Enhanced expression of Mcm proteins in cancer cells derived
from uterine cervix
Yukio Ishimi
1
, Isao Okayasu
2
, Chieko Kato
1
, Hyun-Ju Kwon
1
, Hiroshi Kimura
3
, Kouichi Yamada
4
and Si-Young Song
1
1
Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo;
2
Department of Pathology, Kitasato University School of Medicine,
Sagamihara, Kanagawa;
3
Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University,
Tokyo;
4
National Institute of Health and Nutrition, Tokyo Japan
Minichromosome maintenance proteins (Mcm) 2–7 play
essential roles in eukaryotic DNA replication. Several
reports have indicated the usefulness of Mcm proteins as
markers of cancer cells in histopathological diagnosis.
However, their mode of expression and pathophysiological

helicase in vitro [10–13], suggesting that the Mcm4/6/7
complex acts as a DNA-unwinding enzyme in the replica-
tion. The exact biochemical function of Mcm2, 3, and 5
remains to be determined, but it has been shown that these
proteins can inhibit the helicase activity of Mcm4/6/7 by
disassembling this hexamer [12,14,15], indicating a regula-
tory role. In vivo findings suggest that Mcm2–7 proteins act
as a replicative helicase that is responsible for fork
movement [5,7]. Thus, it is likely that the Mcm2–7 complex
is involved in DNA replication as a DNA helicase, and an
activated form of the Mcm2–7 complex is a Mcm4/6/7
hexamer.
Mcm proteins were identified as a component of the
DNA replication licensing system by which a single round
of DNA replication in a cell cycle is ensured [16–18]. It has
been shown that cyclin-dependent kinase plays a central role
in preventing over-replication [19]. Cdc6, involved in
loading Mcm proteins onto chromatin, is one of the targets
of regulation by the kinase [3]. Recently it has been shown
that deregulation of Cdc6, ORC (origin recognition com-
plex) and Mcm, all of which are targets of phosphorylation
by cyclin-dependent kinase, leads to over-replication in
Saccharomyces cerevisiae [20]. These findings indicate that
Mcm proteins play a role in regulating the replication of
DNA. The gene amplification that has been detected in
various cancer cells [21] is probably generated by the over-
replication of a genomic locus containing replication origins
[22,23]. These notions suggest that the deregulation of DNA
replication contributes to the development of malignant
transformation of cells.

Mcm2-beads prepared by fixing Mcm2 protein to CNBr-
activated Sepharose (Pharmacia). After the loading of
antiserum onto the beads, the antibodies were eluted with
0.2
M
glycine (pH 2.5) and 0.15
M
NaCl. The solution was
neutralized by adding 2
M
Tris/HCl (pH 8.0) to a final
concentration of 100 m
M
. Rabbit anti-Mcm3 serum was
obtained as reported [34] and affinity-purified for immuno-
staining. Anti-Mcm4 Ig were affinity-purified using beads
conjugated with the fragment of mouse Mcm4 (amino acids
683–862) that had been used for immunizing rabbits [34].
Rabbit anti-Mcm5 serum was obtained as reported [35] and
specific antibodies were affinity-purified. Rabbit anti-Mcm6
(sc-9843), mouse anti-Mcm7 (sc-9966) and mouse anti-
PCNA (sc-56) IgG were purchased from Santa Cruz
Biotechnology Inc. Rabbit anti-Ki67 Ig were purchased
from DAKO. Anti-ORC2 Ig were produced as reported [36].
Cells
HeLa cells were cultured in DMEM (Dulbecco’s modified
Eagle’s medium) supplemented with 10% calf serum. WI-38
cells obtained from RIKEN GenBank and SV40-trans-
formed human fibroblasts (GM00637) purchased from
Coriell Cell Repositories were cultured in DMEM supple-

sonicated for 20 s to shear chromosomal DNA before
being loaded onto the SDS gel. Thus, it contained total
cellular proteins. To obtain chromatin-bound proteins, cells
(4 · 10
6
cells) were lysed with solution A as described above
and placed on ice for 15 min. The cell suspension was
centrifuged, and the recovered precipitate was washed once
with solution A. The precipitate was suspended in 0.1 mL of
solution A and then mixed with 0.05 mL of concentrated
SDS sample buffer.
Total cellular proteins and chromatin-bound proteins
were electrophoresed on a 10% acrylamide gel contain-
ing SDS and transferred to a membrane (Immobilon,
Millipore). The membrane was incubated at 37 °Cfor1h
with primary antibodies in a blocking solution (Blockace,
Dai-nippon Pharmaceuticals). After being washed with Tris
buffered saline (TBS; 50 m
M
Tris/HCl, pH 7.5, and 0.15
M
NaCl) plus 0.1% Triton X-100, the membrane was incu-
bated with peroxidase-conjugated anti-rabbit or anti-mouse
secondary antibodies (Bio-Rad). The immunoreacted pro-
teins were detected using a chemiluminescence system
(SuperSignal West Pico or Femto Maximum Sensitivity
Substrate, Pierce), and the level of reactivity was quantified
(Cool Saver AE-6935, Atto).
Immunostaining of cells
Cells cultured on eight-well chambers (Falcon) were pulse-

depleted of methionine (Sigma). Ten microlitres of
[
35
S]methionine (10 mCiÆmL
)1
) was added to the medium,
and incubation was continued for given periods. For pulse
and chase experiments, the cells labeled with [
35
S]methionine
for 2 h were cultured further for different periods in normal
growth medium. The cells were lysed in solution A, and the
precipitate after centrifugation was re-suspended with
solution A. DNase I (Takara) was added to the solution
at a final concentration of 700 unitsÆmL
)1
and the mixture
was incubated at 30 °C for 30 min. After centrifugation, the
supernatant was combined with the first supernatant and
incubated with 1 lg of anti-Mcm4 Ig for 1.5 h at 4 °C.
Protein G-Sepharose (30 lL) was added to the solution,
and the incubation was continued overnight at 4 °C. After
being spun down, the Sepharose beads were washed four
timeswithRIPAbuffer(150m
M
NaCl, 0.5% Nonidet
P-40, 1% Na-deoxycholate, 0.1% SDS, and 50 m
M
Tris/
HCl, pH 7.5) containing proteinase inhibitors and then

1 · 10
5
3 10(8–12) 11(7–15)
4 5(4–7) 6(6–7) 2.5 · 10
6
5.2 · 10
5
57 ND
6 10(5–15) 10(4–14) 2 · 10
6
1.8 · 10
5
7 9(6–10) 13(10–16) 1.5 · 10
6
2.1 · 10
5
ORC2 8
PCNA 1.5(1.4–1.9)
Histones 1.5–2
Fig. 2. Immunostaining of HeLa and WI-38 cells with anti-Mcm4 Ig. Logarithmically growing HeLa (A and B) and WI-38 cells (C and D) in one
section that had been pulse-labeled with BrdU for 15 min were fixed and detected with anti-Mcm4 Ig (A and C) or anti-BrdU Ig (B and D).
1092 Y. Ishimi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Ó FEBS 2003 Mcm expression in cancer cells (Eur. J. Biochem. 270) 1093
Pharmacia Biotech). The cDNA was prepared by RT-PCR
using random primers (ThermoScript RT-PCR System,
GibcoBRL). For the amplification of human Mcm2 cDNA,
5¢-AGACGAGATAGAGCTGACTG-3¢ as a forward
primer and 5¢-CACCACGTACCTTGTGCTTG-3¢ as a
reverse primer were used. Primers for the amplification
of the human glyceraldehyde-3-phosphate dehydrogenase

M
citrate buffer (pH 6.0) at
95 °C for 10 min using a microwave oven to facilitate
antigen retrieval. Following washes in deionized water and
NaCl/P
i
(0.01
M
phosphate buffer pH 7.2 with 0.9% NaCl),
the endogenous peroxidase activity was quenched by
incubation in 0.3% hydrogen peroxide in methanol for
30 min. Sections were then washed in NaCl/P
i
, blocked with
10% normal swine serum in NaCl/P
i
for 30 min and
incubated with each of the following primary antibodies at
4 °C overnight: rabbit anti-Mcm3, anti-Mcm4 and anti-
Ki-67, and mouse anti-PCNA IgG. These antibodies were
diluted in NaCl/P
i
containing 2% normal swine serum
(DAKO). The slides were washed in NaCl/P
i
,andthe
subsequent immunostaining was performed by the labeled
streptavidin biotin-peroxidase method using a kit (LSAB2
kit/HRP, DAKO) following the manufacturer’s protocol.
The coloring reaction was performed using a ready-made

Mcm proteins as a standard, it was calculated that
approximately 1.5–2.5 · 10
6
molecules of Mcm2, 4, 6 and
7 proteins are present in a single HeLa cell on average and
0.1–0.5 · 10
6
molecules in a single WI-38 cell (Table 1).
While total cellular proteins from these cells appeared to
be detected at a comparable level (Fig. 1), the amount
of histone was slightly enriched in HeLa cells compared to
WI-38 cells, which may be consistent with evidence of
increased chromosomal ploidy in HeLa cells. The level of
PCNA, a factor required for processivity of DNA poly-
merase d and e, was comparable between HeLa cells and
WI-38 cells. ORC2, a subunit of the ORC1-6 complex that
is required for loading Mcm proteins onto chromatin, was
detected at eight times the level in HeLa cells as in WI-38
cells. These results suggest that Mcm2–7 and ORC2
proteins are expressed at particularly high levels in HeLa
cells compared to WI-38 cells. As the differences of Mcm
expression between HeLa and WI-38 cells could be due to
their different growth rates, we examined the percentage of
Fig. 3. Mcm proteins in SV40-transformed human fibroblast
(GM00637) and WI-38 cells. Mcm2–7 proteins, ORC2 and PCNA
were detected in total cellular proteins (A) and the chromatin-bound
fraction (B) from GM and WI-38 cells as in Fig. 1. Total proteins
including histones were also detected by Coomassie Brilliant Blue
staining.
Table 2. Quantitation and comparison of proteins in GM and WI-38

-negative HeLa cells, more intense staining was observed in
BrdU-positive WI-38 cells. These data suggest that the level
of Mcm4 protein was maximized at the S phase in WI-38
cells and that the Mcm4 expression in HeLa cells is
maintained irrespective of cell cycle.
Next, we compared the concentration of Mcm proteins in
cell lysate prepared from SV40-transformed human fibro-
blasts (GM00637) and normal human fibroblast WI-38 cells
(Fig. 3). Mcm2–7 proteins were detected in GM cells at 3–9
times the level found in WI-38 cells for total cellular proteins
and at 5–12 times the level for chromatin-bound proteins
(Fig. 3 and Table 2). In contrast, the amounts of total
Fig. 4. Abundance of Mcm2 mRNA in HeLa and WI-38 cells. (A) Total mRNA was purified from HeLa and WI-38 cells, and the concentration of
Mcm2 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was determined by RT-PCR. Increasingly larger volumes of the mRNA
fractions were added to the reaction as indicated. The amplified glyceraldehyde-3-phosphate dehydrogenase cDNA fragment was detected by
staining with ethidium bromide, and the Mcm2 cDNA fragment was detected by hybridizing with the same labeled fragment. These fragments are
indicated. (B) Total RNA was purified from HeLa and WI-38 cells and analyzed by Northern blot analysis. Increasing volumes (0.7, 1.5 and 3 lL
each) of the total RNA were loaded onto the gel. Mcm2 and G3PDH mRNA were detected with specific probes using the same filter. The
electrophoresed RNA (2 lL each) was stained with ethidium bromide (EtBr) to detect ribosomal RNAs.
Ó FEBS 2003 Mcm expression in cancer cells (Eur. J. Biochem. 270) 1095
cellular proteins and core histones were comparable
between these two cells (Fig. 3), in spite of evidence that
half of the GM cells were tetraploids. ORC2 and PCNA
were detected in GM cells at two and four times the levels of
those in WI-38 cells, respectively. Thus, on comparing the
two human fibroblasts, it was found that Mcm proteins are
expressed at higher levels in transformed GM cells than in
normal WI-38 cells. We also observed a higher level of Mcm
expression in SV40-transformed WI-38 cells (VA-13 cells)
(2–3 times the level), human osteosarcoma (approximately

samples loaded on the gel indicated that Mcm2 RNA is 4–8
times more abundant in HeLa cells than in WI-38 cells. In
contrast, the concentrations of glyceraldehyde-3-phosphate
dehydrogenase mRNA and ribosomal RNA were compar-
able in these two fractions. These results indicate that Mcm2
mRNA is approximately four times more abundant in
HeLa than in WI-38 cells.
Southern blot analysis of genomic DNA purified from
HeLa and WI-38 cells was performed using a probe from an
exon of the Mcm2 gene that codes for amino acids 214–287.
Expected bands were detected from HeLa and WI-38 DNA,
although the intensity of a BamH1 fragment decreased and
an additional EcoRI fragment was detected in WI-38 DNA
(data not shown). The results suggest that the Mcm2 gene is
not amplified in HeLa cells but there is some alteration of
the Mcm2 gene structure in WI-38 cells.
Protein synthesis of Mcm4
To further clarify why Mcm proteins are more abundant in
HeLa cells than in WI-38 cells, we compared the synthesis of
Mcm4 protein between the two cell lines. After pulse-
labeling with [
35
S]methionine, Mcm4 protein was immuno-
precipitated. The immunoprecipitates were stringently
washed, and bound proteins were analyzed on SDS gel
Fig. 5. Synthesis and stability of Mcm4 protein. (A) HeLa and WI-38
cells were labeled with [
35
S]methionine for 0.5 or 2 h in medium
depleted of methionine. The total cell extracts were prepared, immu-

medium for 2, 4, 7 and 22 h. The cell extracts were prepared
and immunoprecipitated using anti-Mcm4 Ig, and bound
proteins were analyzed on a SDS gel (Fig. 5B). Constant
amounts of Mcm4 protein were recovered from different
cell extracts (data not shown). The intensity of the Mcm4
band was clearly decreased at 22 h chase in both HeLa and
WI-38 cells, indicating the presence of turnover of the
protein. Quantitation of the Mcm4 band suggested that the
protein stability of Mcm4 does not greatly differ between
HeLa and WI-38 cells (Fig. 5C).
Expression of Mcm3 and 4 proteins
in malignant tissues
Next, we compared the expression of Mcm4 protein in cells
from malignant tissues (Fig. 6). The expression of Mcm4 as
well as two other proliferation marker proteins, PCNA and
Ki67 [39], was examined in five cases of human uterine
cervical cancer by immunohistochemical techniques, and
the results were compared with the findings obtained by
hematoxylin and eosin staining. We examined the expres-
sion of these proteins in a cancer-free layer of squamous
epithelial cells, carcinoma in situ (CIS) and invasive cancer,
all of which were observed in the same section. In the
cancer-free squamous cell epithelial layer, the expression of
Mcm4, PCNA and Ki67 was observed mainly in the basal
cell layer (Fig. 6A). The immunoreactivity varied among the
cells, and strongly immunopositive cells were scattered
along the epithelial layer. Ki67-immunopositive cells were
scarce compared with Mcm4- or PCNA-immunopositive
cells. In cells of the CIS lesion, the expression of Mcm4 and
PCNA was diffuse, and almost all cancer cells were

cell epithelial layer (Fig. 7). The ubiquitous nature of the
expression of both Mcm proteins in cancer cells was also
clear compared with the localized expression in dysplastic
lesions. These results suggest that Mcm3 and 4 proteins are
expressed more ubiquitously in cancer cells than in prolif-
erative cells from normal uterine cervix or cells with
dysplasia. Their expression is probably more abundant in
cancer cells than in normal proliferative cells, although it is
difficult to compare these results quantitatively.
Discussion
We showed that Mcm2–7 proteins were expressed at higher
levels in HeLa cells and SV40-transformed human fibro-
blasts than in normal human fibroblast WI-38 cells, albeit
the total proteins and histone were present at comparable
levels. The higher level of Mcm expression was detected not
only in total cellular proteins but also in chromatin-bound
proteins. The Mcm proteins bound to chromatin probably
function in DNA replication, but the role of the proteins not
tightly bound to chromatin remains to be determined.
Consistent with the results at the protein level, semiquan-
titative PCR and Northern blot analyses suggest that Mcm2
mRNA is approximately four times more abundant in
HeLa cells than in WI-38 cells. Synthesis of Mcm4, 6 and 7
proteins was accelerated in HeLa cells compared to WI-38
cells, but the turnover rate of the Mcm4 protein within 22 h
did not differ between these two cells. Thus, the enhanced
expression of Mcm proteins in HeLa cells may be explained
by the abundance of mRNA. However, it should also be
noted that the level of Mcm4 protein varies among
logarithmically growing WI-38 cells (Fig. 2), suggesting

noted that the results suggest that Mcm proteins are
expressed at high levels in cancer cells derived from
uterine cervix and in HeLa cells.
The expression of Mcm genes in growth-arrested cells is
induced by addition of serum [43]. There are several E2F
binding sites in the promoter region of the Mcm5, 6,and7
genes, and transcription of the genes seems to be regulated
by an E2F transcription factor [44–46]. In HeLa cells, the
human papilloma E7 oncogene product binds to hyper-
phosphorylated Rb to destabilize the Rb/E2F complex ([47]
and references therein), and thereby several genes, including
Mcm, whose expression is dependent on E2F may be
activated [43]. Thus, it is possible that the human papilloma
oncogene products are involved in the higher level of Mcm
expression in HeLa cells and cancer cells from human
uterine cervix.
Although it remains to be determined how enhanced
expression of Mcm proteins affects DNA replication in
cancer cells, the following findings suggest a possible
involvement of Mcm in the malignant transformation.
The human Mcm5 gene has been identified as one of the
cancer-related genes linked to hepatitis B virus-induced
carcinogenesis [48]. The Mcm7 gene has also been identified
as a gene whose expression is up-regulated in colon cancer
metastasis [49]. Recently, it has been reported that Mcm7 in
neuroblastoma is a direct target of the MYCN transcription
factor that binds to an E-box element in the Mcm7
promoter [50]. In conclusion, the present study as well as
previous reports shed light on the biological significance of
Mcm proteins in the aberrant proliferation of cancer cells.

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