evaluating the clinical importance of Mcm7 and Cdc6
expression in oral squamous cell carcinoma (OSCC) and
precancerous lesions. Materials and Methods: RT-PCR and
immunohistochemistry analysis were performed on 47 frozen
samples and 98 paraffin-embedded samples to evaluate the
mRNA and protein expressions of Mcm7 and Cdc6. Results:
RT-PCR and immunohistochemistry indicated positive
expressions of Mcm7 mRNA and protein in normal oral
mucosa, precancerous lesions and OSCC. Significant
differences were found between all the groups. Cdc6 mRNA
and protein had low expressions in normal oral mucosa but
were highly expressed in precancerous lesions and OSCC.
Mcm7 and Cdc6 expressions in the lymph node metastasis
cases were significantly higher than those of the non-
metastatic carcinomas. Conclusion: High expressions of
Mcm7 and Cdc6 are correlated with the development and
metastasis of OSCC and may become a molecular marker for
the early diagnosis and prognosis prediction for OSCC.
Background: The present study aimed at
A major problem in the management of patients with oral
squamous cell carcinoma (OSCC) and precancerous lesions
is the need for early diagnosis and an exact prediction of
transformative process through dysplasia of the normal
differentiated epithelium to malignant cancer is often
presented as overzealous proliferation. It is possible to study
the developmental mechanisms of oral cancer by studying
the activity of cellular proliferation. A key step in the
regulation of cell proliferation is the control of the initiation
behaviour andprognosis. The consistent
of DNA synthesis. Minichromosome maintenance protein 7
(Mcm7) and cell division cycle 6 (Cdc6) are closely related
proteins that are components of the prereplicative complex
(Pre-RC) (1). They are essential for initiating eukaryotic
DNA replication and serve as useful markers of proliferating
cells (2, 3).
Mcm7 is a member of a family of six structurally related
proteins, Mcm2 to Mcm7, that possess helicase activity (4,
5) and which are evolutionarily conserved in all eukaryotes.
The Mcm2-7 proteins appear to form hetero-hexamers and
play important roles in initiation and elongation during DNA
replication. Mcms have been demonstrated in replicating
cells, but not in quiescent, differentiated or senescent cells
(4-7). Cdc6 is essential for the initiation of DNA replication
and, in combination with other regulatory proteins, also
limits cells to a single round of synthesis per cell cycle. Its
best-characterized function is the assembly of Pre-RC at
origins of replication during the G1-phase of the cell division
cycle. Cdc6 also plays important roles in the activation and
maintenance of the checkpoint mechanisms that coordinate
S-phase and mitosis and recent studies have unveiled its
proto-oncogenic activity. In addition, Cdc6 overexpression
in primary cells may promote DNA hyperreplication and
induce a senescence response similar to that caused by
oncogene activation (8, 9). Cdc6 proteins are highly
expressed in various tumour cells and tumorigenesis is
suppressed through suppression of Cdc6 expression (10-12).
Since down-regulation of Mcm7 and Cdc6 proteins is known
to result in loss of cellular proliferation ability, both proteins
have been applied as biomarkers for G1-staged cells in
different fields for their ability to detect “growth potential”
(13-21). Recent studies have shown that Mcm7 and Cdc6
may also be useful proliferation markers in dysplasia and
carcinoma in various tissues (13-19).
previously been assessed in OSCC together (22, 23), the
aim of this study was to assess the expression of both
proteins in a range of oral lesions to determine their
potential value as proliferation and prognosis markers of
Stomatology, Hospital of Stomatology, Sun Yat-Sen University,
Guangzhou 510055, China. Tel: +86 2083862531/13500020072,
Fax: +86 2083822807, e-mail: Drliaoguiqing@hotmail.com
to: Gui-QingLiao, GuanghuaSchool of
Key Words: Oral squamous cell carcinoma(OSCC), precancerous
lesions, Mcm7, Cdc6.
ANTICANCER RESEARCH 28: 3763-3770 (2008)
Expression of Mcm7 and Cdc6 in Oral Squamous
Cell Carcinoma and Precancerous Lesions
CHONG-JIN FENG1, HUI-JUN LI1, JIAN-NA LI2, YONG-JUN LU2and GUI-QING LIAO3
1Department of Stomatology, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou;
2School of Life Sciences, Sun Yat-Sen University, Guangzhou;
3Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-Sen University, Guangzhou, China
oral lesions, especially for OSCC and to determine
whether they are useful in predicting tumour proliferation
rates and prognosis.
Materials and Methods
Experiments using frozen tissues.
Materials. All the experimental samples were procured from the
First Affiliated Hospital’s Oral-Maxillofacial Surgery Department
of Sun Yat-Sen University and included patients diagnosed with
precancerous lesions (leukoplakia, 15 cases) and OSCC (22 cases,
no radiotherapy or chemotherapy was performed before surgery).
Normal oral mucosa samples were collected from peripheral mucosa
tissues of benign oral tumours from ten consenting patients. All the
samples were pathologically substantiated.
RNA extraction and RT-PCR. The primer sequences were as follows:
β-actin (115 bp), F: 5’-CATTGGCAATGAGCGGTTC-3’and R: 5’-
CCACGTCACACTTCATGATG G-3’; Mcm7 (268 bp), F: 5’-
TTCCACGCACAGTT-3’ and Cdc6 (363 bp), F: 5’-TGCTCTTGA
TCAGGCAGTTG A-3’ and R: 5’-GGCCCGAATGTGTAAAGCA
CT-3’. RNA extraction and cDNA synthesis were performed with
frozen samples. The mRNA levels of the target genes were assessed
with the use of a semiquantitative multiplex RT-PCR protocol.
and R: 5’-GTGACGA
Statistical analysis. The genetic expression level was represented by
the relative expression. The relative expression levels of Mcm7 and
Cdc6 equalled the Mcm7 or Cdc6 grey value of each band/β-actin
grey value of each band. SPSS Software 16.0 was used (SPSS Inc.
Chicago, IL, USA) for statistical analysis; t-tests were performed
on the quantitative data and variance analysis and rank-sum tests
were performed for heterogeneous variance.
Experiments using paraffin-embedded tissues.
Materials. Eighty-eight paraffin-embedded samples of oral
precancerous lesions (leukoplakia) and OSCC collected between
2004 and May 2007 by the Pathology Department of the First
Affiliate Hospital of Sun Yat-Sen University were included for
analysis. The specimens were divided into two groups based on the
histological type: 34 specimens of precancerous lesions (mild,
moderate and severe dysplasia 11, 13, and 10 cases, respectively,
representing 15 males and 19 females; ages 17-82, with an average
age of 51.4 years) and 54 patients with OSCC (29 males, 25
females; ages 21-78, with an average age of 53.1 years).
The specimens were separated based on TNM staging criteria
(Union Internationale Contre Cancer, UICC, 2002): stage I: 7 cases,
stage II: 18 cases, stage III: 18 cases and stage IV: 11 cases; lymph
node metastasis: 29 cases and non-lymph node metastases: 25 cases;
pathological criteria: well-differentiated: 30 cases, moderately-
differentiated: 12 cases and poorly-differentiated: 12 cases. All the
samples were collected before laser therapy, radiotherapy or
chemotherapy. Ten specimens of normal oral mucosa were collected
as above and embedded in paraffin.
Immunohistochemistry. Immunohistochemistry was performed using a
standard streptavidin-biotin-peroxidase-diaminobenzidine immuno-
histochemical technique. The primary reagents included mouse anti-
human Mcm7 monoclonal antibody and mouse anti-human Cdc6
monoclonal antibody (Santa Cruz Biology, Santa Cruz, CA USA;
1:100 dilutions), the positive controls were cervical tumour tissue
specimens and PBS was used in lieu of the first antibody as a negative
control. The Mcm7 immunohistochemistry staining results were
interpreted as positive in the presence of brown-yellow granules in the
nucleus. The Cdc6 results were positive in the presence of brown-
yellow granules in the nucleus or yellow stained cytoplasm. At ×400
magnification, three non-overlapping microscopic fields were
examined and the number of positive cells and the total cells in the
field of view were calculated. A labelling index (LI) was calculated
for Mcm7 and Cdc6 expression. The LI equalled the total number of
positive cells divided by the total number of cells times 100.
Statistical analysis. SPSS software 16.0 (SPSS Inc.) was used for
variance analysis and t-tests to evaluate experimental results and
their relationships with clinical and pathological markers. Statistical
significance was established at a p<0.01 threshold.
RT-PCR. Positive expression of Mcm7 mRNA was observed
in the normal oral mucosa, precancerous lesions and OSCC
with expression levels of 1.932±0.104, 2.448±0.103 and
3.123±0.070, respectively. Significant differences were found
between all the groups (p<0.01). Positive expressions of
Cdc6 mRNA were observed in the normal oral mucosa,
precancerous lesions and OSCC with expression levels of
1.798±0.096, 2.448±0.117, and 2.903±0.118, respectively.
Significant differences were found between all the groups
(p<0.01) (Figure 1).
Immunohistochemical staining characteristics of Mcm7 and
Cdc6. Figure 2 depicts positive Mcm7 protein expression in
the nucleus. In the normal oral mucosa epithelium, positive
staining was distributed in the basal layer. Staining
distribution was generally the same with the mild dysplasia
cases. A progressive increase of positive cells along the
epithelium and towards the middle was observed in cases of
moderate dysplasia. In OSCC, strong positive staining was
primarily distributed around cancer nests as differentiated
basal cells and in poorly differentiated cancer cell bands.
Figure 3 depicts positive Cdc6 protein expression. No
expression was noted in the normal oral mucosa. Some
positive cells were observed only in the basal layer and 1-2
layers above the sub-basal layer in mild dysplasia tissues.
Other layers were negative for expression. Positive cells were
heterogeneously expressed at different levels in the various
stages of dysplasia. Several well-differentiated OSCC
showed staining around cancer nests or cornified pearls in
monolayer or multilayer formation. Positive moderate to
poorly differentiated OSCC exhibited loss of regularity, with
Immunohistochemical expressions of Mcm7 and Cdc6. Mcm7
protein-positive expression was observed in the normal oral
mucosa (8/10), precancerous lesions (30/34) and OSCC
ANTICANCER RESEARCH 28: 3763-3770 (2008)
(51/54), with LI values of 3.6%, 22.3% and 45.9%,
respectively. The average LI values differed significantly
between groups (p<0.01). The LI of the moderate dysplasia
group (13/13) and the severe dysplasia group (10/10) was
22.5% and 37.4%, respectively (p<0.01) and both were
statistically significantly different from the mild dysplasia
group (LI=4.4%, p<0.01). In the OSCC group, the LI values
of Mcm7 were correlated with the N-staging of metastasis
of the lymph nodes (r=0.634, p<0.01) and the clinical
staging of combined TNM criteria (r=0.648, p<0.01), but
were not correlated with patient gender, age, T-staging of the
primary lesion, or histological differentiation (Table Ⅰ).
Cdc6 protein was rarely expressed in the normal mucosa
group (0/10) or the mild dysplasia group (3/11), but was
positively expressed in the moderate-severe dysplasia group
(13/23) and the OSCC group (33/54). The LI values for the
mild dysplasia group, the moderate and severe dysplasia
group and for the OSCC group were 1.5%, 24.7% and
31.2%, respectively (p<0.01). In the OSCC group, LI values
for Cdc6 were also correlated with N-staging (r=0.697,
p<0.01) and clinical staging (r=0.707, p<0.01), but not
correlated with patient gender, age, T-staging of the primary
lesion or histological differentiation (Table Ⅰ).
Feng et al: Mcm7 and Cdc6 Expression in OSCC
Figure 1. Expression analysis of Mcm7 (A) and Cdc6 (B) mRNA in normal oral mucosa (N), precancerous lesions (P) and oral squamous cell
carcinoma(C), 3 samples of each. The β-actin lanes demonstrate equal loading.
Table I. Correlations of Mcm7 and Cdc6 expression to clinicopathological
features of OSCC.
Lymph node status
*G1, well-differentiated; G2, moderately differentiated; G3, poorly
differentiated. Status: UICC, 2002.
ANTICANCER RESEARCH 28: 3763-3770 (2008)
Figure 2. In situ immunohistochemical staining of Mcm7 protein. A, Normal oral mucosa epithelium; B, mild dysplasia; C, moderate dysplasia; D,
E, squamous cell carcinoma; F, cervical cancer, positive control. Magnification: ×200.
Feng et al: Mcm7 and Cdc6 Expression in OSCC
Figure 3. In situ immunohistochemical staining of Cdc6 protein. G, Normal oral mucosa; H, severe dysplasia; I, well-differentiated squamous cell
carcinoma cancer nests;J, K, moderate- to poorly differentiated squamous cell carcinomas;L, cervical cancer, positive control. Magnification:×200.
In this study RT-PCR indicated that mRNA of Mcm7 and
Cdc6 were not or only little expressed in normal mucosa, and
precancerous lesions and OSCC, suggesting that both
proteins may be involved in the occurrence and development
process of OSCC.
Mcm proteins have been shown to be expressed locally in
normal cellular proliferation zones, while in dysplasias and
malignant tumours, they are expressed in most cells and
throughout the entire layer of the epithelium (24). In the
present immunohistochemical examination, Mcm7 expression
in the normal oral mucosa epithelium was mainly centralized
in the basal layer and 1-2 layers above the sub-basal layer,
while absent from other layers, indicating the presence of cell
division and proliferation ability in and above the basal layer.
Mcm7 expression was progressively elevated during the
transformation of normal cells to moderate-severe dysplasia
and invasive carcinoma, suggesting an increasing number of
cells entering the proliferation cycle during tumorigenesis.
proliferative activity and thus the detection of Mcm7
expression may diagnostically assist the determination of
OSCC, particular with regard to precancerous lesions. An
earlier immunohistochemical study of 101 cases of oral cell
smears similarly revealed differences in Mcm2 and Mcm5
expression in oral normal mucosa, different levels of
dysplasia and carcinogenesis (22).
The present results also indicated that Mcm7 expression
was correlated with metastasis of lymph nodes and clinical
TNM staging in the patients with oral cancer, suggesting that
highly expressed Mcm7 may indicate a poor prognosis.
Szelachowska et al. (23) in their retrospective study also
indicated the possibility of Mcm2 as a prognostic factor in
Cdc6 protein level, to a certain degree, reflected cellular
proliferation activity, but compared to Mcm7, the Cdc6
protein-positive expression was lower in all the samples.
Even though Cdc6 expression was progressively elevated
during normal-moderate-severe dysplasia and invasive
carcinogenesis, a lack of staining did not necessarily
indicate low proliferative activity due to the high negative
rates of the methodology. The results suggest that it would
be difficult to employ Cdc6 as a marker of OSCC and
precancerous lesions in clinical pathology. However, high
expression of Mcm7 was usually present in most of the
samples with positive Cdc6 expression. Therefore, Cdc6
may be valuable with Mcm7 in the early diagnosis and
prognosis of OSCC. This study also suggested a
correlation between Cdc6 expression and metastasis of
lymph nodes and clinical staging of TNM in the oral
expressionwas significantly up-regulatedin
Mcm7 proteinlevel directlyreflectedcellular
In conclusion, high expressions of Mcm7 and Cdc6 are
suggesting that both may act as important factors in OSCC
and metastasis to the lymph nodes. Expression and LI values
of both proteins may help distinguish normal tissues from
dysplasia and tumour lesions and, to a certain extent, predict
metastasis to the lymph nodes. Therefore, the Mcm7 and
Cdc6 expression may potentially become molecular markers
for the early diagnosis and prognosis prediction of OSCC.
This work was supported in part by the Guang Dong Province
Cooperative Foundation of the School of Life Sciences and the First
Affiliated Hospital, Sun Yat-Sen University (162010).
Project Foundation (2006B60501019) and the
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Received June 30, 2008
Revised September 8, 2008
Accepted September 30, 2008
Feng et al: Mcm7 and Cdc6 Expression in OSCC