Endocrinology Vol. 143, No. 11 4422-4428
Copyright © 2002 by The Endocrine Society
Two-Dimensional Complementary Deoxyribonucleic Acid Electrophoresis Revealing Up-Regulated Human Epididymal Protein-1 and Down-Regulated CL-100 in Thyroid Papillary Carcinoma
Jun-ichi Asakawa,
Mieko Kodaira,
Naofumi Ishikawa,
Yuko Hirai,
Shigenobu Nagataki,
Farhad Moatamed and
Masahiro Sugawara
Radiation Effects Research Foundation (J.A., M.K., Y.H., S.N.), Hiroshima 732-0815, Japan; Ito Hospital (N.I.), Tokyo 150-0001, Japan; Department of Pathology and Laboratory Medicine (F.M.), Molecular Pathology Division, and Department of Medicine (M.S.), Division of Endocrinology and Metabolism, Greater Los Angeles VA Health Care System and University of California, Los Angeles School of Medicine, Los Angeles, California 90073
Address all correspondence and requests for reprints to: Dr. Jun-ichi Asakawa, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732-0815, Japan. E-mail: asakawa{at}rerf.or.jp.
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Abstract
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Two-dimensional cDNA electrophoresis was used to analyze gene expressions in papillary carcinoma and normal tissue of thyroid glands. Pooled thyroid tissues were used to extract mRNA. Complementary DNAs, synthesized with NotI anchor primers, were digested with three restriction enzymes, NotI, EcoRV, and PvuII. The protruding NotI ends were filled in with 32P deoxynucleotide triphosphates, and the radiolabeled cDNA fragments were separated in two dimensions. Approximately 500 cDNA fragments were visualized as discrete spots without probes. A total of 20 spots, 9 up-regulated and 11 down-regulated cDNAs in papillary carcinoma, were selected and cloned for sequencing. This experiment lent itself to a novel discovery of up-regulated human epididymal protein 1 (HE-1) and down-regulated CL-100 genes in thyroid papillary carcinomas confirmed by Northern blot analysis. Immunohistochemical stains showed abundant HE-1 protein in the papillary carcinoma, whereas little or no HE-1 protein was detected in other types of thyroid cancers and normal thyroid tissues. The restricted localization of HE-1 protein to the portions of papillary projections suggests an involvement of HE-1 protein for forming papillary shape. Our study showed that two-dimensional cDNA electrophoresis is a useful method of detecting differentially expressed genes in human diseases as demonstrated for HE-1 and CL-100 in papillary carcinoma.
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Introduction
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CHARACTERIZATION OF GENE expression in thyroid papillary carcinoma should help our understanding of genes involved in the development of this common form of thyroid cancer. Differential display of genes between diseased and normal state has been used for identification of the genes involved in the diseases of interest. To screen many genes already known, DNA array technology is getting more common (1, 2). To identify unknown genes by unbiased gene screening, PCR-based differential display (3), serial analysis of gene expression (4), and/or total gene expression analysis (5) have been used. These three methodologies have limitations because they require time-consuming steps and induction of false-positive clones. One of the authors (J.A.) has developed a method of two-dimensional electrophoresis for end-labeled genomic DNA fragments (6). This method then was modified for application to cDNA for differential display of gene fragments (7, 8). This method visualizes approximately 500 end-labeled cDNA fragments on a single autoradiogram without probes, enabling one to identify not only differentially expressed known genes but also unknown genes (7, 8). To our surprise, this study identified up-regulation of human epididymal 1 protein (HE-1) and down-regulation of CL-100 genes in the papillary carcinoma. Also, we demonstrate that this method is capable of differentially displaying many other genes.
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Materials and Methods
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Human thyroid tissues
For two-dimensional electrophoresis, thyroid tissues were obtained as surgical samples from three patients with papillary carcinoma during the routine course of patient care. Normal portions of the thyroid glands in the goiter tissue specimens were used as normal control. Immediately after surgery, portions of all tissues were frozen and kept at -80 C until use. Surgical samples of papillary carcinoma were pooled together for this experiment. Similarly, normal portions of the nodular goiter samples were pooled.
For immunohistochemistry, formalin-fixed and paraffin-embedded sections were employed. The tissue samples were obtained for routine surgical pathology diagnoses. This study was approved by the Institutional Review Board at Ito Hospital, Tokyo, Japan. All the extracted RNAs were used without identifier.
Two-dimensional cDNA electrophoresis
Steps of two-dimensional cDNA electrophoresis are outlined in Fig. 1
, and the detail procedures are explained below.
1. Extraction of mRNA and cDNA synthesis.
Extraction of mRNA from total RNA was carried out using oligo-dT columns (Invitrogen Co., Carlsbad, CA). Synthesis of cDNA was carried out on approximately 1 µg mRNA employing a cDNA synthesis kit (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen). Synthetic oligonucleotides for the reverse transcriptase were designed to have at least one NotI recognition site (GC-GGCCGC) at the 3' end of synthesized cDNAs. The sequences of the reverse transcription (RT) primers used in this study are shown below:
5'GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTVN3' in which V can be any nucleotide except T and N represents any of the four nucleotides G, A, C, or T. Four RT primers were prepared. Each primer was identified by its last nucleotide in the N position. The synthesized cDNA was treated with RNase ONE ribonucleases (Promega Corp., Madison, WI). After phenol extraction and ethanol precipitation, cDNA was separated from deoxynucleotide triphosphates and RNA molecules by a passage of microspin column (S-400HR, Amersham Pharmacia Biotech, Piscataway, NJ). The cDNA was incubated at 37 C for 2 h with 10 U of each of the restriction enzymes NotI, EcoRV, and PvuII simultaneously. One-fifth of the digest, 2 µl, was mixed with 1 µl of 0.1 M dithiothreitol, 1 µl each of [
-32P]dCTP and [-32P]dGTP (6000 Ci/mmol, Amersham Pharmacia Biotech) and 1 U Sequenase (United States Biochemical Corp., Cleveland, OH). After incubation at 37 C for 30 min, 3 µl solution containing 50% sucrose, 50 mM EDTA, and 0.5% each of bromophenol blue and xylene cyanol was added to stop the reaction.
2. Electrophoresis.
The end-labeled cDNA was mixed with the remaining unlabeled NotI/EcoRV/PvuII digest of cDNA. The first dimension of electrophoresis was carried out on a 61-cm-long 1% agarose gel cast in Teflon tubing (Sanplatec, Osaka, Japan) with an inner diameter of 2.3 mm. The sample was electrophoresed at 120 V for approximately 14 h and at 150 V for approximately 24 h (until bromophenol blue dye migrated 48 cm). The 30-cm portion of the gel containing cDNA fragments approximately 0.2–3 kb in length was expelled from the tube and was incubated with 500 U HinfI for 30 min after equilibration with HinfI buffer. After digestion with the third enzyme, the fragments in the first-dimension gels were separated perpendicularly on a 5.15% polyacrylamide gel (33 x 46 x 0.08 cm) at 150 V for 18 h at room temperature. The gel was dried using a gel dryer (Bio-Rad Laboratories, Inc., Richmond, CA), and autoradiography was performed. Autoradiograms were digitized using a laser densitometer (Abe-Sekkei, Tokyo, Japan) at a resolution of 200 µm with 12-bit values for each pixel. Custom translation algorithms were employed to reduce the images to 8-bit pixels in a format suitable for performing spot detection and quantitation with Bioimage software (BM Luton, Inc., Jackson, MI).
3. Cloning of DNA from spots of interest.
Cloning of the NotI-HinfI DNA fragments was carried out as previously described (9). Briefly, spots of interest were marked on the film, and this marked film was superimposed to the original dried gel. Each spot in the gel was then cut by a sterile knife. The dried gel was resuspended in a Tris-EDTA buffer containing tRNA, and the NotI-HinfI fragments were electroeluted on a diethylaminoethyl paper embedded in 0.7% agarose gel. The diethylaminoethyl paper was rinsed with low-salt buffer (0.1 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA) and eluted with high-salt buffer (1 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA). The eluted DNA was phenol extracted twice and ethanol precipitated. DNA was ligated in a modified vector pBluescript II (PstI site was modified into HinfI site) with T4 DNA ligase at 16 C overnight. The transformation was done by an electroporation with Epicurian Coli XL1-Blue MRF’ electroporation-competent cells (Stratagene, La Jolla, CA). Plasmid DNAs were extracted from positive clones and subjected to sequence analyses. The similarity of sequences of each clone was searched in the GenBank database using the BLAST software.
Northern blot
To confirm gene expression of HE-1 and CL-100 in papillary carcinoma and normal tissues, Northern blot was carried out on surgical samples of papillary carcinoma (n = 3) and normal portions of the thyroid glands (n = 4) using 32P-labeled cDNA probes of HE-1 and CL-100.
Immunohistochemical stains
Four- to six-micrometer-thick formalin-fixed and paraffin-embedded sections were stained for HE-1 using an automated immunoperoxidase method (DAKO Corp., Santa Barbara, CA). The sections included papillary carcinoma (n = 10), follicular carcinoma (n = 5), medullary carcinoma (n = 5), anaplastic carcinoma (n = 3), benign goiter (n = 5), and normal portion of benign goiter specimens (n = 3). A goat polyclonal antibody to human HE-1 protein (10) was kindly provided by Dr. Christiane Kirchhoff (University of Hamburg, Germany). A dilution of 1:100 was made in the appropriate buffer solution and incubated with the tissue sections for 1 h. The staining of the sections was carried out as in previous publications (11, 12).
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Results
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Two-dimensional cDNA electrophoresis
Figures 2 and 3 show the results of two-dimensional cDNA electrophoresis when cDNA preparations were made using RT-primer C and G, respectively. Approximately 500 spots were visualized on each preparation. Eighteen spots showing apparently different intensities between papillary carcinoma and normal tissues were selected for cloning and sequencing. Ten predominant spots in the normal thyroid and eight predominant spots in papillary carcinoma were selected. They are highlighted in Figs. 2 and 3. Table 1 shows identification of genes in those 18 spots derived from Figs. 2 and 3. The spot numbers 8 and 11 showed gene fragments whose DNA sequences did not match any known genes reported previously. Each clone from spots 16 and 17 also contained unknown genes. The submitted GenBank IDs of the novel genes identified in this study are also shown in Table 1. Thyroglobulin and thyroid peroxidase (TPO) genes were down-regulated in cancer tissues. Decreased TPO gene expression in papillary carcinoma also was confirmed by Northern blot in three papillary carcinoma tissues (data not shown). Mitochondrial cox II mRNA for cytochrome c oxidase II subunit was found in spots 3, 9, 12, 13, and 14. Nucleotide sequences of cDNAs cloned from four large spots in papillary carcinoma sample, spots 13–18, were identical or highly homologous to portions of 18S rRNA or 28S rRNA; thus, the four spots seemed to be derived from rRNA.
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Figure 2. Distribution of radiolabeled cDNA fragments. The numbers in fragment spots in normal tissues correspond to those of papillary carcinoma. The cDNAs were synthesized using RT primer C, 5'GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTVC3' (V = A, C, or G). Estimated fragment sizes for each dimension are indicated.
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Figure 3. Distribution of radiolabeled cDNA fragments. The numbers in fragment spots in normal tissues correspond to those of papillary carcinoma. The cDNAs were synthesized using RT primer G, 5'GACTAGTTCTAGATCGCGAGCGGCCGCCGTTTTTTTTTTTTTTTVG3' (V = A, C, or G). Estimated fragment sizes for each dimension are indicated.
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HE-1 and CL-100 genes
When RT-primer A was used for cDNA synthesis, two distinct spots were identified by the two-dimensional cDNA electrophoresis. The DNA sequences from the two spots were identical with CL-100 and HE-1 genes. The HE-1 spot was more intense, and CL-100 was far less intense, in papillary carcinoma than in normal tissues (Fig. 4A). Northern blot analysis in four normal thyroid and three papillary carcinoma tissues also confirmed the above findings (Fig. 4B); conversely, CL-100 gene expression was stronger in normal tissues (three of the four tissues) than in papillary carcinomas (Fig. 4B).
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Figure 4. A, Differentially expressed HE-1 and CL-100 genes in cDNAs synthesized by RT primer A, 5'GACTAGTTCTAGATCGCGAGCGGCCGCCGTTTTTTTTTTTTTTTVA3' (V = A, C, or G). B, Northern blot analyses of HE-1 and CL-100 genes in normal and papillary carcinoma tissues.
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Immunohistochemical stain for HE-1 protein
Immunohistochemical staining of the cells for HE-1 protein was consistently fine granular in the cytoplasm of the cells. The reactions in all 10 papillary carcinomas were very intense. In normal tissues, the reaction was scarce as it was in five follicular and three anaplastic carcinomas but slightly variable in the latter. Five medullary carcinoma tissues were all negative. In the five benign goiters, however, the reaction was intense in which papillary projections were present. Examples of HE-1 protein expression are depicted in Fig. 5 with papillary carcinoma showing an intense cytoplasmic staining of HE-1 protein, compared with other types of thyroid cancers and normal tissues. A papillary formation in benign goiter reflects an intense cytoplasmic staining for HE-1 protein (Fig. 5G).
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Figure 5. HE-1 protein expression is shown by immunohistochemical stain in different types of thyroid cancers and normal thyroid tissue. A, Strong expression in a small focus of papillary carcinoma with surrounding normal thyroid tissue is shown. The focus indicated by an arrow is magnified in B indicating strong granular cytoplasmic reactions shown by the arrows. Follicular carcinoma (C) and anaplastic carcinoma (D) show minimal granular reactions in the cytoplasm, indicated by the arrows. Follicular colloids frequently show a positive reaction, which represents nonspecific reaction. Medullary carcinoma (E) shows no reaction for HE-1 protein, with arrows pointing to the amyloid matrix of the tumor. F, Normal thyroid tissue with sparse cytoplasmic reaction for the protein is depicted. G, Focal papillary formations in a benign goiter. Note the intense reaction in which papillary projections are developing in the follicles. The reaction, however, is very weak in the flat portion of the follicle.
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Discussion
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Thyroid papillary carcinoma is the most common type of thyroid cancer, accounting for 80% of all thyroid malignancies (13). The mechanism of developing this type of cancer appears to involve multiple oncogenes, growth factors, and abnormal DNA methylation (13), but chromosome aneuploidy is relatively uncommon (14). Our present study using two-dimensional cDNA electrophoresis successfully detected up-regulated and down-regulated genes in thyroid papillary carcinoma (Table 1
). The advantages of this method over other differential display techniques are that up- and down-regulated gene fragments are visible and the entire procedure, including cloning of cDNA from spots of interest and identification of the genes, can be accomplished within 2 wk. DNA array methods are probably the best for screening many already-known genes (1, 2). The merit of two-dimensional cDNA electrophoresis, in comparison to DNA array methods, is the capability of detecting novel genes and mutated genes with altered size in cancer tissues. This method requires enough tissue to obtain at least about 1 µg mRNA for cloning of cDNA fragments of interest. If the experiment is designed only for visualization of the cDNA fragments, a small piece of tissue sample containing about 0.2 µg mRNA is sufficient. Outcome of two-dimensional electrophoresis also depends on the multiple choices of RT primers and combinations of restriction enzymes. If only one RT primer is used and the synthesized cDNA is treated with only one set of restriction enzymes, then the result may not reflect the majority of up- and down-regulated gene fragments; for instance, up-regulated HE-1 and down-regulated CL-100 genes in papillary carcinoma were detected only with RT primer A (Fig. 5) but not with other primers (Figs. 2 and 3).
To increase the number of visible gene fragments, all four RT primers and different combinations of restriction enzymes may be used. For instance, one can use several six-base and four-base cutters besides EcoRV, PvuII, and HinfI. DNA polymorphisms should be taken into consideration for interpretation of gene fragment distribution, particularly when multiple tissue samples are pooled for cDNA preparation. If DNA polymorphism is present at the recognition sites of the used restriction enzymes, gene fragments (NotI fragments) from the same genes may appear in different spots or multiple spots. The intensity of each spot should also be interpreted carefully; for instance, if one spot contains a single gene fragment, the intensity of radioactivity represents its level of gene expression. If a spot consists of multiple gene fragments, it is necessary to examine which gene is specifically up-regulated or down-regulated using other methods such as Northern blot or RT-PCR.
Down-regulated thyroglobulin and TPO genes in papillary carcinoma tissues are in concordance with previous reports (15, 16). As shown in Table 1, cDNA spots derived from human rRNA species are characteristic in papillary carcinoma, although the biological significance of this finding is unknown. It is also possible that rRNA content was greater in cancer tissues than in normal tissues, despite passage of RNA samples through the oligo-dT column twice. Appearance of multiple spots derived from mitochondrial cox II mRNA for cytochrome c oxidase II subunit is intriguing. The fragments of this gene were intensified in spots 3 and 9 in normal thyroid tissue and spots 12, 13, and 14 in papillary carcinoma tissue. The DNA sequences of spots 3 and 9 were 9 bp shorter than those of the other spots. It is speculated that different splicing, modification of the gene, or presence of polymorphisms is responsible for the appearance of mitochondrial cox II mRNA fragments in normal and cancer tissues.
A remarkable new finding in this study is the discovery of up-regulated HE-1 and down-regulated CL-100 genes in papillary carcinomas. HE-1 is the major secretory protein in the human epididymis (10). Its function includes sperm maturation (10), cholesterol binding (17), and reduction of cholesterol accumulation in Niemann-Pick disease 2C (18). Takano et al. (19) used the serial analysis of gene expression method to screen gene profiles in human thyroid cancers, including papillary carcinoma, but failed to detect HE-1 expression. Recently, Huang et al. (20) compared gene expression profiles in eight papillary carcinoma tissues and corresponding normal thyroid tissues using the microarray technique. In their study, 12,000 genes were screened in each array, and 226 genes were uniquely altered in the cancer tissues. However, the change of HE-1 gene expression was not identified by their approach (20). It is conceivable that the chip they used might have not contained HE-1 gene. In our study, up-regulation of the HE-1 gene in papillary carcinoma detected by two-dimensional cDNA analysis was confirmed at protein level by immunohistochemical staining as increased HE-1 protein expression was observed in all 10 examined cases as shown in the example of Figs. 5, A and B. Normal tissues and other types of thyroid cancers showed a much lower intensity of HE-1 staining than that of papillary carcinoma. Interestingly, portions of benign goiters also showed a strong staining for HE-1 protein, particularly in the areas of papillary projections (Fig. 5G). This finding suggests that HE-1 protein may be involved in the formation of papillae, although this hypothesis has yet to be proved.
Identification of down-regulated CL-100 in three of the four papillary carcinomas in this report was in good accordance with the microarray study by Huang et al. (20); they found down-regulation of CL-100 in six of the eight papillary carcinoma samples. The activation of MAPK, locating downstream of the ras and raf oncogene cascade, is the early event of most epithelial carcinomas, and the CL-100 inactivates MAPK (21, 22). Up-regulated CL-100, therefore, keeps MAPK inactive in normal tissues to prevent active mitosis by inhibiting phosphorylation of critical proteins, including transcriptional factors. It is reasonable to conclude that down-regulation of CL-100 is involved in carcinogenesis of papillary carcinomas. The first application of two-dimensional cDNA electrophoresis has revealed characteristic expression of the genes in papillary carcinomas. HE-1 and CL-100 are prime examples. Finally, we believe that our two-dimensional cDNA method is superior to two-dimensional genomic DNA electrophoresis because we failed to identify the current genetic changes by the latter method (23). Two-dimensional cDNA electrophoresis is a powerful and rapid method for unbiased gene screening, discovery of novel genes, and identification of genes differently spliced in diseased states.
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Acknowledgments
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We thank Akiko Miura, Junko Kaneko, Satomichi Kaneoka, Takahiro Tsuji, Shuji Mishima, Masaaki Imanaka, and Hiroshi Haba for their technical assistance. We are indebted to Dr. Christiane Kirchhoff (Hamburg, Germany) for providing polyclonal antibody to human HE-1 and Nazlin Sharif for performing the immunohistochemistry on the tissue sections. This publication is based on research performed at Radiation Effects Research Foundation (RERF) in Hiroshima and Nagasaki, Japan. RERF is a private nonprofit foundation funded equally by the Japanese Ministry of Health, Labor, and Welfare and the U.S. Department of Energy through the National Academy of Science.
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Footnotes
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This work was supported in part by Radiation Effects Research Foundation in Hiroshima and Nagasaki and Greater Los Angeles Veterans Affairs Medical Center.
Abbreviations: HE-1, Human epididymal 1 protein; RT, reverse transcription; TPO, thyroid peroxidase.
Received May 28, 2002.
Accepted for publication July 30, 2002.
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Abstract
Introduction
