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RESEARCH REPORT |
1 Department of Oral and Maxillofacial Surgery, Institute of Dentistry, University of Turku, Lemminkäisenkatu 2, FIN-20520 Turku, Finland;
2 Department of Medical Genetics, Haartman Institute and Helsinki University Central Hospital, FIN-00014 University of Helsinki, Finland; and
3 Haartman Institute, Department of Pathology, FIN-00014 University of Helsinki, Finland;
4 corresponding author, heikinhe{at}netlife.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: gene expression • cDNA • microarray • odontogenic tumor • RT-PCR • tooth germ.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Ameloblastoma is believed to arise from epithelial cells of the developing tooth, including cells of the dental lamina and enamel organ (reviewed in Melrose, 1999). The differentiation level of ameloblastoma cells remains at the cap/bell stage of tooth development. Follow-up and cell-proliferation studies of ameloblastoma confirm its slow growth rate (Jääskeläinen et al., 2002). Other clinical characteristics include preference for the molar area of the mandible, an infiltrative growth pattern, and a substantial tendency for recurrence without potential to metastasize (Vickers and Gorlin, 1970; Kramer et al., 1992). Previous gene expression studies have shown both similarities and differences with the developing human tooth (Heikinheimo, 1993). However, genetic changes in ameloblastoma are as yet poorly understood (Jääskeläinen et al., 2002).
The cDNA microarray is a powerful tool in the analysis and classification of human tumors (Duggan et al., 1999; Ross et al., 2000; Todd and Wong, 2002). We compared gene expression patterns in ameloblastoma and cap/bell-stage human tooth germs in cDNA microarray. The aims of our study were to characterize the gene expression profile of ameloblastoma, and to identify candidate genes that may be involved in the origination and progression of this tumor.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Tumors
Eight fresh ameloblastoma specimens (3 follicular, 4 plexiform, and 1 of the acanthomatous type) were obtained during surgery in the Department of Oral Diseases, Turku University Central Hospital, Turku, Finland, from six men and one woman (age range from 18 to 73 yrs, mean 51 yrs). The specimens were snap-frozen in liquid nitrogen and stored at -70°C. Five tumors were primary and 3 were recurrent ameloblastomas of the mandible. The Ethical Committee of the Medical Faculty, University of Turku, Finland, had approved the study.
RNA Isolation
Total RNA was isolated from fresh-frozen tissues with use of the RNeasy Total RNA kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. To remove genomic DNA contamination, we treated RNA with RNase-free DNase 1 (Clontech Laboratories Inc., Palo Alto, CA, USA). The quality and integrity of the RNA were checked by spectrophotometry and agarose-gel electrophoresis.
cDNA Probe Labeling and Hybridization
A single-pass reverse-transcription reaction was used for preparation of labeled cDNA from total RNA, with the use of SuperScript reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD, USA). 33P-dATP was used as a labeled nucleotide according to the manufacturer’s recommendations (www.clontech.com). Atlas Human Cancer cDNA Expression Array Filters containing 588 cancer-related human cDNA fragments were purchased from Clontech Laboratories Inc. Pre-hybridization, hybridization, and post-hybridization washes were performed according to Clontech instructions. The membranes were then exposed to Fuji BAS-MS image plates (Fuji, Nakanuma, Japan), with an intensifying screen, at room temperature for 4 days. For repeated use, membranes were stripped by being boiled in a 0.5% SDS (sodium dodecyl sulphate) solution and scanned for residual hybridization.
The hybridization experiments were repeated for 3 of 9 samples (normal control tissue and 2 tumors) with the use of newly synthesized probes from original total RNA under the conditions described above.
Image Analysis
The array image plates (Fuji) were scanned by means of a BAS-2500, Fuji Bio-Imaging Analyzer (Fuji). Images obtained (in TIFF format) were imported for examination with Atlas Image 1.5 analysis software (Clontech). We compared the reference image with tumor images using global normalization, subtracting the average of the intensity differences of the genes in one array from each intensity difference value, thus standardizing the sample average in all arrays to zero. Genes exhibiting intensity ratios above 3 or less than 0.4 in 5 or more samples were classed as differentially expressed. We chose these limits to improve the selectivity of gene identification in our array experiments from less than 1% obtained with the software using a ratio of 2.0 for overexpression and 0.5 for underexpression. All differentially expressed spots on filters were also confirmed visually.
Statistical Analyses Relating to Confirmation of Gene Expression
XY scatterplots were used in parallel with global normalization. Preparation of scatterplots is a powerful method of analyzing gene expression levels in two independent array experiments. In a scatterplot, the coordinates of each dot are the gene expression values obtained in each experiment. Regression lines (y = ax + b) were drawn and R2 values derived with the use of Microsoft Excel 2.0. R2 is the Pearson correlation coefficient of the least-squares regression line. The standard deviation of mean distance between points and the least-squares regression line was calculated in Microsoft Excel 2.0. An area bounded by lines representing the standard deviation multiplied by 1.96 on both sides of the least-squares regression line was drawn on the XY scatterplot to define a 95% confidence interval. Genes outside this area were classed as differentially expressed.
Quantitative Real-time RT-PCR
To obtain comparative information on gene expression profile, we performed real-time reverse-transcription/polymerase chain-reaction (RT-PCR) on the same tumor-derived RNA samples. Primers were designed and prepared for 3 genes overexpressed (FOS, COL8A, and TNFRSF1A) and for 3 genes underexpressed (CDH11, SHH, and TGFB) in the microarray by TIB Molbiol (Berlin, Germany) (Table 1
). A total of 500 ng RNA from each tumor studied and normal control tissue was reverse-transcribed into cDNA with the use of an oligo-p(dT)6 primer from the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche Diagnostics Corp., Indianapolis, IN, USA). PCR reactions were performed simultaneously for all tumor samples to minimize the effect of variation in amount and quality of the cDNA among samples. The real-time RT-PCR was performed with the use of a LightCycler rapid thermal cycler system (Roche Diagnostics GmbH, Mannheim, Germany). The reaction volume was 10 µL containing 1 µL "Hot Start" reaction mixture from the LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics GmbH), 2.5 mM MgCl2, 0.5 mM of each primer, and 1 µL of diluted cDNA (1:10). Initial denaturation was carried out at 95°C for 7 min, with denaturation at 95°C for 15 sec and annealing at 60-65°C for 5 sec, followed by 48 cycles of elongation at 72°C for 10 sec. To verify the amplification specificity, we performed melting curve analyses using an initial denaturation at 95°C for 10 sec, followed by 20 sec at 55°C, and then heated the samples at 95°C at a slow rate of 0.1°C/sec with continuous fluorescence detection. Each patient sample was run in parallel with the control normal tissue as well as a negative control. In addition, we obtained standard curves to calculate the relative gene expression by running a dilution series of the ß globulin gene (LightCycler-Control Kit DNA; Roche Diagnostics GmbH) in each assay according to the manufacturer’s instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Sixty genes were differentially expressed in at least 2 of the tumors (25%) when compared with respective normal control tissue (Fig. 1A). Eleven genes were overexpressed more than three-fold, and 23 genes were underexpressed to below 40% of the control level. The most highly overexpressed gene was FOS (from eight- to 14-fold, Fig. 2), followed by tumor-necrosis-factor-receptor 1 (TNFRSF1A). Genes for SHH, ARHGAP4, DCC (encoding mediators or receptors of cell signaling), CDH 12, and CDH 13 (encoding calcium-dependent cell-adhesion molecules), and TDGF1 and TGFB1 (encoding growth factors) were underexpressed in all cases (Fig. 1A).
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). R2 values can be considered as quantifying similarity between results of the experiments concerned. Two aliquots of pooled RNA derived from 20 tooth germs were used for control experiments. A very high R2 value of 0.986 was obtained. High degrees of similarity in relation to gene expression patterns were also found between tumor cases (R2 = 0.919).
Gene Expression by Image Analysis and XY Scatterplots
We obtained similar gene expression profiles using image analysis software (Clontech) with global normalization and XY scatterplots. These two methods identified 34 genes differentially expressed in most tumors (5 or more) in comparison with the reference. We also identified 60 differentially expressed genes in at least 2 tumors by means of global normalization analysis and 65 (the additional 5 being GIP3, DSC1, EFNB2, BMP5, and CDH6) by means of XY scatterplots (data not shown).
Gene Expression by Real-time RT-PCR
We compared relative gene expression change obtained by cDNA microarray with that by real-time RT-PCR methods to substantiate the reproducibility of our cDNA microarray results. The logarithm ratio between the gene expression levels of tumor samples and normal control tissue samples was calculated for 6 selected genes (Table 1). Genes for FOS, COL8A, and TNFRSF1A were found to be overexpressed and CDH11, SHH, and TGFB underexpressed by quantitative real-time RT-PCR in all 5 ameloblastomas studied (Fig. 1C). Although the degree of the relative gene expression change in the selected genes varied somewhat between the two methods used, we prefer to ascribe this to differences in sensitivity between the two methods. This is supported by the consequent pattern of lesser changes in the gene expression level detected in cDNA microarray compared with real-time RT-PCR (Fig. 1C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The second most highly overexpressed gene was tumor-necrosis-factor-receptor 1A (TNFR1A), which is involved in the induction of AP-1 activity (for review, see Baud and Karin, 2001). Signal transduction is mediated by TNF-
, a potent cytokine produced by many cell types. It plays an important role in inflammation, and in control of cell proliferation, differentiation, and apoptosis. Recently, TNFs and their receptors have been shown to be involved in the development of ectodermally derived organs such as teeth (Laurikkala et al., 2001). Mutations in some TNF family members may result in human ectodermal dysplasia, causing abnormalities, e.g., in teeth (Monreal et al., 1999). Consequently, overexpression of FOS and TNFR1A may be important in the oncogenic transformation pathway of ameloblastoma.
Among the other overexpressed genes, it has been suggested that matrix metalloproteinases (MMPs), which degrade various ECM components, play important roles in organogenesis, tissue remodeling, and tumor invasion (Sato and Seiki, 1996; Ha et al., 2001). Overexpression of these genes has been reported in several malignancies, including hepatocellular carcinoma, colon carcinoma, and breast carcinoma (Gorrin Rivas et al., 1998; Jacobs et al., 1999). On the other hand, type-VIII collagen (encoded by COL8A) is an ECM protein anchored to the plasma membrane via a transmembrane segment. It has been suggested that it mediates cell adhesion (Shuttleworth, 1997). Distribution of type-VIII collagen in tissues is limited to endothelial cell-basement membranes. Overexpression of such a protein could have a bearing on processes involved in tumor angiogenesis.
Several genes in our study exhibited marked underexpression. A few of them also are known to be expressed aberrantly in other jaw diseases. Sonic hedgehog (SHH), which is expressed during early murine tooth development, is released by dental epithelium and induces expression of the transcription factor PTCH in mesenchyme (Hardcastle et al., 1998). It has been suggested that dysregulation of the PTCH/SHH signaling pathway in epithelial-mesenchymal interactions plays a role in the formation of odontogenic keratocysts (Lench et al., 1997; Barreto et al., 2000). This condition, although presumably not neoplastic, is clinically problematic because of its frequent recurrence, even after long symptom-free periods.
Many of the genes that were found to be underexpressed in the study reported here are involved in the regulation of cell adhesion, cell shape, and angiogenesis. For instance, CDHs, KRT7, NOTCH, and TGFB1 may be involved in disturbances of cell-to-cell adherence junctions and cell-to-cell communication (Pepper, 1997; Kawamura-Kodama et al., 1999; Pishvaian et al., 1999; Blobe et al., 2000; Bessho et al., 2001). This suggests that gap-junction communication may be low and cell adhesion lost in ameloblastomas, as described for many types of neoplasia. Such alterations in cell-membrane environment could also increase the locally aggressive growth potential of ameloblastomas.
Analyses of results of replicate experiments by two statistical methods gave essentially similar gene expression profiles. R2 values of over 90% were obtained, which suggested a satisfactory level of reproducibility. When results obtained by image analysis, global normalization, and XY scatterplots were compared, discrepancies were seen in relation to 5 genes. This level of discrepancy, relating to just 5 of the 588 genes (0.85%), was not considered significant, although any one of these genes could play an important role in connection with an individual tumor. Real-time PCR experiments gave similar relative gene expression changes, further supporting our microarray cDNA results. We also undertook cluster analysis as another means of assessing significances of differences relating to gene expression in each case and of relationships between cases. The results obtained reveal no association with known parameters, e.g., the histopathological pattern of the tumor. This finding corresponds to previous observations relating to ameloblastoma obtained by other methods (Melrose, 1999).
To conclude, we have demonstrated that several genes are over- or underexpressed in ameloblastoma compared with the tooth germ, its putative tissue of origin. Our findings provide a new basis for further studies intended to improve understanding of the molecular pathway of ameloblastoma development. These may reveal new targets relating to tumor diagnosis, prognosis, and intervention. In our study, several previously unidentified genes in the human tooth germ were also detected. These may be significant in relation to the regulation of normal tooth development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received August 27, 2001; Last revision March 25, 2002; Accepted May 13, 2002
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