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

We com-paredthe level of expression of Mcm proteins among human HeLa uterine cervical carcinoma cells, SV40-transformed human fibroblast GM00637 cells andnormal human fibro-blast WI-38 cel

Enhanced expression of Mcm proteins in cancer cells derived

from uterine cervix

Yukio Ishimi1, Isao Okayasu2, Chieko Kato1, Hyun-Ju Kwon1, Hiroshi Kimura3, Kouichi Yamada4

and Si-Young Song1

1

Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo;2Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa;3Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo;4National 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-paredthe level of expression of Mcm proteins among human

HeLa uterine cervical carcinoma cells, SV40-transformed

human fibroblast GM00637 cells andnormal human

fibro-blast WI-38 cells All the proteins examinedwere detectedin

HeLa andGM cells at 6–10 times the level foundin WI-38

cells on average This increase was observedboth in total

cellular proteins andin the chromatin-boundfraction

Consistently, Mcm2 mRNA was enrichedin HeLa cells to approximately four times the level in WI-38 cells, andthe synthesis of Mcm4, 6 and7 proteins was acceleratedin HeLa cells Immunohistochemical studies of surgical materials from human uterine cervix showedthat Mcm3 and4 are ubiquitously expressedin cancer cells Further, the positive rate andlevel of Mcm3 and4 expression appearedto be higher in cancer cells than in normal proliferating cells of the uterine cervix anddysplastic 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 formedat 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,

and7 form a hexameric complex andfunction 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, and5

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 involvedin DNA replication as a DNA helicase, andan

activatedform 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) andMcm, 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 generatedby the over-replication of a genomic locus containing over-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 reportedthat Mcm proteins are more frequently detected in cells from malig-nant tissues than those from normal tissues [24–33] This phenomenon was also observedin dysplastic cells, suggest-ing that Mcm proteins are a goodindicator of proliferative

or cancer cells in malignant tissues [28] Elucidation of how the expression of Mcm protein changes relates to malignant transformation of cells andthe pathophysiological signifi-cance of those changes awaits further studies In this paper,

we report that Mcm proteins are expressedat higher levels

in the transformedcell lines than in normal fibroblasts We also foundmore frequent andhigher-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: yukio@libra.ls.m-kagaku.co.jp

Abbreviations: BrdU, bromodeoxyuridine; CIS, carcinoma in situ;

Mcm, minichromosome maintenance protein; ORC, origin

recognition complex.

(Received24 September 2002, revised16 December 2002,

accepted20 December 2002)

Eur J Biochem 270, 1089–1101 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03440.x

Mcm3 and4 in cancer cells than in normal proliferating cells

of human uterine cervix anddysplastic cells The results

suggest that enhancedexpression of Mcm proteins plays a

role in the malignant transformation of cells

Materials and methods

Antibodies

Rabbit anti-Mcm2 serum was preparedusing mouse Mcm2

protein as an antigen, andthe antibodies were purifiedwith

Mcm2-beads prepared by fixing Mcm2 protein to

CNBr-activatedSepharose (Pharmacia) After the loading of

antiserum onto the beads, the antibodies were eluted with

0.2Mglycine (pH 2.5) and0.15MNaCl The solution was

neutralized by adding 2M Tris/HCl (pH 8.0) to a final

concentration of 100 mM Rabbit anti-Mcm3 serum was

obtainedas reported[34] andaffinity-purifiedfor

immuno-staining Anti-Mcm4 Ig were affinity-purifiedusing beads

conjugatedwith the fragment of mouse Mcm4 (amino acids

683–862) that hadbeen usedfor immunizing rabbits [34]

Rabbit anti-Mcm5 serum was obtainedas reported[35] and

specific antibodies were affinity-purified Rabbit anti-Mcm6

(sc-9843), mouse Mcm7 (sc-9966) andmouse

anti-PCNA (sc-56) IgG were purchasedfrom Santa Cruz

Biotechnology Inc Rabbit anti-Ki67 Ig were purchased

from DAKO Anti-ORC2 Ig were producedas reported[36]

Cells

HeLa cells were culturedin DMEM (Dulbecco’s modified

Eagle’s medium) supplemented with 10% calf serum WI-38

cells obtainedfrom RIKEN GenBank

andSV40-trans-formedhuman fibroblasts (GM00637) purchasedfrom

Coriell Cell Repositories were culturedin DMEM

supple-mentedwith 10% fetal calf serum WI-38 cells at a

population doubling level of 33–38 were used for

experi-ments andhadalmost stoppedproliferating at

approxi-mately 41 population doubling level

Fractionation of cell extracts and Western-blot analysis

of Mcm proteins

Cells (4· 106 cells) were lysedin 0.2 mL of CSK buffer

(10 mMPipes, pH 6.8, 100 mMNaCl, 1 mMMgCl2, 1 mM

EGTA, 1 mMdithiothreitol and 1 mM

phenylmethanesulfo-nyl fluoride) containing 0.1% Triton X-100, 1 mM ATP

andproteinase inhibitors (Pharmingen; solution A) [37]

The suspension was mixedwith 0.1 mL of 3· concentrated

sample buffer for SDS gel electrophoresis andthen

sonicatedfor 20 s to shear chromosomal DNA before

being loadedonto the SDS gel Thus, it containedtotal

cellular proteins To obtain chromatin-boundproteins, cells

(4· 106cells) were lysedwith solution A as describedabove

andplacedon ice for 15 min The cell suspension was

centrifuged, andthe recoveredprecipitate was washedonce

with solution A The precipitate was suspended in 0.1 mL of

solution A andthen mixedwith 0.05 mL of concentrated

SDS sample buffer

Total cellular proteins andchromatin-boundproteins

were electrophoresedon a 10% acrylamide gel

contain-ing SDS andtransferredto a membrane (Immobilon,

Millipore) The membrane was incubatedat 37C for 1 h with primary antibodies in a blocking solution (Blockace, Dai-nippon Pharmaceuticals) After being washedwith Tris bufferedsaline (TBS; 50 mMTris/HCl, pH 7.5, and0.15M NaCl) plus 0.1% Triton X-100, the membrane was incu-batedwith peroxidase-conjugatedanti-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), andthe level of reactivity was quantified (Cool Saver AE-6935, Atto)

Immunostaining of cells Cells culturedon eight-well chambers (Falcon) were pulse-labeledwith 20 lMbromodeoxyuridine (BrdU) for 15 min After being washedwith phosphate-bufferedsaline (Dul-becco’s NaCl/PBS–, Nissui), the cells were fixedby incuba-tion with 4% paraformaldehyde in NaCl/Pifor 5 min at room temperature The cells were washedwith NaCl/Pi, and then permeabilizedandblockedby incubation with 0.1% Triton X-100, 0.02% SDS and2% nonfat driedmilk in NaCl/Pifor 1 h at 37C Incubation of the cells with anti-Mcm4 rabbit Ig was performedfor 1 h at 37C in the above blocking solution The cells were washedwith the same solution andthen incubatedwith Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) for 1 h in the blocking solution Then, they were re-fixed, treated with 4M HCl for 30 min at room temperature andincubatedwith rat anti-BrdU Ig (Harlan Sera Laboratory, Clone BU1/75) followedby the incubation with FITC-conjugatedanti-rat IgG (Cappel) Positive immunoreactivities were detected with fluorescence microscopy (AX-80, Olympus)

Cell labeling and immunoprecipitation HeLa andWI-38 cells that hadbeen platedin dishes (30 mm in diameter) were cultured for 30 min in medium depleted of methionine (Sigma) Ten microlitres of [35S]methionine (10 mCiÆmL)1) was added to the medium, andincubation was continuedfor given periods For pulse andchase experiments, the cells labeledwith [35S]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)1andthe mixture was incubatedat 30C for 30 min After centrifugation, the supernatant was combinedwith the first supernatant and incubatedwith 1 lg of anti-Mcm4 Ig for 1.5 h at 4C Protein G-Sepharose (30 lL) was added to the solution, andthe incubation was continuedovernight at 4C After being spun down, the Sepharose beads were washed four times with RIPA buffer (150 mM NaCl, 0.5% Nonidet P-40, 1% Na-deoxycholate, 0.1% SDS, and 50 mM Tris/ HCl, pH 7.5) containing proteinase inhibitors andthen mixedwith 30 lL of 2· concentratedSDS sample buffer RT-PCR

mRNA was purifiedfrom HeLa andWI-38 cells using a kit (QuickPrep Micro mRNA Purification Kit, Amersham

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 separatedin SDS-polyacrylamid e gel, transferredto a membrane andanalyzedby immunoblotting using anti-Mcm andanti-PCNA Ig Total proteins were analyzedon 15 : 25% gradient gel andstainedwith Coomassie Brilliant Blue Core histones are indicated (B) Chromatin-bound proteins (4 · 10 4

cellsÆlL)1) were analyzedby immunoblotting using anti-Mcm andanti-ORC2 Ig Chromatin-boundproteins were analyzedon 15 : 25% gradient gel andstainedwith Coomassie Brilliant Blue.

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 comparedbetween HeLa andWI-38 cells with respect to their two fractions, total cellular protein andchromatin-boundprotein After the bandintensities were quantitatedin each protein, the concentrations of these proteins in total andchromatin-boundfraction 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 presentedwith a range of the values (in parenthesis) ND, not determined The total number of molecules in a single HeLa andWI-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, and7 proteins purifiedfrom HeLa cells by histone-column chromatography.

Numbers of HeLa/WI Numbers of molecules (total)

1.8 · 10 5

PCNA 1.5(1.4–1.9)

Fig 2 Immunostaining of HeLa and WI-38 cells with anti-Mcm4 Ig Logarithmically growing HeLa (A andB) andWI-38 cells (C andD) in one section that hadbeen pulse-labeledwith BrdU for 15 min were fixedanddetectedwith anti-Mcm4 Ig (A andC) or anti-BrdU Ig (B andD).

FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1093

Pharmacia Biotech) The cDNA was preparedby RT-PCR

using random primers (ThermoScript RT-PCR System,

GibcoBRL) For the amplification of human Mcm2 cDNA,

5¢-AGACGAGATAGAGCTGACTG-3¢ as a forward

primer and5¢-CACCACGTACCTTGTGCTTG-3¢ as a

reverse primer were used Primers for the amplification

of the human glyceraldehyde-3-phosphate dehydrogenase

cDNA were purchasedfrom Toyobo

Northern blot analysis

Total RNA was extractedfrom HeLa andWI-38 cells with

phenol, andelectrophoresedon 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-fiedby RT-PCR were labeledat the 5¢ endwith

poly-nucleotide kinase in the presence of [c-32P]ATP The blotted

membrane was incubatedwith the labeledprobe in 1%

dextran sulphate, 1% SDS and 1MNaCl in the presence of

salmon sperm DNA (50 lgÆmL)1) It was then washedtwice

with 2· NaCl/Cit at room temperature for 5 min, twice with

2· NaCl/Cit plus 1% SDS at 60 C for 30 min andfinally

once with 0.1· NaCl/Cit at room temperature The

radioactivity on the membrane was detectedandquantified

with an Image Analyzer (FLA2000, Fuji)

Immunostaining of human tissues

Paraffin-embedded surgical material from uterine cervical

cancer was cut 4-lm thick, andsections were placedon

aminopropyltriethoxysilane-coatedslides These sections

were dewaxedin xylene, andtreatedwith a series of

decreasing concentrations of ethanol, then with deionized water They were boiledin 0.01Mcitrate buffer (pH 6.0) at

95C for 10 min using a microwave oven to facilitate antigen retrieval Following washes in deionized water and NaCl/Pi(0.01Mphosphate 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 washedin NaCl/Pi, blockedwith 10% normal swine serum in NaCl/Pi for 30 min and incubatedwith each of the following primary antibodies at

4C overnight: rabbit anti-Mcm3, anti-Mcm4 andanti-Ki-67, andmouse anti-PCNA IgG These antibodies were diluted in NaCl/Pi containing 2% normal swine serum (DAKO) The slides were washed in NaCl/Pi, and the subsequent immunostaining was performedby 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, clearedin xylene andcoverslipped

Results

Enhanced expression of Mcm proteins in cancer cells Total cellular proteins (2· 107cellsÆmL)1) andproteins boundto chromatin (4· 107cellsÆmL)1) were prepared from logarithmically growing HeLa uterine cervical carci-noma cells andhuman normal fibroblast WI-38 cells They were analyzedby Western blotting using anti-Mcm2, 3, 4, 5,

6 and7 Ig (Fig 1) On comparing the amounts of Mcm in total cellular proteins andchromatin-boundproteins, approximately one-thirdof total Mcm protein was found

to be recoveredin the chromatin-boundfraction 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 andare 6–13 times for chromatin-boundproteins (Fig 1 andTable 1) Using purifiedhuman Mcm proteins as a standard, it was calculated that approximately 1.5–2.5· 106molecules of Mcm2, 4, 6 and

7 proteins are present in a single HeLa cell on average and 0.1–0.5· 106molecules in a single WI-38 cell (Table 1) While total cellular proteins from these cells appearedto

be detected at a comparable level (Fig 1), the amount

of histone was slightly enrichedin HeLa cells comparedto WI-38 cells, which may be consistent with evidence of increasedchromosomal ploidy in HeLa cells The level of PCNA, a factor requiredfor 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 requiredfor 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 andORC2 proteins are expressedat particularly high levels in HeLa cells comparedto WI-38 cells As the differences of Mcm expression between HeLa andWI-38 cells couldbe 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 andPCNA

were detected in total cellular proteins (A) and the chromatin-bound

fraction (B) from GM andWI-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 andPCNA in total protein andchromatin-bound

protein were comparedbetween GM andWI-38 as d escribedin

Table 1.

GM/WI Total Chromatin

cell population in the S phase in logarithmically growing

cells anddoubling time The percentage of BrdU-positive

cells was approximately 32% for HeLa cells and35% for

WI-38 cells Measurements of growth curves suggestedthat

the doubling time was approximately 21 h for HeLa cells

and25 h for WI-38 cells These results indicatedthat the

difference in the level of Mcm expression between these two

cells cannot be attributedto the difference in the growth

rate Then we performeddouble immunostaining of HeLa

andWI-38 cells with anti-Mcm4 andanti-BrdU Ig (Fig 2)

to gain insight into the correlation between the expression of

Mcm4 andthe cell cycle In both cells, clear nuclear staining

was observedwith anti-Mcm4 Ig Although the staining

intensity was relatively constant within BrdU-positive and -negative HeLa cells, more intense staining was observedin BrdU-positive WI-38 cells These data suggest that the level

of Mcm4 protein was maximizedat the S phase in WI-38 cells andthat the Mcm4 expression in HeLa cells is maintainedirrespective of cell cycle

Next, we comparedthe concentration of Mcm proteins in cell lysate preparedfrom SV40-transformedhuman fibro-blasts (GM00637) andnormal human fibroblast WI-38 cells (Fig 3) Mcm2–7 proteins were detected in GM cells at 3–9 times the level foundin WI-38 cells for total cellular proteins andat 5–12 times the level for chromatin-boundproteins (Fig 3 andTable 2) In contrast, the amounts of total

Fig 4 Abundance of Mcm2 mRNA in HeLa and WI-38 cells (A) Total mRNA was purifiedfrom HeLa andWI-38 cells, andthe 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, andthe Mcm2 cDNA fragment was detectedby hybridizing with the same labeledfragment These fragments are indicated (B) Total RNA was purifiedfrom HeLa andWI-38 cells andanalyzedby Northern blot analysis Increasing volumes (0.7, 1.5 and3 lL each) of the total RNA were loadedonto the gel Mcm2 andG3PDH mRNA were detectedwith specific probes using the same filter The electrophoresedRNA (2 lL each) was stained with ethidium bromide (EtBr) to detect ribosomal RNAs.

FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1095

cellular proteins andcore 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 detectedin GM cells at two andfour times the levels of

those in WI-38 cells, respectively Thus, on comparing the

two human fibroblasts, it was foundthat Mcm proteins are

expressedat higher levels in transformedGM cells than in

normal WI-38 cells We also observeda higher level of Mcm

expression in SV40-transformedWI-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) andWI-38 cells

Expression of the humanMcm2 gene

To understandthe mechanism behindthe enhanced expression of Mcm proteins in HeLa cells, the concentra-tions of Mcm2 mRNA in the mRNA fracconcentra-tions purified from HeLa andWI-38 cells was comparedby RT-PCR (Fig 4A) A 300-base fragment of expectedsize was amplifiedfrom 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 enrichedin HeLa cells 2–4 times more than in WI-38 cells Next, the abundance of Mcm2 mRNA was examinedby Northern blot analysis Total RNA was preparedfrom HeLa andWI-38 cells, andthe RNA transferredto a membrane was probedwith a labeled Mcm2 fragment (Fig 4B) RNA of the expectedsize (approximately 3 kb) was detected on electrophoresis of the mRNA preparedfrom 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 purifiedfrom HeLa andWI-38 cells was performedusing a probe from an exon of the Mcm2 gene that codes for amino acids 214–287 Expectedbands were detectedfrom HeLa andWI-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 amplifiedin 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 comparedthe synthesis of Mcm4 protein between the two cell lines After pulse-labeling with [35S]methionine, Mcm4 protein was immuno-precipitated The immunoprecipitates were stringently washed, andboundproteins were analyzedon SDS gel

Fig 5 Synthesis and stability of Mcm4 protein (A) HeLa andWI-38

cells were labeledwith [35S]methionine for 0.5 or 2 h in medium

depleted of methionine The total cell extracts were prepared,

immu-noprecipitatedwith anti-Mcm4 Ig andthe immunoprecipitates were

analyzedon SDS gel (B) HeLa andWI-38 cells were labeledfor 2 h as

describedabove andincubatedin the growth medium for 0, 2, 4, 7 and

22 h Immunoprecipitation with anti-Mcm4 Ig was performedas

described above (C) The radioactivity in Mcm4 in (B) was quantitated

anddisplayedas a course of chase periods WI-38 (squares, solidline),

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) and a region with deep invasion (right) were selected from adjacent regions in the same section to avoidstaining artifacts Four consecutive sections were stainedby hematoxylin andeosin (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 stainedwith HE, anti-Mcm4, anti-PCNA and anti-Ki67 Ig as indicated.

FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1097

(Fig 5A) In addition to Mcm4 protein, Mcm6 and Mcm7

proteins tightly boundto Mcm4 were

coimmunoprecipi-tatedfrom HeLa cell extracts Newly synthesizedMcm4

was also detected in the immunoprecipitates from WI-38

cells, but the intensity of the bandwas much weaker

(approximately one tenth) than that from HeLa cells Thus,

the synthesis of Mcm4 was acceleratedin HeLa cells in

comparison to WI-38 cells

Next, a pulse andchase experiment was performedto

compare the protein stability of Mcm4 in these two cells

After 2-h pulse labeling, the cells were chasedin normal

medium for 2, 4, 7 and 22 h The cell extracts were prepared

andimmunoprecipitatedusing anti-Mcm4 Ig, andbound

proteins were analyzedon a SDS gel (Fig 5B) Constant

amounts of Mcm4 protein were recoveredfrom different

cell extracts (data not shown) The intensity of the Mcm4

bandwas clearly decreasedat 22 h chase in both HeLa and

WI-38 cells, indicating the presence of turnover of the

protein Quantitation of the Mcm4 bandsuggestedthat the

protein stability of Mcm4 does not greatly differ between

HeLa andWI-38 cells (Fig 5C)

Expression of Mcm3 and 4 proteins

in malignant tissues

Next, we comparedthe 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 examinedin five cases of human uterine

cervical cancer by immunohistochemical techniques, and

the results were comparedwith the findings obtainedby

hematoxylin andeosin staining We examinedthe

expres-sion of these proteins in a cancer-free layer of squamous

epithelial cells, carcinoma in situ (CIS) andinvasive cancer, all of which were observedin the same section In the cancer-free squamous cell epithelial layer, the expression of Mcm4, PCNA andKi67 was observedmainly in the basal cell layer (Fig 6A) The immunoreactivity variedamong the cells, andstrongly immunopositive cells were scattered along the epithelial layer Ki67-immunopositive cells were scarce comparedwith 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 and7 proteins are more frequently detected in cells from malignant tissues than those from normal tissues [24–33] Mcm4-immunoreactivity was enhancedin 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 observedin cancer cells with deep invasion To further characterize the expression of Mcm4, PCNA andKi67 in cancer cells, a section that contains a boundary region of CIS (CIN3 of FIGO classification) anddysplasia (CIN1) was immunostained(Fig 6B) Mcm4- andPCNA-immu-noreactivity showed a more diffuse distribution in dysplasia than in the normal squamous cell epithelial layer, but was still localizedcomparedwith 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 attainedwith anti-Mcm4 Ig: more intense Mcm3- andMcm-4 immunoreactivities are

Fig 7 Immunohistochemical detection of Mcm3and Mcm4 in normal, dysplastic and malignant cells of uterine cervix Mcm3- andMcm-4 immu-noreactivities were examinedin the normal squamous cell epithelial layer (Normal) andinvasive cancer lesion (Cancer), both of which were foundin adjacent regions in the same section Mcm3- andMcm-4 immunoreactivities were also examinedin a section containing the boundary of CIS (left portion) and dysplasia (right portion, Cancer/dysplasia).