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Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano (T.G., G.S., L.P., A.F., F.C., M.S.), Università Federico II, 80131 Naples, Italy; Dipartimento di Oncologia (P.F., R.G., F.B.), Università di Pisa, 56126 Pisa, Italy; and Instituto de Patologia e Imunologia Molecular da Universidade do Porto (G.G.-R.), 4200-465 Porto, Portugal
Address all correspondence and requests for reprints to: Massimo Santoro, Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Facoltá di Medicina e Chirurgia, via S. Pansini 5, 80131 Naples, Italy. E-mail: masantor{at}unina.it.
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Abstract |
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Objective: In an attempt to cast light on the mechanisms governing ATC, we evaluated MCM5 and MCM7 expression in human normal, papillary (PTC), and anaplastic thyroid samples, as well as in primary culture cells and transgenic mouse models.
Results: MCM5 and MCM7 expression was high in 65% of ATC and negligible in normal thyroid tissue and papillary thyroid carcinomas. In ATC, high MCM5 and MCM7 expression was paralleled by high levels of MCM2 and MCM6. An analysis of human ATC primary cell cultures and of a transgenic mouse model of ATC confirmed these findings. An increased transcription rate accounted for MCM7 up-regulation, because the activity of the MCM7 promoter was more than 10-fold higher in ATC cells compared with normal thyroid cells. Adoptive overexpression of wild-type p53, but not of its inactive (R248W and R273H) mutants, strongly down-regulated transcription from the MCM7 promoter, suggesting that p53 knock-out contributes to MCM7 up-regulation in ATC. Treatment with small inhibitory duplex RNAs, which decrease MCM7 protein levels, reduced the rate of DNA synthesis in ATC cells.
Conclusion: MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.
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Introduction |
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Differently from WDC, ATC have a high proliferation rate and marked genetic instability. This is reflected in the rapid tumor growth and dissemination that characterize the clinical course of ATC. The combination of a short cell-doubling time and a low apoptotic rate results in one of the most rapid growth rates in any solid tumor, and ATC tumors may double in size within 1 wk (2, 3). The molecular mechanisms underlying the anaplastic transformation of thyroid follicular cells are largely unknown. Structural rearrangements of the receptor tyrosine kinases RET and TRKA (tyrosine kinase), which are common in PTC, and of PPAR
(peroxisome proliferator-activated receptor ), typical of FTC, are rarely seen in ATC (4). Point mutations in RAS small GTPases (5) and in the BRAF (BRCA2-associated factor) serine/threonine kinase have been detected in ATC (6), and, at variance with WDC, ATC is associated with a high prevalence of p53 gene mutations (7, 8). Mutations in ß-catenin are also frequent in ATC (9). In general, ATC are characterized by a high degree of aneuploidy and complex chromosomal alterations (10); in particular, loss of 16p is common in ATC cell lines (11).
Minichromosome maintenance proteins (MCM2–MCM7) are required for DNA replication in eukaryotic cells. They were originally discovered in yeast and subsequently identified in Drosophila melanogaster, Xenopus laevis, mice, and humans (12). MCM proteins form a heterohexamer that is part of the prereplication complex (pre-RC). pre-RC factors bind to chromatin in a highly ordered sequence that triggers DNA synthesis. The process begins with the loading of the origin recognition complex. Cdc6 (cell division control protein 6) is then recruited to the site, and this allows loading of the MCM complex. As cells enter S-phase, Cdc6 is released and other factors are loaded onto the replication origin to initiate DNA synthesis. The process requires the S-phase-specific kinases CDK2 (cyclin-dependent kinase)-cyclin E and Dbf4 (DNA binding factor)-Cdc7. Dissociation of the pre-RC from chromatin ensures that each region of DNA is replicated only once during a single cell cycle (12). Being one of the helicases involved in DNA unwinding at the replication forks, MCM proteins are required throughout DNA elongation (13, 14).
The expression of MCM proteins depends on the proliferation state of the cell. Quiescent and differentiated cells do not express MCM, whereas proliferating cells and cells with proliferation potential do (15). In normal or reactive tissues, MCM expression is restricted to proliferative compartments. In contrast, in dysplastic and malignant tissues, MCM proteins are present in most cells, and their expression level inversely correlates with the degree of tumor differentiation (15). Uterine cervix, prostate, and esophageal dysplasia are positive to anti-MCM antibodies, and staining increases in neoplastic lesions (16, 17, 18, 19). MCM assay has a high specificity and sensitivity in detecting cancerous cells in feces (colorectal carcinoma) and urine (bladder carcinoma) (20, 21).
We have investigated the expression of MCM proteins in normal thyroid tissue, PTC, and ATC tumors in mice and humans. We show that MCM2, MCM5, MCM6, and MCM7 are expressed at a high level in ATC, and that MCM7 is involved in the proliferation of ATC cells.
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Materials and Methods |
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Tumors were retrieved from the files of the Pathology Departments of the Hospital Central de Asturias (Oviedo University, Asturias, Spain) and the Hospital Clinico Universitario (Santiago de Compostela University, Galicia, Spain) and the Department of Oncology of the University of Pisa (Pisa, Italy). In all cases, the study protocols were approved by the respective institutional review boards. Cases were chosen randomly among those for whom detailed clinical and follow-up data were available. For each case, the sex and age of the patient at diagnosis, tumor size, extrathyroidal extension, vascular invasion, lymph node or distant metastases, stage, and survival were recorded. Normal thyroid tissue samples were also retrieved from the files of the Department of Oncology of the University of Pisa. Sections of paraffin-embedded samples were stained with hematoxylin and eosin for histological examination to ensure that the samples fulfilled the diagnostic criteria (22). Formalin-fixed and paraffin-embedded 3- to 5-µm-thick tumor sections were deparaffinized, placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 min, and treated with blocking serum for 20 min. The slides were incubated overnight with mouse monoclonal antibodies against MCM5 (MCA 1860; Serotec, Oxford, UK), MCM7 (141.2, sc-9966; Santa Cruz Biotechnology, Santa Cruz, CA), or proliferating cell nuclear antigen (PCNA) (MAB424; Chemicon, Temecula, CA). After incubation with biotinylated antimouse secondary antibody for 15 min, slides were stained with premixed avidin-biotin complex (Vectostain ABC kits; Vector Laboratories, Burlingame, CA). Sections were developed for 5 min with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Dako, Carpinteria, CA) and 0.01% hydrogen peroxide in 0.05 M Tris-HCl buffer (pH 7.6), counterstained with hematoxylin, dehydrated, and mounted. Negative controls were performed in each case by incubating tissue slides with preimmune serum.
Cell cultures
Primary cell cultures of normal thyroid (S11N and S63N), follicular adenoma (HTU31 and HTU42), PTC (HTU56 and HT59), and ATC (HTU8 and S11T) were kindly donated by F. Curcio (University of Udine, Udine, Italy) and H. Zitzelsberger (Forschungszentrum fur Umwelt und Gesundheit, Neuherberg, Germany). The ARO cell line was established from a human ATC by Guy J. Juillard (University of California, Los Angeles, Los Angeles, CA) (7). Cells were grown in RPMI supplemented with 20% fetal bovine serum (Invitrogen, Paisley, PA), 2 mM L-glutamine, and 100 U/ml penicillin–streptomycin (Invitrogen). For the measurement of MCM5 and MCM7 half-life, cells were grown to subconfluence and then treated with 10 µg/ml cycloheximide (Sigma, St. Louis, MO) for up to 48 h. For bromodeoxyuridine (BrdU) incorporation, cells were seeded on glass coverslips. Forty-eight hours after transfection, BrdU (Sigma) was added to the cell culture media at a final concentration of 100 µg/ml for 2 h before harvest. Cells were fixed with paraformaldehyde (4%) and permeabilized with Triton X-100 (0.2%) before staining. Coverslips were incubated with anti-BrdU mouse monoclonal antibody and then with a fluorescein isothiocyanate-conjugated antimouse antibody (Roche Molecular Biochemicals, Mannheim, Germany). Coverslips were counterstained in PBS containing Hoechst 33258 (final concentration, 1 µg/ml; Sigma), rinsed in PBS, and mounted in Moviol on glass slides. The fluorescent signal was visualized with an epifluorescent microscope (Axiovert 2; Zeiss, Oberkochen, Germany) interfaced with the KS300 image analyzer software (Zeiss).
Protein analyses
Protein lysates were prepared according to standard procedures. Briefly, cells or tumor tissues were lysed in a buffer containing 50 mM HEPES (pH 7.5), 1% (vol/vol) Triton X-100, 50 mM NaCl, 5 mM EGTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin. Lysates were clarified by centrifugation at 10,000 x g for 15 min. Lysates containing comparable amounts of proteins, estimated by a modified Bradford assay (Bio-Rad, Munich, Germany), were subjected to Western blot. Immune complexes were detected with the enhanced chemiluminescence kit (Amersham Biosciences, Little Chalfont, UK). The image was saved by the Typhoon 8600 laser scanning system (Amersham Biosciences). The density and width of each band was quantified using the ImageQuant 5.0 software (Amersham Biosciences). Anti-MCM2 (N-19; sc-9839), anti-MCM6 (C-20; sc-9843), and anti-MCM7 (141.2; sc-9966) were from Santa Cruz Biotechnology. Anti-MCM5 (MCA 1860) was from Serotec. Anti-
-tubulin was from Sigma. Secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology. Triton X-100-extracted nuclei were prepared as described previously (23). Briefly, cells were cultured in 100-mm plates; then, they were washed three times with ice-cold PBS and lysed for 10 min on ice with 1 ml ice-cold CSK buffer [10 mM 1,4-piperazinediethanesulfonic acid (pH 6.8), 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin] containing 0.5% Triton X-100. After low-speed centrifugation (3000 rpm, 3 min at 4 C), nuclei were washed once with 1 ml ice-cold 0.5% Triton X-100 in CSK, centrifuged, and directly resuspended in 1x SDS sample buffer. Protein amounts of Triton X-100-soluble and -insoluble fractions deriving from comparable number of cells were loaded on SDS-PAGE and subjected to Western blotting.
Northern blot
Cells were harvested and stored frozen until RNA was extracted. Total RNA was isolated by the RNeasy Kit (QIAGEN, Crawley, West Sussex, UK) and subjected to on-column DNase digestion with the RNase-free DNase set (QIAGEN) according to the instructions of the manufacturer. RNA was analyzed by electrophoresis through 1% agarose gel and visualized with ethidium bromide. Total RNA (20 µg) was size fractionated on a denaturing formaldehyde agarose gel and blotted onto nylon filters (Hybond-N; Amersham Biosciences). The MCM5 probe was a 503-bp RT-PCR product obtained using the following amplimers: forward (mcm5f), 5'-ACTCAAGCGGCATTACAACC-3'; reverse (mcm5r), 5'-GTCTGGAAGTCCACGCATTT-3'.
The MCM7 probe was a 174-bp RT-PCR product obtained using the following amplimers: forward (mcm7f), 5'-TGAACTCGGGAAGAAGCA-3'; reverse (mcm7r), 5'-TGTACGGCATCAGCAAAGAG-3'.
We used the random oligonucleotide primer kit (Amersham Biosciences) to label the probe. Hybridizations and washings were performed under stringent conditions. Autoradiography was performed with Eastman Kodak (Rochester, NY) XAR films at –70 C for 7 d with intensifying screens. The image was saved by the Typhoon 8600 laser scanning system, and the density and width of each band was quantified with the ImageQuant 5.0 software.
Reporter gene assay
A 440-bp PCR fragment of the human MCM7 promoter spanning from –500 to –60 relative to the transcription start (24) was cloned into the pGL3 Basic vector (Promega, Madison, WI) carrying the luciferase reporter gene, to obtain the pGL3-MCM7LUC plasmid. PCR amplification was performed on normal human genomic DNA with the following primers: forward, 5'- TAGATCTCAGCCCCAAGGGTCTAGG-3'; reverse, 5'- TAAGCTTGGGAAGCTGAGAATCTTCCG-3'.
The obtained construct was controlled by DNA sequencing. Cells were transfected by using the LipofectAMINE Reagent (Invitrogen) according to the instructions of the manufacturer. Cells were transfected with the different expression plasmids together with 500 ng of the reporter plasmid DNA and 100 ng pRL-null DNA (a plasmid expressing the enzyme Renilla luciferase from Renilla reniformis) as an internal control. In all of the cases, the total amount of transfected plasmid DNA was normalized by adding empty vector DNA. After 16 h from transfection, Firefly and Renilla luciferase activities present in cellular lysates were assayed using the Dual-Luciferase Reporter System (Promega), and light emission was quantified using the Lumat LB9507 luminometer (EG Berthold, Bundoora, Australia), as specified by the manufacturer. Each experiment was performed in triplicate. Expression vectors for wild-type p53, p53 R248W, and p53 R273H mutant cDNAs, cloned in the pCMV6 vector, have been described previously (25).
RNA silencing
Premade (Smart Pool) small inhibitory duplex RNAs (siRNA) for MCM7 were purchased from Dharmacon (Chicago, IL). Another set of anti-MCM7 siRNA was designed with a program available online (http://jura.wi.mit.edu/siRNAext/) and synthesized by Proligo (Boulder, CO). The sense strand of the second MCM7 siRNA duplex was 5'-AAGAUGUCCUGGACGUUUACAdTdT-3'. The anti-laminA/C siRNA (Proligo) used as control was 5'-CUGGACUUCCAGAAGAACAdTdT-3'. Scrambled control siRNA (Proligo) was 5'-ACCGUCGAUUUCACCCGGdTdT-3. S11T cells were grown under standard conditions. The day before transfection, cells were plated in six-well dishes at 50–60% confluency. Transfection was performed with 5 µg siRNA and 6 µl oligofectamine reagent (Invitrogen, Groningen, The Netherlands), as described previously (26). Cells were harvested at 72 h after transfection.
Statistical analysis
Significance was determined by the two-tailed Fisher’s test (Statistica 6.0; StatSoft, Tulsa OK). P < 0.05 was considered statistically significant.
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Results |
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Using immunohistochemistry with specific anti-MCM5 and anti-MCM7 monoclonal antibodies, we evaluated MCM5 and MCM7 expression levels in 30 normal thyroid samples, 20 PTC samples, and 52 ATC samples. In 65% of ATC samples, more than 10% of cells (on average, 25% of the cells) were positive for MCM5, and, in 73% of the samples, more than 10% (on average, 30% of the cells) were positive for MCM7 expression. In both cases, the signal was exclusively nuclear. Virtually no MCM5 and MCM7 expression (<2% of cells) was detected in normal and PTC samples (Fig. 1
and Table 1). Expression of PCNA, a conventional proliferation marker, was also examined. PCNA expression was significantly correlated (P < 0.01) with MCM5 and MCM7 expression being detected mainly in ATC samples (65%). Only two PTC samples contained a few PCNA-positive cells (<5% of cells) (Table 1). Normal tissue was invariably PCNA negative. Representative examples of the expression of MCM5, MCM7, and PCNA are shown in Fig. 1. These findings suggested that MCM5 and MCM7 overexpression may be related to the high mitotic activity of ATC. We next separated pure ATC samples from ATC samples containing well-differentiated (PTC or Hurthle-cell variant FTC) areas identified by morphological features. The expression of MCM5 and MCM7 was more prevalent in pure ATC (P < 0.05) and in ATC with coexisting Hurthle-cell areas than in mixed PTC-ATC samples (Table 2). In parallel, pure ATC samples were also more frequently PCNA positive than mixed PTC-ATC samples (P = 0.054) (Table 2). More importantly, when MCM7, MCM5, and PCNA were detected in mixed PTC-ATC tumors, their expression was invariably confined to the ATC area (data not shown).
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and data not shown). Phosphor imaging quantitation showed that MCM5 and MCM7 were induced, on average, by 4 ± 1-fold and 8 ± 3-fold, respectively, in ATC with respect to normal samples.
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). Finally, we examined primary cultures derived from two samples each of normal thyroid, follicular adenoma, PTC, and ATC. As shown in Fig. 2C, MCM protein expression was higher in ATC cells than in normal or in differentiated tumor cells. MCM5 and MCM7 are overexpressed at mRNA level in ATC
To obtain a semiquantitative estimate of the extent of MCM5 and MCM7 overexpression, we compared protein levels in ATC cells (HTU8 and S11T) with those in normal cells (S11N) by loading increasing amounts of total protein extracts on SDS-PAGE (Fig. 3A) and compared band intensity by phosphor imaging. MCM5 and MCM7 expression was about five times greater in ATC cells than in normal cells (Fig. 3A).
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). MCM5 and MCM7 had a half-life of 24 h in both ATC and normal cells, which indicates that posttranslational mechanisms were not involved in MCM protein overexpression.
We next measured MCM5 and MCM7 mRNA levels in normal and ATC cells by Northern blot (Fig. 3C). MCM mRNA levels were higher in ATC than in normal cells. Importantly, the increase in mRNA (
5-fold) was similar to the increase in protein, which indicates that changes at the mRNA level caused the enhanced MCM5 and MCM7 expression. Increased mRNA levels may be due to gene amplification, increased transcription, or, less frequently, an increased mRNA half-life. Amplification of MCM genes has been described in hypopharyngeal carcinomas (30). Hence, we determined the MCM2–MCM7 gene copy number by semiquantitative PCR on genomic DNA in human samples (tissue samples and primary cell cultures) listed in Fig. 2. There was no gene amplification in any sample (data not shown), which suggests that enhanced MCM expression results from increased transcription.
The human MCM7 gene transcriptional promoter has been characterized (24). To prove our hypothesis, we cloned the MCM7 promoter (–500 to –60 relative to the transcription start site) upstream to the luciferase reporter gene (pGL3-MCM7LUC). The pGL3-MCM7LUC construct (or the empty vector) was transiently transfected in triplicate in S11N and ARO cells, and luciferase activity was measured. The MCM7 promoter displayed a significantly higher (>10-fold) activity in anaplastic cells compared with normal ones (Fig. 3D, top). MCM7 expression is positively controlled by the E2F (E2 promoter binding factor) transcriptional factor (31), and the MCM7 promoter contains binding sites for E2F (24). By stimulating increased levels of the CDK inhibitor p21WAF1, p53 is able to reduce pRb phosphorylation levels and, in turn, E2F transcriptional activity, potentially keeping MCM7 expression under check (32). Thus, increased MCM7 gene transcription in ATC could be due to the inactivation of p53, a genetic hallmark of this tumor type (7, 8). We addressed this possibility by measuring p53 effects on pGL3-MCM7LUC transcription. As shown in Fig. 3D (bottom), adoptive overexpression of wild-type p53, but not of two inactive p53 mutants (R248W and R273H), decreased by almost 3-fold pGL3-MCM7LUC activity in ARO cells.
MCM7 overexpression is required for DNA synthesis in ATC cells
In mammalian cells, MCM proteins are present in the nuclei in two forms: one soluble form that can be extracted by non-ionic detergents and one insoluble form (23). The insoluble form is the active one, associated with prereplication chromatin as part of the pre-RC. The soluble form is considered inactive and no longer capable of binding to chromatin. We differentially extracted soluble and insoluble MCM7 from ATC and normal cell nuclei. Although the ratio chromatin-loaded/chromatin-unloaded MCM7 was slightly reduced in ATC with respect to normal cells, the absolute amount of chromatin-bound MCM7 was definitely increased in ATC cells (4-fold by phosphor imaging) (Fig. 4A).
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). Finally, we studied the ability of MCM7-silenced cells to replicate DNA, by measuring BrdU incorporation rate. Decreased MCM7 protein levels reduced the rate of BrdU incorporation by about 50% in S11T cells, which suggests that MCM7 expression is required to sustain the proliferative capacity of ATC cells (Fig. 4B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data indicate that increased gene transcription results in MCM overexpression in ATC. This fits nicely with the molecular events described in ATC. Indeed, MCM gene transcription is controlled by the E2F/pRb axis (24, 31), which, in turn, is controlled by p53. p53 stimulates increased levels of the CDK inhibitor p21WAF1, thereby inhibiting pRb phosphorylation and blocking E2F transcriptional activity (32). This pathway is probably disrupted in ATC because of their high prevalence of p53 mutations (7, 8). Accordingly, we have shown that the p53-null ARO ATC cells display a high rate of transcription from the MCM7 promoter compared with normal cells and that reintroduction of a wild-type p53 gene into ARO cells decreases the activity of the promoter. The mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) pathways are also known to stimulate MCM expression (34, 35). Both pathways are probably hyperactive in ATC due to gain-of-function mutations in Ras (5), BRAF (6), and AKT/PKB (protein kinase B) (36) and might contribute to MCM up-regulation, as well.
It is the prevailing view that ATC represents a terminal dedifferentiation of a preexisting PTC or FTC. Comparison of the histology of fatal PTC with tumors of patients who survived their disease reveals a high incidence of foci of spindle and giant cell metaplasia that are indicative of transition into ATC (2, 3). Although histologic examination of different samples within the same thyroid tumor is sufficient for diagnostic identification of ATC, MCM testing could be used to identify ATC foci in otherwise well-differentiated carcinomas.
Finally, ATC is refractory to most treatment strategies and is almost always fatal. Current chemotherapeutic agents do not induce consistent beneficial therapeutic responses. Local tumor control with surgery and radiotherapy merely provides palliation and delays death. MCM proteins promote DNA synthesis thanks to their helicase function that is essential for DNA replication fork unwinding (14). Our data raise the possibility of molecular therapy of ATC based on targeting MCM proteins by interfering with their expression or blocking their helicase function with small inhibitors.
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Acknowledgments |
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Footnotes |
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First Published Online May 17, 2005
Abbreviations: ATC, Anaplastic thyroid carcinomas; BrdU, bromodeoxyuridine; FTC, follicular thyroid carcinomas; MCM, minichromosome maintenance protein; PCNA, proliferating cell nuclear antigen; pre-RC, prereplication complex; PTC, papillary thyroid carcinoma; siRNA, small inhibitory duplex RNA; WDC, well-differentiated thyroid carcinomas.
Received December 16, 2004.
Accepted May 10, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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