 min, and the super-
natants were collected as the chromatin fraction.
Immunoprecipitation. To analyze MDC1 and its
potential interactive proteins, immunoprecipitation was
carried out in nucleoprotein samples and the whole cell
extracts (WCE) of TE-1 cells stably expressing HA-tagged
MDC1. The nucleoprotein samples were extracted from
the TE-1 cells using an EpiQuik Nuclear Extraction Kit
(Epigentek, USA) according to the supplier’s instruc-
tions. Immunoprecipitation was performed with a Co-
Immunoprecipitation Kit (Pierce, USA) according to the