Tài liệu Báo cáo khoa học: Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix docx

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

significance in cancer cells remain to be clarified. We com-
pared the level of expression of Mcm proteins among human
HeLa uterine cervical carcinoma cells, SV40-transformed
human fibroblast GM00637 cells and normal human fibro-
blast WI-38 cells. All the proteins examined were detected in
HeLa and GM cells at 6–10 times the level found in WI-38
cells on average. This increase was observed both in total
cellular proteins and in the chromatin-bound fraction.
Consistently, Mcm2 mRNA was enriched in HeLa cells to
approximately four times the level in WI-38 cells, and the
synthesis of Mcm4, 6 and 7 proteins was accelerated in HeLa
cells. Immunohistochemical studies of surgical materials
from human uterine cervix showed that Mcm3 and 4 are
ubiquitously expressed in cancer cells. Further, the positive
rate and level of Mcm3 and 4 expression appeared to be
higher in cancer cells than in normal proliferating cells of the
uterine cervix and dysplastic cells, suggesting that they can be
useful markers to distinguish these cells.
Keywords: cancer cells; DNA replication; Mcm; protein
expression; uterine cervix.
The entire Mcm family (Mcm2–7) is essential for eukaryotic
DNA replication [1–4], playing roles in the initiation and
elongation of DNA replication [5]. Mcm2–7 proteins
constitute the prereplicative complex that is formed at the
replication origin [6,7]. Among several Mcm complexes,
only the Mcm2–7 hexamer has the ability to induce DNA
replication in Xenopus egg extracts [8]. All family members
have a DNA-dependent ATPase motif in the central
domain [9]. However, it has been reported that Mcm4, 6,
and 7 form a hexameric complex and function as a DNA

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.

Recently, several groups have reported that Mcm
proteins are more frequently detected in cells from malig-
nant tissues than those from normal tissues [24–33]. This
phenomenon was also observed in dysplastic cells, suggest-
ing that Mcm proteins are a good indicator of proliferative
or cancer cells in malignant tissues [28]. Elucidation of how
the expression of Mcm protein changes relates to malignant
transformation of cells and the pathophysiological signifi-
cance of those changes awaits further studies. In this paper,
we report that Mcm proteins are expressed at higher levels
in the transformed cell lines than in normal fibroblasts. We
also found more frequent and higher-level expression of
Correspondence to Y. Ishimi, Mitsubishi Kagaku Institute of Life
Sciences, 11 Minamiooya, Machida, Tokyo 194–8511, Japan.
Fax: + 81 42 724 6314, Tel.: + 81 42 724 6266,
E-mail:
Abbreviations: BrdU, bromodeoxyuridine; CIS, carcinoma in situ;
Mcm, minichromosome maintenance protein; ORC, origin
recognition complex.
(Received 24 September 2002, revised 16 December 2002,
accepted 20 December 2002)
Eur. J. Biochem. 270, 1089–1101 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03440.x
Mcm3 and 4 in cancer cells than in normal proliferating cells
of human uterine cervix and dysplastic cells. The results
suggest that enhanced expression of Mcm proteins plays a
role in the malignant transformation of cells.
Materials and methods
Antibodies
Rabbit anti-Mcm2 serum was prepared using mouse Mcm2
protein as an antigen, and the antibodies were purified with

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-

mented with 10% fetal calf serum. WI-38 cells at a
population doubling level of 33–38 were used for experi-
ments and had almost stopped proliferating at approxi-
mately 41 population doubling level.
Fractionation of cell extracts and Western-blot analysis
of Mcm proteins
Cells (4 · 10
6
cells) were lysed in 0.2 mL of CSK buffer
(10 m
M
Pipes, pH 6.8, 100 m
M
NaCl, 1 m
M
MgCl
2
,1m
M
EGTA, 1 m
M
dithiothreitol and 1 m
M
phenylmethanesulfo-
nyl fluoride) containing 0.1% Triton X-100, 1 m
M
ATP
and proteinase inhibitors (Pharmingen; solution A) [37].
The suspension was mixed with 0.1 mL of 3· concentrated
sample buffer for SDS gel electrophoresis and then

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-

labeled with 20 l
M
bromodeoxyuridine (BrdU) for 15 min.
After being washed with phosphate-buffered saline (Dul-
becco’s NaCl/PBS–, Nissui), the cells were fixed by incuba-
tion with 4% paraformaldehyde in NaCl/P
i
for 5 min at
room temperature. The cells were washed with NaCl/P
i
,and
then permeabilized and blocked by incubation with 0.1%
Triton X-100, 0.02% SDS and 2% nonfat dried milk in
NaCl/P
i
for 1 h at 37 °C. Incubation of the cells with anti-
Mcm4 rabbit Ig was performed for 1 h at 37 °Cinthe
above blocking solution. The cells were washed with the
same solution and then incubated with Cy3-conjugated
anti-rabbit IgG (Jackson ImmunoResearch) for 1 h in the
blocking solution. Then, they were re-fixed, treated with 4
M
HCl for 30 min at room temperature and incubated with rat
anti-BrdU Ig (Harlan Sera Laboratory, Clone BU1/75)
followed by the incubation with FITC-conjugated anti-rat
IgG (Cappel). Positive immunoreactivities were detected
with fluorescence microscopy (AX-80, Olympus).
Cell labeling and immunoprecipitation
HeLa and WI-38 cells that had been plated in dishes
(30 mm in diameter) were cultured for 30 min in medium

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

mixed with 30 lLof2· concentrated SDS sample buffer.
RT-PCR
mRNA was purified from HeLa and WI-38 cells using a kit
(QuickPrep Micro mRNA Purification Kit, Amersham
1090 Y. Ishimi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 1. Mcm proteins in total cell extracts and chromatin-bound fraction from HeLa and WI 38 cells. (A) Different volumes (0.7–12 lL) of total
cellular proteins (equivalent to 2 · 10
4
cellsÆlL
)1
) were separated in SDS-polyacrylamide gel, transferred to a membrane and analyzed by
immunoblotting using anti-Mcm and anti-PCNA Ig. Total proteins were analyzed on 15 : 25% gradient gel and stained with Coomassie Brilliant
Blue. Core histones are indicated. (B) Chromatin-bound proteins (4 · 10
4
cellsÆlL
)1
) were analyzed by immunoblotting using anti-Mcm and anti-
ORC2 Ig. Chromatin-bound proteins were analyzed on 15 : 25% gradient gel and stained with Coomassie Brilliant Blue.
Ó FEBS 2003 Mcm expression in cancer cells (Eur. J. Biochem. 270) 1091
Table 1. Quantitation and comparison of Mcm amounts. From the data in Fig. 1 and others, the concentrations of Mcm proteins, ORC2 and PCNA
were compared between HeLa and WI-38 cells with respect to their two fractions, total cellular protein and chromatin-bound protein. After the
band intensities were quantitated in each protein, the concentrations of these proteins in total and chromatin-bound fraction were determined. The
relative ratio (HeLa/WI) in the concentration was calculated. When several experiments were performed in each Mcm, the average of the values
(ratio) was presented with a range of the values (in parenthesis). ND, not determined. The total number of molecules in a single HeLa and WI-38 cell
was calculated by determining the Mcm concentration in total cellular protein using a standard curve which was determined by immunoblotting
human Mcm2, 4, 6, and 7 proteins purified from HeLa cells by histone-column chromatography.
Numbers of HeLa/WI Numbers of molecules (total)
Total Chromatin HeLa WI
Mcm2 14(13–16) 12 1.6 · 10
6

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

cDNA were purchased from Toyobo.
Northern blot analysis
Total RNA was extracted from HeLa and WI-38 cells with
phenol, and electrophoresed on agarose gel containing
formaldehyde [38]. The RNA on the gel was transferred to a
nylon membrane (Hybond-N plus, Amersham). Mcm2 and
glyceraldehyde-3-phosphate dehydrogenase probes ampli-
fied by RT-PCR were labeled at the 5¢ end with poly-
nucleotide kinase in the presence of [c-
32
P]ATP. The blotted
membrane was incubated with the labeled probe in 1%
dextran sulphate, 1% SDS and 1
M
NaCl in the presence of
salmon sperm DNA (50 lgÆmL
)1
). It was then washed twice
with 2· NaCl/Cit at room temperature for 5 min, twice with
2· NaCl/Cit plus 1% SDS at 60 °C for 30 min and finally
once with 0.1 · NaCl/Cit at room temperature. The
radioactivity on the membrane was detected and quantified
with an Image Analyzer (FLA2000, Fuji).
Immunostaining of human tissues
Paraffin-embedded surgical material from uterine cervical
cancer was cut 4-lm thick, and sections were placed on
aminopropyltriethoxysilane-coated slides. These sections
were dewaxed in xylene, and treated with a series of
decreasing concentrations of ethanol, then with deionized
water. They were boiled in 0.01

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

substrate solution of diaminobenzidine (Stable DAB,
Pharma). The slides were then lightly counterstained with
methyl green, dehydrated in a graded series of ethanol,
cleared in xylene and coverslipped.
Results
Enhanced expression of Mcm proteins in cancer cells
Total cellular proteins (2 · 10
7
cellsÆmL
)1
) and proteins
bound to chromatin (4 · 10
7
cellsÆmL
)1
)wereprepared
from logarithmically growing HeLa uterine cervical carci-
noma cells and human normal fibroblast WI-38 cells. They
were analyzed by Western blotting using anti-Mcm2, 3, 4, 5,
6 and 7 Ig (Fig. 1). On comparing the amounts of Mcm in
total cellular proteins and chromatin-bound proteins,
approximately one-third of total Mcm protein was found
to be recovered in the chromatin-bound fraction in HeLa
cells (data not shown). Thus, a considerable portion of Mcm
protein is present in the nucleoplasm and/or easily released
from chromatin. Titration of these two fractions in the
Western blot analysis shows that Mcm2–7 proteins are 5–14
times more abundant in HeLa cells than in WI-38 cells for
the total cellular proteins and are 6–13 times for chromatin-
bound proteins (Fig. 1 and Table 1). Using purified human

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

cells. From the data in Fig. 2 and others, the concentrations of Mcm
proteins, ORC2 and PCNA in total protein and chromatin-bound
protein were compared between GM and WI-38 as described in
Table 1.
GM/WI
Total Chromatin
Mcm2 6(5–7) 12(5–20)
3 4(2–6) 6(4–7)
435
5910
646
7 4(3–7) 5(5–6)
ORC2 2
PCNA 4
Histones 1
1094 Y. Ishimi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
cell population in the S phase in logarithmically growing
cells and doubling time. The percentage of BrdU-positive
cells was approximately 32% for HeLa cells and 35% for
WI-38 cells. Measurements of growth curves suggested that
the doubling time was approximately 21 h for HeLa cells
and 25 h for WI-38 cells. These results indicated that the
difference in the level of Mcm expression between these two
cells cannot be attributed to the difference in the growth
rate. Then we performed double immunostaining of HeLa
and WI-38 cells with anti-Mcm4 and anti-BrdU Ig (Fig. 2)
to gain insight into the correlation between the expression of
Mcm4 and the cell cycle. In both cells, clear nuclear staining
was observed with anti-Mcm4 Ig. Although the staining
intensity was relatively constant within BrdU-positive and

-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

four times), human Burkitt’s lymphoma (Raji cells) and
human colorectal COLO 320 DM cells than in WI-38 cells
(data not shown). In contrast, we observed a comparable
level of Mcm expression in normal human fibroblasts from
umbilical cord (HUC-F cells) and WI-38 cells.
Expression of the human
Mcm2
gene
To understand the mechanism behind the enhanced
expression of Mcm proteins in HeLa cells, the concentra-
tions of Mcm2 mRNA in the mRNA fractions purified
from HeLa and WI-38 cells was compared by RT-PCR
(Fig. 4A). A 300-base fragment of expected size was
amplified from both mRNA fractions. Titration of these
two fractions indicated that Mcm2 mRNA is 4–8 times
more abundant in the HeLa mRNA fraction than in the
WI-38 fraction. In contrast, the concentration of glyceral-
dehyde-3-phosphate dehydrogenase mRNA (1000 nucleo-
tides), which is known to be relatively constant among
various cells, was at most doubled in the HeLa mRNA
fraction. Thus, the RT-PCR experiment suggests that the
level of mRNA is enriched in HeLa cells 2–4 times more
than in WI-38 cells. Next, the abundance of Mcm2 mRNA
was examined by Northern blot analysis. Total RNA was
prepared from HeLa and WI-38 cells, and the RNA
transferred to a membrane was probed with a labeled
Mcm2 fragment (Fig. 4B). RNA of the expected size
(approximately 3 kb) was detected on electrophoresis of
the mRNA prepared from HeLa cells, but only a faint band
was detected from the WI-38 mRNA. Titration of the

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-

noprecipitated with anti-Mcm4 Ig and the immunoprecipitates were
analyzed on SDS gel. (B) HeLa and WI-38 cells were labeled for 2 h as
described above and incubated in the growth medium for 0, 2, 4, 7 and
22 h. Immunoprecipitation with anti-Mcm4 Ig was performed as
described above. (C) The radioactivity in Mcm4 in (B) was quantitated
and displayed as a course of chase periods. WI-38 (squares, solid line),
HeLa (diamonds, dashed line).
Fig. 6. Immunohistochemical detection of Mcm4, PCNA and Ki67 in
normal, dysplastic and malignant cells of uterine cervix. The cancer-free
squamous cell epithelium layer (left), carcinoma in situ (CIS; middle)
andaregionwithdeepinvasion(right)wereselectedfromadjacent
regions in the same section to avoid staining artifacts. Four consecutive
sections were stained by hematoxylin and eosin (HE; first), anti-Mcm4
(second), anti-PCNA (third) and anti-Ki67 (fourth) Ig. (B) Sections
containing part of the boundary of CIS (left portion) and dysplasia
(CIN1 of FIGO classification, right portion) were stained with HE,
anti-Mcm4, anti-PCNA and anti-Ki67 Ig as indicated.
1096 Y. Ishimi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Ó FEBS 2003 Mcm expression in cancer cells (Eur. J. Biochem. 270) 1097
(Fig. 5A). In addition to Mcm4 protein, Mcm6 and Mcm7
proteins tightly bound to Mcm4 were coimmunoprecipi-
tated from HeLa cell extracts. Newly synthesized Mcm4
was also detected in the immunoprecipitates from WI-38
cells, but the intensity of the band was much weaker
(approximately one tenth) than that from HeLa cells. Thus,
the synthesis of Mcm4 was accelerated in HeLa cells in
comparison to WI-38 cells.
Next, a pulse and chase experiment was performed to
compare the protein stability of Mcm4 in these two cells.
After 2-h pulse labeling, the cells were chased in normal

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

immunopositive. These results are consistent with the
observation that Mcm2, 3, 5 and 7 proteins are more
frequently detected in cells from malignant tissues than
those from normal tissues [24–33]. Mcm4-immunoreactivity
was enhanced in the larger nuclei of cells of CIS compared
with the cancer-free squamous cell epithelial layer. The
increase in PCNA- or Ki67-immunoreactivity in cancer cells
was not so marked, and Ki67-immunopositivity was
detected in some of the cancer cells. All these findings were
also observed in cancer cells with deep invasion. To further
characterize the expression of Mcm4, PCNA and Ki67 in
cancer cells, a section that contains a boundary region of
CIS (CIN3 of FIGO classification) and dysplasia (CIN1)
was immunostained (Fig. 6B). Mcm4- and PCNA-immu-
noreactivity showed a more diffuse distribution in dysplasia
than in the normal squamous cell epithelial layer, but was
still localized compared with the distribution in CIS. Mcm4-
immunoreactivity was stronger in cancer cells (CIS) than in
cells with dysplasia. PCNA-immunoreactivity was slightly
increased, but Ki67-immunoreactivity did not significantly
change in the cancer cells. The results of anti-Mcm3 immu-
nostaining was similar to those attained with anti-Mcm4 Ig:
more intense Mcm3- and Mcm-4 immunoreactivities are
Fig. 7. Immunohistochemical detection of Mcm3 and Mcm4 in normal, dysplastic and malignant cells of uterine cervix. Mcm3- and Mcm-4 immu-
noreactivities were examined in the normal squamous cell epithelial layer (Normal) and invasive cancer lesion (Cancer), both of which were found in
adjacent regions in the same section. Mcm3- and Mcm-4 immunoreactivities were also examined in a section containing the boundary of CIS (left
portion) and dysplasia (right portion, Cancer/dysplasia).
1098 Y. Ishimi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
ubiquitously expressed in the larger nuclei of cancer cells
compared with those in basal cells of the normal squamous

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

the presence of regulated turnover of Mcm4 protein in
WI-38 cells. A higher level of Mcm expression was observed
in several other transformed cell lines, although the extent of
the enhancement varied among the cells. The enhanced level
of Mcm expression in these transformed cell lines would
contribute to the growth of cancer cells by facilitating
genome replication. As Mcm proteins bound to chromatin
are present at higher levels in these cells, whether the
number of replication initiation sites increases and/or the
rate of the replication fork movement is accelerated,
compared to WI-38 cells, deserves to be examined. It is
also possible that the enhanced expression of Mcm proteins
in these transformed cells contributes to cell growth by
facilitating transcription [40,41] or through interaction with
the Rb (retinoblastoma) protein [42].
It has been reported that the frequency of Mcm
expression is much higher in malignant tissues than
normal tissues [24–33]. Consistent with these findings we
observed that Mcm3 and 4 are ubiquitously expressed in
cancer cells from human uterine cervix (Figs 6 and 7). It
has also been indicated that Mcm5 protein is expressed at
a similar level in normal and cancer cells from uterine
cervix, breast and large intestine [28]. However, we suggest
that the amounts of Mcm3 and 4 protein were increased
in cancer cells compared to proliferating cells from normal
tissue. Further quantitative experiments are required to
confirm whether Mcm proteins are expressed at higher
levels in cancer cells, but the present immunohistochemical
findings on uterine cervical cancer are consistent with an
enhanced expression in transformed cell lines. It should be

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.

In addition, Mcm3 and 4 proteins seem to be useful as a
marker to discriminate cancer cells and dysplastic cells in the
uterine cervix, as was revealed in the present work.
Acknowledgements
We thank Yuki Komamura-Kohno for assistance. This work is in part
supported by a grant-in-aid for scientific research on priority area from
the Ministry of Education, Science, Sports and Culture of Japan.
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