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

Eur J Biochem 270, 1089–1101 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03440.x 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 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 compared the level of expression of Mcm proteins among human HeLa uterine cervical carcinoma cells, SV40-transformed human fibroblast GM00637 cells and normal human fibroblast 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, and proteins was accelerated in HeLa cells Immunohistochemical studies of surgical materials from human uterine cervix showed that Mcm3 and are ubiquitously expressed in cancer cells Further, the positive rate and level of Mcm3 and 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 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 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 replication The exact biochemical function of Mcm2, 3, and 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 regulatory 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 complex) 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 overreplication 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 malignant tissues than those from normal tissues [24–33] This phenomenon was also observed in dysplastic cells, suggesting 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 significance 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: yukio@libra.ls.m-kagaku.co.jp 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) Keywords: cancer cells; DNA replication; Mcm; protein expression; uterine cervix Ó FEBS 2003 1090 Y Ishimi et al (Eur J Biochem 270) Mcm3 and 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 CNBractivated 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 M Tris/HCl (pH 8.0) to a final concentration of 100 mM Rabbit anti-Mcm3 serum was obtained as reported [34] and affinity-purified for immunostaining 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 antiPCNA (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-transformed human fibroblasts (GM00637) purchased from Coriell Cell Repositories were cultured in DMEM supplemented with 10% fetal calf serum WI-38 cells at a population doubling level of 33–38 were used for experiments and had almost stopped proliferating at approximately 41 population doubling level Fractionation of cell extracts and Western-blot analysis of Mcm proteins Cells (4 · 106 cells) were lysed in 0.2 mL of CSK buffer (10 mM Pipes, pH 6.8, 100 mM NaCl, mM MgCl2, mM EGTA, mM dithiothreitol and mM phenylmethanesulfonyl fluoride) containing 0.1% Triton X-100, mM 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 · 106 cells) were lysed with solution A as described above and placed on ice for 15 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 containing SDS and transferred to a membrane (Immobilon, Millipore) The membrane was incubated at 37 °C for h with primary antibodies in a blocking solution (Blockace, Dai-nippon Pharmaceuticals) After being washed with Tris buffered saline (TBS; 50 mM Tris/HCl, pH 7.5, and 0.15 M NaCl) plus 0.1% Triton X-100, the membrane was incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Bio-Rad) The immunoreacted proteins 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 pulselabeled with 20 lM bromodeoxyuridine (BrdU) for 15 After being washed with phosphate-buffered saline (Dulbecco’s NaCl/PBS–, Nissui), the cells were fixed by incubation with 4% paraformaldehyde in NaCl/Pi for at room temperature The cells were washed with NaCl/Pi, and then permeabilized and blocked by incubation with 0.1% Triton X-100, 0.02% SDS and 2% nonfat dried milk in NaCl/Pi for h at 37 °C Incubation of the cells with antiMcm4 rabbit Ig was performed for h at 37 °C in the above blocking solution The cells were washed with the same solution and then incubated with Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) for h in the blocking solution Then, they were re-fixed, treated with M HCl for 30 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 in medium depleted of methionine (Sigma) Ten microlitres of [35S]methionine (10 mCiỈmL)1) was added to the medium, and incubation was continued for given periods For pulse and chase experiments, the cells labeled with [35S]methionine for 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 unitsỈmL)1 and the mixture was incubated at 30 °C for 30 After centrifugation, the supernatant was combined with the first supernatant and incubated with lg of anti-Mcm4 Ig for 1.5 h at °C Protein G-Sepharose (30 lL) was added to the solution, and the incubation was continued overnight at °C 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 and then mixed with 30 lL of 2· concentrated SDS sample buffer RT-PCR mRNA was purified from HeLa and WI-38 cells using a kit (QuickPrep Micro mRNA Purification Kit, Amersham Ó FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1091 Fig 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 · 104 cellsỈlL)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 · 104 cellsỈlL)1) were analyzed by immunoblotting using anti-Mcm and antiORC2 Ig Chromatin-bound proteins were analyzed on 15 : 25% gradient gel and stained with Coomassie Brilliant Blue Ó FEBS 2003 1092 Y Ishimi et al (Eur J Biochem 270) Table Quantitation and comparison of Mcm amounts From the data in Fig 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 proteins purified from HeLa cells by histone-column chromatography Numbers of HeLa/WI Total Mcm2 ORC2 PCNA Histones 14(13–16) 10(8–12) 5(4–7) 10(5–15) 9(6–10) 1.5(1.4–1.9) Numbers of molecules (total) Chromatin 12 11(7–15) 6(6–7) ND 10(4–14) 13(10–16) HeLa WI · 105 2.5 · 106 5.2 · 105 · 106 1.5 · 106 1.8 · 105 2.1 · 105 1.6 · 10 1.5–2 Fig 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 were fixed and detected with anti-Mcm4 Ig (A and C) or anti-BrdU Ig (B and D) Ó FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1093 Ó FEBS 2003 1094 Y Ishimi et al (Eur J Biochem 270) Table Quantitation and comparison of proteins in GM and WI-38 cells From the data in Fig 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 GM/WI Total Mcm2 ORC2 PCNA Histones Chromatin 6(5–7) 4(2–6) 4(3–7) 12(5–20) 6(4–7) 10 5(5–6) 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 amplified by RT-PCR were labeled at the 5¢ end with polynucleotide kinase in the presence of [c-32P]ATP The blotted membrane was incubated with the labeled probe in 1% dextran sulphate, 1% SDS and M NaCl in the presence of salmon sperm DNA (50 lgỈmL)1) It was then washed twice with 2· NaCl/Cit at room temperature for min, twice with 2· NaCl/Cit plus 1% SDS at 60 °C for 30 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 Fig 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 Total proteins including histones were also detected by Coomassie Brilliant Blue staining 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 using a microwave oven to facilitate antigen retrieval Following washes in deionized water and NaCl/Pi (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 Sections were then washed in NaCl/Pi, blocked with 10% normal swine serum in NaCl/Pi for 30 and incubated with each of the following primary antibodies at °C overnight: rabbit anti-Mcm3, anti-Mcm4 and antiKi-67, and mouse 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 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 · 107 cellsỈmL)1) and proteins bound to chromatin (4 · 107 cellsỈmL)1) were prepared from logarithmically growing HeLa uterine cervical carcinoma cells and human normal fibroblast WI-38 cells They were analyzed by Western blotting using anti-Mcm2, 3, 4, 5, and 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 chromatinbound proteins (Fig and Table 1) Using purified human Mcm proteins as a standard, it was calculated that approximately 1.5–2.5 · 106 molecules of Mcm2, 4, and proteins are present in a single HeLa cell on average and 0.1–0.5 · 106 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 polymerase 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 Ó FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1095 Fig 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 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 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 fibroblasts (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 and Table 2) In contrast, the amounts of total Ó FEBS 2003 1096 Y Ishimi et al (Eur J Biochem 270) Expression of the human Mcm2 gene Fig Synthesis and stability of Mcm4 protein (A) HeLa and WI-38 cells were labeled with [35S]methionine for 0.5 or h in medium depleted of methionine The total cell extracts were prepared, immunoprecipitated with anti-Mcm4 Ig and the immunoprecipitates were analyzed on SDS gel (B) HeLa and WI-38 cells were labeled for h as described above and incubated in the growth medium for 0, 2, 4, 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) 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 To understand the mechanism behind the enhanced expression of Mcm proteins in HeLa cells, the concentrations 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 glyceraldehyde-3-phosphate dehydrogenase mRNA (1000 nucleotides), 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 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 comparable 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 pulselabeling with [35S]methionine, Mcm4 protein was immunoprecipitated The immunoprecipitates were stringently washed, and bound proteins were analyzed on SDS gel Fig 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 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 Ó FEBS 2003 Mcm expression in cancer cells (Eur J Biochem 270) 1097 1098 Y Ishimi et al (Eur J Biochem 270) Ó FEBS 2003 Fig Immunohistochemical detection of Mcm3 and Mcm4 in normal, dysplastic and malignant cells of uterine cervix Mcm3- and Mcm-4 immunoreactivities 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) (Fig 5A) In addition to Mcm4 protein, Mcm6 and Mcm7 proteins tightly bound to Mcm4 were coimmunoprecipitated 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, 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 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 expression 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, and 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-immunoreactivity 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 Mcm4immunoreactivity 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 immunostaining was similar to those attained with anti-Mcm4 Ig: more intense Mcm3- and Mcm-4 immunoreactivities are 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 proteins are expressed more ubiquitously in cancer cells than in proliferative 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 fibroblasts 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, semiquantitative 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, and 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 are ubiquitously expressed in cancer cells from human uterine cervix (Figs 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 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 Mcm expression in cancer cells (Eur J Biochem 270) 1099 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, and 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 hyperphosphorylated 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 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 References Kearsey, S.E & Labib, K (1998) MCM proteins: evolution, properties, and role in DNA replication Biochim Biophys Acta 1398, 113–136 Tye, B.K (1999) MCM proteins in DNA replication Annu Rev Biochem 68, 649–686 Kelly, T.J & Brown, G.W (2000) Regulation of chromosome replication Annu Rev Biochem 69, 829–880 Bell, S.P & Dutta, A (2002) DNA replication in eukaryotic cells Annu Rev Biochem 71, 333–374 Labib, K., Tercero, J.A & Diffley, J.F (2000) Uninterrupted MCM2-7 function required for DNA replication fork progression Science 288, 1643–1647 Tanaka, T., Knapp, D & Nasmyth, K (1997) Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs Cell 90, 649–660 Aparicio, O.M., Weinstein, D.M & Bell, S.P (1997) Components and dynamics of DNA replication complexes in S cerevisiae: redistribution of MCM proteins and Cdc45p during S phase Cell 91, 59–69 1100 Y Ishimi et al (Eur J Biochem 270) Thommes, P., Kubota, Y., Takisawa, H & Blow, J.J (1997) The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides EMBO J 16, 3312–3319 Koonin, E.V (1993) A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication Nucleic Acids Res 21, 2541–2547 10 Ishimi, Y (1997) A DNA helicase activity is associated with an MCM4,-6, and -7 protein complex J Biol Chem 272, 24508– 24513 11 You, Z., Komamura, Y & Ishimi, Y (1999) Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity Mol Cell Biol 19, 8003–8015 12 Lee, J.-K & Hurwitz, J (2000) Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe J Biol Chem 275, 18871–18878 13 Lee, J.-K & Hurwitz, J (2001) Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and complex requires forked DNA structures Proc Natl Acad Sci USA 98, 54–59 14 Ishimi, Y., Komamura, Y., You, Z & Kimura, H (1998) Biochemical function of mouse minichromosome maintenance protein J Biol Chem 273, 8369–8375 15 Sato, M., Gotow, T., You, Z., Komamura-Kohno, Y., Uchiyama, Y., Yabuta, N., Nojima, H & Ishimi, Y (2000) Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex J Mol Biol 300, 421–431 16 Kubota, Y., Mimura, S., Nishimoto, S.I., Takisawa, H & Nojima, H (1995) Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor Cell 81, 601–609 17 Chong, P.J., Mahbubani, H.M., Khoo, C.Y & Blow, J.J (1995) Purification of an MCM-containing complex as a component of the DNA replication licensing system Nature 375, 418–421 18 Madine, M.A., Khoo, C.Y., Mills, A.D & Laskey, R.A (1995) MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells Nature 375, 421–425 19 Lygerou, Z & Nurse, P (2000) Controlling S-phase onset in fission yeast Biological responses to DNA damage Symposium on Quantitative Biology, 65, 323–332 20 Nguyen, Y.Q., Co, C & Li, J.J (2001) Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms Nature 411, 1068–1073 21 Brodeur, G.M & Hogarty, M.D (1998) Gene amplification in human cancers: biological and clinical significance In The Genetic Basis of Human Cancer (Vogelstein, B & Kinzler, K.W., eds), pp 161–172 McGraw-Hill, Health Professions Division 22 Schimke, R.T (1988) Gene amplification in cultured cells J Biol Chem 263, 5989–5992 23 Stark, G.R., Debatisse, M., Giulotto, E & Wahl, G.M (1989) Recent progress in understanding mechanisms of mammalian DNA amplification Cell 57, 901–908 24 Hiraiwa, A., Fujita, M., Nagasaka, T., Adachi, A., Ohashi, M & Ishibashi, M (1997) Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth Int J Cancer 74, 180–184 25 Hiraiwa, A., Fujita, M., Adachi, A., Ono, H., Nagasaka, T., Matsumoto, Y., Ohashi, M., Tomita, Y & Ishibashi, M (1998) Specific distribution patterns of hCDC47 expression in cutaneous diseases J Cutan Pathol 25, 285–290 26 Williams, G.H., Romanowski, P., Morris, L., Madine, M., Mills, A.D., Stoeber, K., Marr, J., Laskey, R.A & Coleman, N (1998) Improved cervical smear assessment using antibodies against proteins that regulate DNA replication Proc Natl Acad Sci USA 95, 14932–14937 Ó FEBS 2003 27 Todorov, I.T., Werness, B.A., Wang, H.-Q., Buddharaju, L.N., Todorova, P.D., Slocum, H.K., Brooks, J.S & Huberman, J.A (1998) HsMCM2/BM28: a novel proliferation marker for human tumors and normal tissues Laboratory Invest 78, 73–78 28 Freeman, A., Morris, L.S., Mills, A.D., Stoeber, K., Laskey, R.A., Williams, G.H & Coleman, N (1999) Minichromosome maintenance proteins as biological markers of dysplasia and malignancy Clin Cancer Res 5, 2121–2132 29 Stoeber, K., Halsall, I., Freeman, A., Swinn, R., Doble, A., Morris, L., Coleman, N., Bullock, N., Laskey, R.A., Hales, C.N & Williams, G.H (1999) Immunoassay for urothelial cancers that detects DNA replication protein Mcm5 in urine Lancet 354, 1524–1525 30 Stoeber, K., Tisty, T.D., Happerfield, L., Thomas, G.A., Romanov, S., Bobrow, L., Williams, E.D & Williams, G.H (2001) DNA replication licensing and human cell proliferation J Cell Sci 114, 2027–2041 31 Wharton, S.B., Chan, K.K., Anderson, J.R., Stoeber, K & Williams, G.H (2001) Replicative Mcm2 protein as a novel proliferation marker in oligodendrogliomas and its relationship to Ki67 labelling index, histological grade and prognosis Neuropath Appl Neurobiol 27, 305–313 32 Endl, E., Kausch, I., Baak, M., Knippers, R., Gerdes, J & Scholzen, T (2001) The expression of Ki-67, MCM3, and p27 defines distinct subsets of proliferating, resting, and differentiated cells J Pathol 195, 457–462 33 Tan, D.-F., Huberman, J.A., Hyland, A., Loewen, G.M., Brooks, J.S.J., Beck, A.F., Todorov, I.T & Bepler, G (2001) MCM2- a promising marker for premalignant lesions of the lung: a cohort study BMC Cancer 1, 34 Kimura, H., Ohtomo, T., Yamaguchi, M., Ishii, A & Sugimoto, K (1996) Mouse MCM proteins: complex formation and transportation to the nucleus Genes Cells 1, 977–993 35 Kimura, H., Takizawa, N., Nozaki, N & Sugimoto, K (1995) Molecular cloning of cDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between P1 (MCM3) and CDC46 proteins Nucleic Acids Res 23, 2097–2104 36 Fujita, M., Ishimi, Y., Nakamura, H., Kiyono, T & Tsurumi, T (2002) Nuclear organization of DNA replication initiation proteins in mammalian cells J Biol Chem 277, 10354–10361 37 Fujita, M., Kiyono, T., Hayashi, Y & Ishibashi, M (1997) In vivo interaction on of human MCM heterohexameric complexes with chromatin Possible involvement of ATP J Biol Chem 272, 10928–10935 38 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, Vol 3, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 39 Scholzen, T & Gerdes, J (2000) The Ki-67 protein: from the known and the unknown J Cell Physiol 182, 311–322 40 DeFonseca, C.J., Shu, F & Zhang, J.J (2001) Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-c Proc Natl Acad Sci USA 98, 3034–3039 41 Yankulov, K., Todorov, I., Romanowski, P., Licatalosi, D., Cilli, K., McCracken, S., Laskey, R & Bentley, D.L (1999) MCM proteins are associated with RNA polymerase II holoenzyme Mol Cell Biol 19, 6154–6163 42 Sterner, J.M., Dew-Knight, S., Musahl, C., Kornbluth, S & Horowitz, J.M (1998) Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with Mcm7 Mol Cell Biol 18, 2748–2757 43 Leone, G., DeGregori, J., Yan, Z., Jakoi, L., Ishida, S., Williams, R.S & Nevins, J.R (1998) E2F3 activity is regulated during the cell cycle and is required for the induction of S phase Genes Dev 12, 2120–2130 Ó FEBS 2003 44 Tsuruga, H., Yabuta, N., Hosoya, S., Tamura, K., Endo, Y & Nojima, H (1997) HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5 Genes Cells 2, 381–399 45 Suzuki, S., Adachi, A., Hiraiwa, A., Ohashi, M., Ishibashi, M & Kiyono, T (1998) Cloning and characterization of human MCM7 promoter Gene 216, 85–91 46 Ohtani, K., Iwanaga, R., Nakamura, M., Ikeda, M., Yabuta, N., Tsuruga, H & Nojima, H (1999) Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F Oncogene 18, 2299–2309 47 Goodwin, E.C & DiMaio, D (2000) Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways Proc Natl Acad Sci USA 97, 12513–12518 Mcm expression in cancer cells (Eur J Biochem 270) 1101 48 Gozuacik, D., Murakami, Y., Saigo, K., Chami, M., Mugnier, C., Lagorce, D., Okanoue, T., Urashima, T., Brechot, C & PaterliniBrechot, P (2001) Identification of human cancer-related genes by naturally occurring hepatitis B virus DNA tagging Oncogene 20, 6233–6240 49 Saha, S., Bardelli, A., Buckhaults, P., Velculescu, V.E., Rago, C., Croix, B.S., Romans, K.E., Choti, M.A., Lengauer, C., Kinzler, K.W & Vogelstein, B (2001) A phosphatase associated with metastasis of colorectal cancer Science 294, 1343–1346 50 Shohet, J.M., Hicks, M.J., Plon, S.E., Burlingame, S.M., Stuart, S., Chen, S.-Y., Brenner, M.K & Nuchtern, J.G (2002) Minichromosome maintenance protein MCM7 is a direct target of the MYCN transcription factor in neuroblastoma Cancer Res 62, 1123–1128  ... 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, ... significance of Mcm proteins in the aberrant proliferation of cancer cells In addition, Mcm3 and proteins seem to be useful as a marker to discriminate cancer cells and dysplastic cells in the uterine cervix, ... Biochem 270) Mcm3 and 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