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Molecular Biology and Genetics |
Human Cancer Genetics Program [M. A. A., M. E. G-P., C. D. M., A. P. P., C. P., C. E.], Clinical Cancer Genetics Program [M. A. A., M. E. G-P., C. D. M., C. E.], Comprehensive Cancer Center [M. A. A., M. E. G-P., C. D. M., S. M. J., C. P., C. E.], Division of Human Cancer Genetics, Department of Molecular Virology, Immunology, and Medical Genetics [M. A. A., M. E. G-P., C. P., C. E.]; Division of Human Genetics, Departments of Internal Medicine [C. D. M., C. E.], Pathology [C. D. M.], and Physiology and Cell Biology [S. M. J.], The Ohio State University, Columbus, Ohio 43210; Department of Surgery [O. G., C. H-V., H. D.] and Institute of Pathology [U. K.], Martin-Luther University, Halle-Wittenberg, Halle/Saale, Germany; Division of Medical Genetics, University of Leicester, Leicester, United Kingdom [M. A. A.]; and Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge, United Kingdom [C. E.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15 of 19 FTC tumors (79%) by semiquantitative reverse transcription-PCR. Caveolin-1 showed preferential down-regulation of its ß-isoform at both the mRNA and protein level, suggesting a distinct function for this isoform. Caveolin-1 is of particular functional interest because it has been shown to interact with PTEN, the tumor suppressor gene mutated in Cowden syndrome, an inherited multiple hamartoma syndrome that includes predisposition to FTC. Immunohistochemical analysis of 141 thyroid tumors of various histological types showed significantly fewer caveolin-1-positive tumors in FTCs, including insular type tumors, and Hurthle cell carcinomas in comparison with normal thyroid. PTC and anaplastic thyroid carcinomas did not show significant down-regulation, and thus, caveolin-1 may become a useful molecular marker to differentiate the various histologies of thyroid malignancies. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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at 3p25 in 53–62.5% of a total of 32 FTCs in three series (10, 11, 12)
and hypermethylation of the RASSF1A promoter at 3p21 in 70% of FTCs (13)
. To further elucidate the etiology of sporadic FTC, we conducted microarray expression profiling of FTC tumors compared with normal thyroid tissue. Differentially regulated genes were prioritized for analysis by targeting those in regions of LOH reported previously. In this way, we have identified five genes in three chromosomal locations which may represent novel tumor suppressor genes commonly down-regulated in sporadic FTCs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fusion transcript was assayed as described previously (10)
. The two positive tumors (FC5 and 1989T) were then used for microarray analysis, together with seven negative tumors and six normal thyroid samples. Total RNA (8 µg) was reverse transcribed, in vitro translated into biotinylated cRNA, and hybridized to U95A version 2 GeneChips (Affymetrix, Inc., Santa Clara, CA) following the manufacturer’s protocols. Duplicate experiments could not be performed because of limited RNA availability. However, our past and continuing experience using replicates of two and three per sample has demonstrated excellent reproducibility. Furthermore, to control for any variability between chips, data for each sample were normalized to a mean intensity value of 1500. Data were processed with Microarray Suite v5.0 and Data Mining Tool software packages (Affymetrix, Inc.). Because the majority of tumor samples did not have paired normal tissue available, multiple pairwise comparisons were made between all normal and tumor samples, a total of 54 comparisons. These data were then ranked to select genes showing >2-fold change across 70% of comparisons. cDNA for follow-up analyses was synthesized from 1-µg aliquots of RNA pretreated with DNase I (Invitrogen), then reverse transcribed using SuperScript II (Invitrogen) primed with random hexamers. Semiquantitative RT-PCR was conducted in a duplex reaction with GAPDH primers as an internal amplification control, except the chordin-like transcript (CHRDL), for which the ß-actin competimer kit (Ambion, Austin, TX) was used at a ratio of 4:6 amplimer:competimer. GDF10, GPC3, and CHRDL were amplified for 27–29 cycles in a 20-µl reaction, and the entire sample was loaded onto a 1.5% agarose gel. For more quantitative assessment, caveolin-1 and -2 were amplified with GAPDH for only 24 cycles with 6FAM-labeled primers. Reactions were then dried down and resuspended in 3 µl of formamide loading buffer with ROX400 size standard, and a 1.5-µl aliquot was loaded onto a ABI377 genotyping gel (Applied Biosystems, Foster City, CA), allowing accurate measurement of peak heights for each transcript. All primer sequences are available on request.
Isoform-specific quantitative real-time PCR was conducted with the QuantiTect SYBR Green RT-PCR system (Qiagen) on an ABI7700 instrument (Applied Biosystems) according to the manufacturers’ recommendations using the standard curve quantitation method. Standards were prepared from a mixture of RNA from four normal thyroids and analyzed in duplicate. Primer sequences were: CAV1
5'-AACCTCCTCACAGTTTTCATCC and 5'-CTTGTTGTTGGGCTTGTAGATG, CAV1ß 5'-TTGCATTTTTCCTCCCACC and 5'-CTTGTAGATGTTGCCCTGTTCC, CAV2 5'-ACGACTCCTACAGCCACCACAG and 5'-AGATGCGAGTTGAGCCGGT, CAV2ß 5'-ACGTCCTTCCCTCCTGCTTC and 5'-AGTGCGTAGTCACCGGCTCT, and GAPDH 5'-GGGCTGCTTTTAACTCTGGTAA and 5'-ATGGGTGGAATCATATTGGAAC. Test samples were analyzed in triplicate, and an average quantity was derived for each transcript, which was then normalized to the average quantity of GAPDH for that sample.
Microsatellite markers for LOH analysis were selected from Genethon and Marshfield genetic maps according to their current physical locations and analyzed as described previously (3) . These were D7S486, D7S522, D7S523, and D7S2406 on chromosome 7 and D10S220, D10S1766, and D10S1793 on chromosome 10. Mutation analysis was conducted by single-strand conformation analysis or Big Dye terminator sequencing (Applied Biosystems) of PCR amplified products according to the manufacturer’s recommendations. Primers for exon amplification are available on request. All PCR reactions consisted of 35–40 cycles using HotStar mastermix with 1x Q-solution (Qiagen), 1 µM primers, and annealing at 55°C, except GDF10 reactions, which used Platinum Taq polymerase and buffer (Invitrogen).
Methylation analysis of promoter regions was conducted on bisulfite-modified genomic DNA (ref). Controls included nontreated DNA, bisulfite-modified normal thyroid DNA, and bisulfite modified DNA, in which all CpG dinucleotides were methylated by SssI-methylase treatment (New England Biolabs, Beverly, CA). Two regions of the CAV1 promoter region were analyzed as described previously (14) . Additionally, primers 5'-AGGTGAGATTGAGTTTTAGGAT and 5'-CCACACCCCTCTATACTCTACA were designed for a CpG-rich region in intron 1. In all three cases, amplified PCR products were cloned into pCR2.1TOPO (Invitrogen), and plasmid DNA was prepared from multiple individual colonies with QIAprep spin miniprep kit (Qiagen) and sequenced by Big Dye terminator chemistry (Applied Biosystems) using M13 primers. Methylation status of GDF10 and GPC3 was assayed by methylation-specific PCR. For GPC3, primers specific for methylated DNA (5'-CGTATTGTTTTCGTTCGGTTTT and 5'-TTTCCTCGCAACTACCTAAACG) and unmethylated DNA (5'-AGAGTGGTTGTGAGTGGGTAGT and 5'-CTCAACAAACCTAACAATAACCC) were combined in a duplex reaction annealing at 55°C. GDF10 primers analyzed a region between -578 and -413 relative to the translation start site. The methylation-specific reaction used primers 5'-CGGCGTCGATATATAGGAGTC and 5'-AAATCGTCCCTAACCCGACT for 35 cycles annealing at 61°C, whereas the assay specific for nonmethylated DNA was conducted for 38 cycles annealing at 56°C with primers 5'-TGGTGTTGATATATAGGAGTT and 5'-AAATCATCCCTAACCCAACT.
For caveolin-1 immunohistochemistry, a total of 150 sections was analyzed, as detailed in Table 3
, including 17 of the 19 FTCs used for RT-PCR analysis, additional independent cases accrued through the Department of Pathology, The Ohio State University, and a commercial human thyroid cancer tissue array (IMH-319; Imgenex Corp., San Diego, CA). Controls were derived from normal postmortem thyroid specimens and normal tissue adjacent to tumors. After antigen retrieval for 30 min in citric acid buffer at 94°C, the antibody (clone 2297; BD Transduction Laboratories, Palo Alto, CA) was incubated at a dilution of 1:500 for 1 h at 37°C. Slides were blocked for endogenous biotin activity using a biotin blocking system (DAKO Corp., Carpinteria, CA) followed by antibody detection with the LSAB2 streptavidin-horseradish peroxidase system (DAKO Corp.). Slides were counterstained with hematoxylin I and scored for the presence or absence of specific staining in the plasma membrane or Golgi. The presence of strong colloidal staining in some samples was not scored. Results were analyzed for statistically significant differences using two-tailed Fisher’s exact test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 74 genes showed a 2-fold down-regulation in 70% of pairwise comparisons, of which 24 were down-regulated by 4-fold by the same criteria. A notable exception was RASSF1A, which although reported to be hypermethylated in a high proportion of FTCs (13)
, showed no change in expression across all nine tumors. Genes were prioritized for further study as follows. Firstly, three of the most consistently down-regulated genes localized to regions of LOH reported previously in follicular thyroid neoplasia and were therefore candidate tumor suppressor genes, namely caveolin-1 (CAV1) and caveolin-2 (CAV2) at 7q31.1 and bone morphogenetic protein 3b (GDF10) at 10q11.1. Additionally, two down-regulated X-linked genes, glypican-3 (GPC3) and a novel chordin-like gene (hereafter referred to as CHRDL), were examined because of their functional links with the bone morphogenetic protein family and, thus, a possible relationship to GDF10. PPAR was found to be up-regulated in two tumors known to carry a PAX8-PPAR translocation but was down-regulated in most of the nontranslocation tumors and is the subject of a separate study (15)
. In unsupervised cluster analysis, the translocation-positive tumors could not be distinguished from the nontranslocation tumors (data not shown), but in view of the very small numbers involved, no firm conclusions could be drawn as to whether they have distinct expression profiles.
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. For both genes, statistical analysis with the t test demonstrated a significant difference between the mean ratio in the FTC group compared with controls (CAV1: normals 1.52 ± 0.6, FTCs 0.33 ± 0.55, P < 0.001; CAV2: normals 1.83 ± 0.64, FTCs 0.39 ± 0.67, P < 0.001, both with 30 degrees of freedom). A cutoff of 2 SD below the normal mean was applied as a definition of down-regulation (i.e., ratio < 0.33 for CAV1 and <0.54 for CAV2). By these criteria, 15 of 19 FTCs examined (79%) were coordinately down-regulated for both caveolin genes, the remaining four tumors being in the normal range for both transcripts (Table 2)
. Analysis with flanking microsatellites indicated LOH in 6 of 17 tumors tested (35%), but no mutations were identified on direct sequencing of the coding region of the two genes.
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. This pattern was absent in the majority of FTC sections examined, whereas nearby vessel endothelial cells continued to stain strongly. Analysis of 141 thyroid tumors of various histological type by tissue microarray immunohistochemistry showed significantly fewer tumors positive for caveolin-1 staining compared with normal thyroid in all FTC and Hurthle cell groups, including insular type and minimally invasive tumors, but not in PTC or anaplastic thyroid carcinomas (Table 3)
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and ß Isoforms of Caveolins 1 and 2 Are Encoded by Different Transcripts that Are Differentially Down-Regulated in Some FTCs. and ß, the -isoform having an additional 31 amino acids at the NH2 terminus in both cases. It was originally thought that these were derived from the same transcript, with the ß-isoform representing an alternative translational start site in exon 2 (16)
. However, recent evidence in mouse shows that the CAV1ß is predominantly encoded by an alternative transcript derived from a promoter in intron 1 (17)
. We hypothesized that the same mechanism might be operative in humans, particularly in view of a putative CpG island in intron 1 (18)
. RT-PCR experiments in normal thyroid using a forward primer positioned in intron 1 of CAV1 and a reverse primer in exon 3 amplified a transcript of the predicted size (Fig. 3A)
, confirming that there is transcription from the alternative promoter. Furthermore, similar experiments with caveolin-2 showed that the same mechanism operates for its ß-isoform (Fig. 3A)
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-isoform of caveolin-1 (20)
, whereas we chose an antibody detecting both isoforms. We therefore hypothesized that the discrepancy might be attributable to significantly higher expression of CAV1ß compared with CAV1 in normal thyroid. To test this, quantitative real-time RT-PCR analysis was used to examine the relative abundance of the different transcripts in a subset of normal and tumor specimens. For caveolin-1, we found that both isoforms were expressed in normal thyroid and approximately equal amounts (Table 4)
. In the FTCs known to be down-regulated for total CAV1, both isoforms were individually confirmed to be down-regulated in comparison with GAPDH. However, the ß-isoform was consistently down-regulated to a slightly greater extent than the , leading to a small shift in the :ß ratio. This was apparent even for tumor 49T, which did not show significant down-regulation for overall CAV1 expression, whereas one down-regulated tumor, 02E108, showed a highly skewed ratio. The difference between the mean :ß ratio in tumors (6.6 ± 7.05) and normals (1.02 ± 0.49) was statistically significant (t = 2.24, P < 0.05 with 13 degrees of freedom). Similar experiments with caveolin-2 showed no significant difference between the mean :ß ratio in tumors (1.89 ± 0.66) compared with normals (1.2 ± 0.84).
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. In addition, although normal thyroid and tumor FC7 showed approximately equal amounts of the two isoforms, the ß-isoform was greatly reduced in 02E108 compared with the , thus demonstrating a direct correlation between mRNA and protein levels for the two isoforms.
Methylation of the Caveolin-1 Promoter Does Not Correlate with Expression Status.
Methylation analysis of the caveolin-1 promoter region was carried out by bisulfite sequencing of two PCR amplicons. Region A contains 7 CpG dinucleotides and was the most extensively analyzed, because it has been implicated previously in caveolin-1 gene silencing (14)
. The results are summarized in Fig. 4
. The first four CpGs were fully or partially methylated in most samples, including normal thyroid tissue from two FTC patients and a commercial (Promega) control lymphocyte DNA, which is derived from blood pooled from several anonymous donors. CpG number 5 was unmethylated in lymphocyte DNA. There was some suggestion of a correlation with CAV1 expression levels, because this residue becomes methylated in four of four clones from 1989T, a down-regulated tumor, compared with only one of eight clones from paired normal adjacent thyroid tissue. However, this residue was only methylated in one of five clones from 02E108, which is strongly down-regulated for CAV1. The final two CpGs in this amplicon were unmethylated in lymphocyte DNA and tumors 49T and 1928T but were partially methylated in both normal adjacent thyroids and all remaining tumors. Tumor 1928T was hypomethylated in comparison with normal adjacent tissue, despite being down-regulated for CAV1 expression. Thus, overall, this region showed a high level of methylation in most samples, but this did not correlate with CAV1 expression levels.
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:ß ratio but is not down-regulated for CAV1; and (b) 02E108, which shows extreme down-regulation of CAV1ß. Bisulfite sequencing of five clones from each tumor showed no methylation in 02E108 and methylation at one CpG in only two of five clones for 49T. Therefore, tumor-specific down-regulation of CAV1ß must be caused by mechanisms other than methylation or mutation.
Three BMP-related Genes Are Down-Regulated in the Majority of FTCs.
Consistent with data from our exploratory microarray analyses, semiquantitative RT-PCR analysis demonstrated down-regulation of three BMP-related genes, GDF10, CHRDL, and GPC3, in 16, 15, and 17 of 19 FTCs, respectively, by agarose gel analysis (Table 2)
. As with the caveolins, LOH, mutation, and promoter hypermethylation were possible mechanisms by which these genes are down-regulated. LOH was observed at 10q11 in 3 of 17 FTCs (18%). LOH was not examined for GPC3 and CHRDL because these genes are both X-linked, and 10 of the 19 samples are from males. Mutation analysis was conducted for the coding regions of all three genes by single-stranded conformation analysis or direct sequencing of genomic DNA. Only one mutation was identified, a heterozygous 3-bp deletion of GDF10 in tumor 1066T. This mutation (c.61–63delCTG) leads to deletion of one leucine residue in a run of six leucines, and because it is in-frame, the functional consequences are unclear. Furthermore, no normal tissue was available to determine whether or not this mutation had arisen somatically in the tumor. All other sequence changes were judged to be polymorphisms, and no abnormal splice products were detected on RT-PCR. Abnormal methylation of the GDF10 or GPC3 promoter was only detected in a small minority of tumors (Table 2)
. The promoter region of CHRDL is not well defined and so was not examined for methylation status.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chromosomal region 7q31.1, particularly in the vicinity of the microsatellite marker D7S522, shows frequent LOH in a variety of different cancers, including FTC (5 , 6) . It was, therefore, of considerable interest to find that caveolin-1 and -2, which lie immediately adjacent to D7S522, were down-regulated in FTC microarray studies. Caveolins are the major structural protein components of caveolae, small invaginations in the plasma membrane of cells. In addition, caveolins interact with signaling molecules of a number of key pathways, including endothelial nitric oxide synthase, src family tyrosine kinases, ras, and protein kinase A (reviewed in Ref. 21 ). Caveolin-1 has already been widely implicated as a tumor suppressor gene. Down-regulation has been reported in various types of primary tumors and cancer cell lines, whereas in vitro studies have demonstrated that caveolin-1 inhibits anchorage-independent growth and colony forming ability of several different tumor cell lines (22, 23, 24, 25) . In addition, caveolin-1 knockout mice exhibit generalized hyperproliferation (26) , and, most notably, a dominant-negative missense mutation in the caveolin-1 gene has been reported in 16% of invasive human scirrhous breast cancers (27 , 28) . Paradoxically, overexpression of caveolin-1 in tumors can be correlated with a worse prognosis (29, 30, 31, 32) . These apparently contradictory results may reflect the relative importance of the structural versus the cell signaling roles of caveolin-1 in different tumor types or at different stages of tumor progression.
We have demonstrated that caveolin-1 and -2 are coordinately down-regulated in 15 of 19 (79%) FTCs by RT-PCR, whereas caveolin-1 was absent in 49 of 58 (84%) FTCs by immunohistochemistry. However, the molecular mechanisms underlying this down-regulation are unclear. LOH contributes in a significant proportion of tumors, but no mutations were identified in either gene, and methylation status of the caveolin-1 promoter did not correlate with expression status. It is notable that the caveolin-1 knockout mouse also shows greatly reduced caveolin-2 protein levels (26
, 33)
. However, this is because of increased proteasomal degradation of caveolin-2 rather than reduced transcription. In contrast, we have demonstrated a primary down-regulation at the mRNA level, suggesting there may be a common transcriptional regulatory pattern for the two genes in thyroid that is perturbed in FTC. For both genes, we showed that the ß-isoform is transcribed from an alternative promoter in intron 1. Isoform-specific, real-time quantitative RT-PCR revealed a shift in the :ß ratio for caveolin-1 in FTCs compared with normal thyroid, with the ß-isoform down-regulated to a greater extent. For one tumor with a highly skewed ratio, this result was confirmed at the protein level by Western blot analysis. The ratios for caveolin-2 were not significantly different between normal and tumor groups. Any functional differences that might exist between the two isoforms have not been characterized. The ß-isoform of both proteins contains all of the major domains, including the scaffolding domain, but lacks a src-phosphorylation site that is present near the NH2 terminus of the -isoform. Our data suggest that, at least for caveolin-1, the ß-isoform might have a distinct role in FTC. Thus, a series of additional experiments are indicated, firstly to confirm that caveolin-1 and -2 act as functional tumor suppressors in FTC cell lines, as already demonstrated for caveolin-1 in other tumor types (discussed above), and then to determine any functional differences between the two isoforms of caveolin-1 and their respective roles in thyroid neoplasia.
GDF10 localizes to 10q11.1, another region of LOH in FTCs. Additionally known as BMP3b, this gene has putative tumor suppressor functions, being hypermethylated and down-regulated in lung cancers (34) . We therefore chose to analyze GDF10 in detail, together with two other BMP-related genes: (a) glypican-3 (GPC3), which has been implicated in the pathogenesis of several tumor types (35, 36, 37, 38) , and (b) a novel chordin-like gene. We demonstrated down-regulation of all three genes in 79–89% of 19 FTCs. However, as with the caveolins, no clearly pathogenic mutations were identified in any of the three genes, and there was little evidence of hypermethylation of the promoter regions for GDF10 or GPC3.
All five of these genes are down-regulated in the majority of FTCs analyzed and, thus, are not obviously correlated with histological subtype of FTC or clinical history. Indeed, we have preliminary evidence that GDF10, GPC3, and CHRDL are all down-regulated in benign thyroid conditions, including follicular adenoma, nodular goiter, and thyroid hyperplasia, and so do not distinguish benign from malignant neoplasms. Rather, they could instead represent genes down-regulated as the earliest events in both benign and malignant thyroid neoplasia. In support of this, GPC3 is also known to be down-regulated in a proportion of PTCs (39) . Caveolin-1 does, however, help distinguish between FTCs and PTCs. Although caveolin-1 has been reported as up-regulated in PTCs based on immunohistochemistry alone (19) , we found both isoforms to be unchanged in five PTCs by real-time PCR (data not shown). In addition, our immunohistochemical analysis showed that caveolin-1 is significantly down-regulated in FTC and Hurthle cell carcinomas but not PTC or anaplastic cell carcinomas when compared with normal thyroid. Thus, given the current paucity of molecular markers that characterize FTC, down-regulation of caveolin-1, in particular, its ß-isoform, will be useful in distinguishing between different histological types of thyroid carcinoma and investigating their etiological pathways.
The mechanisms by which down-regulation of these five genes contribute to tumorigenesis must be largely speculative at present. No functional link is known between the caveolins and glypican-3 or GDF-10, although recycling of glypican-1, a glycosylphosphatidylinositol-linked cell-surface proteoglycan closely related to glypican-3, takes place in caveolin-1-containing endosomes (40)
. Pathways that link caveolin-1 with two other proteins known to be involved in follicular thyroid neoplasia, namely PPAR and PTEN, are clearer. We have recently shown that PPAR is down-regulated at both mRNA and protein levels in the majority of FTCs (15)
. Because overexpression of PPAR can induce expression of adipocyte marker genes, including caveolin-1 and aP2/FABP4 (41)
, both of which were down-regulated in our microarray analysis, their down-regulation in FTCs might be secondary events that follow from down-regulation of PPAR. PTEN protein is commonly present at reduced levels in sporadic follicular thyroid tumors and mostly localized in the cytoplasm, in contrast to normal thyroid, where nuclear localization predominates (42)
. Despite this observation, mutations in the PTEN gene are rare in sporadic FTCs (1
, 2)
, suggesting other mechanisms of down-regulation are operative. Cell fractionation and coimmunoprecipitation experiments have recently demonstrated that a proportion of PTEN protein localizes to lipid rafts and interacts with caveolin-1 (43)
. Down-regulation of caveolin-1 may therefore be one of the mechanisms directly contributing to dysregulation of PTEN function in follicular thyroid tumors (Fig. 5)
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by an Advanced Training Fellowship from the Wellcome Trust, United Kingdom (to M. A. A.). C. Eng is a recipient of the Doris Duke Distinguished Clinical Scientist Award. This work was partially funded by National Cancer Institute Grant P30CA16058 to The Ohio State University Comprehensive Cancer Center and a generous gift from the Brown family in memory of Welton D. Brown (to C. E.). 
2 To whom requests for reprints should be addressed, at Human Cancer Genetics Program, The Ohio State University, 420 West 12th Avenue, Suite 690 TMRF, Columbus, OH 43210. Phone: (614) 292-2347; Fax: (614) 688-3582; E-mail: eng-1{at}medctr.osu.edu
3 The abbreviations used are: FTC, follicular thyroid carcinoma; LOH, loss of heterozygosity; PTC, papillary thyroid carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BMP, bone morphogenetic protein; PPAR, peroxisome proliferator-activated receptor; RT-PCR, reverse transcription-PCR.
Received 1/ 3/03. Accepted 3/27/03.
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F. Weber, L. Shen, M. A. Aldred, C. D. Morrison, A. Frilling, M. Saji, F. Schuppert, C. E. Broelsch, M. D. Ringel, and C. Eng Genetic Classification of Benign and Malignant Thyroid Follicular Neoplasia Based on a Three-Gene Combination J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2512 - 2521. [Abstract] [Full Text] [PDF]
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 T. M. Williams and M. P. Lisanti Caveolin-1 in oncogenic transformation, cancer, and metastasis Am J Physiol Cell Physiol, March 1, 2005; 288(3): C494 - C506. [Abstract] [Full Text] [PDF]
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 B. Jarzab, M. Wiench, K. Fujarewicz, K. Simek, M. Jarzab, M. Oczko-Wojciechowska, J. Wloch, A. Czarniecka, E. Chmielik, D. Lange, et al. Gene Expression Profile of Papillary Thyroid Cancer: Sources of Variability and Diagnostic Implications Cancer Res., February 15, 2005; 65(4): 1587 - 1597. [Abstract] [Full Text] [PDF]
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M. Eszlinger, K. Krohn, K. Berger, J. Lauter, S. Kropf, M. Beck, D. Fuhrer, and R. Paschke Gene Expression Analysis Reveals Evidence for Increased Expression of Cell Cycle-Associated Genes and Gq-Protein-Protein Kinase C Signaling in Cold Thyroid Nodules J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1163 - 1170. [Abstract] [Full Text] [PDF]
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F. Weber, M. A. Aldred, C. D. Morrison, C. Plass, A. Frilling, C. E. Broelsch, K. A. Waite, and C. Eng Silencing of the Maternally Imprinted Tumor Suppressor ARHI Contributes to Follicular Thyroid Carcinogenesis J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1149 - 1155. [Abstract] [Full Text] [PDF]
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 M. A. Aldred, Y. Huang, S. Liyanarachchi, N. S. Pellegata, O. Gimm, S. Jhiang, R. V. Davuluri, A. de La Chapelle, and C. Eng Papillary and Follicular Thyroid Carcinomas Show Distinctly Different Microarray Expression Profiles and Can Be Distinguished by a Minimum of Five Genes J. Clin. Oncol., September 1, 2004; 22(17): 3531 - 3539. [Abstract] [Full Text] [PDF]
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 W. Tang, S. B. Chang, and M. E. Hemler Links between CD147 Function, Glycosylation, and Caveolin-1 Mol. Biol. Cell, September 1, 2004; 15(9): 4043 - 4050. [Abstract] [Full Text] [PDF]
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H. Kogo, T. Aiba, and T. Fujimoto Cell Type-specific Occurrence of Caveolin-1{alpha} and -1{beta} in the Lung Caused by Expression of Distinct mRNAs J. Biol. Chem., June 11, 2004; 279(24): 25574 - 25581. [Abstract] [Full Text] [PDF]
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M. J. Costa, Y. Song, P. Macours, C. Massart, M. C. Many, S. Costagliola, J. E. Dumont, J. Van Sande, and V. Vanvooren Sphingolipid-Cholesterol Domains (Lipid Rafts) in Normal Human and Dog Thyroid Follicular Cells Are Not Involved in Thyrotropin Receptor Signaling Endocrinology, March 1, 2004; 145(3): 1464 - 1472. [Abstract] [Full Text] [PDF]
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 R. D. Irwin, G. A. Boorman, M. L. Cunningham, A. N. Heinloth, D. E. Malarkey, and R. S. Paules Application of Toxicogenomics to Toxicology: Basic Concepts in the Analysis of Microarray Data Toxicol Pathol, January 1, 2004; 32(1_suppl): 72 - 83. [Abstract] [PDF]
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, methylated residues; 
, unmethylated residues.