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Departments of Medicine [W. E-R., M. K. A., J. H., C. Y., S. M. P.] and Pathology [C. A. M., H. F. F.], University of Virginia Health System, Charlottesville, Virginia 22908, and Duke University Medical Center, Department of Pathology, Durham, North Carolina 27710 [G. J. R.]
| ABSTRACT |
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| Introduction |
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| Materials and Methods |
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SAGE was performed using SAGE protocol version 1.0e, June 23, 2000, which includes a few modifications of the standard protocol (8) . The NlaIII restriction site used in library construction defined all SAGE tags. A detailed protocol and schematic of the method is available.4 Two thousand clones were sequenced for each case by the Cancer Genome Anatomy Project. Tags were extracted from the raw sequence data using SAGE2000 analysis software version 4.12 developed by Dr. Kenneth Kinzler. This software automatically delimits each tag from its adjacent tag and abutting linker sequence and counts the number of copies of each tag (thus, of each mRNA species). The resulting tag libraries were compared with UniGene cluster to SAGE tag "reliable" mapping database,5 and the statistical analyses were performed using the SAGE software. Significant changes in levels of expression (P < 0.01) were determined. Sequence data from our SAGE libraries are publicly available6 with Geo Accession Omnibus numbers GSM757 for GC and GSM784 for normal stomach. The Cancer Genome Anatomy Project maintains the SAGE public database for gene expression in human cancer (16) .
Quantitative Real-Time PCR.
For quantitative real-time PCR, 20 primary GCs and 13 normal gastric epithelial samples were collected. All tumors were dissected from frozen tissue specimens and had at least 80% tumor cell content with the least possible amount of contaminating necrotic, normal inflammatory, and stromal cells. The normal gastric mucosal epithelial tissues were carefully examined and were devoid of any inflammatory or necrotic contaminating cells. The collected tumors ranged from well-differentiated to poorly differentiated, stages I to IIIa, and there was a mix of intestinal and diffuse-type tumors. The mRNA was isolated using RNeasy kit (Qiagen). Single-stranded cDNA was synthesized using Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA). Quantitative PCR was performed using iCycler (Biol-Rad, Hercules, CA), and threshold cycle number was determined using iCycler software version 2.3. Reactions were performed in triplicate, and threshold cycle numbers were averaged. Gene-specific primers for S100A2, S100A7, S100A8, S100A9, and S100A10 were designed. The primers used for RT-PCR were obtained from GeneLink (Hawthorne, NY), and their sequences are available on request. The results were normalized to ß-amyloid precursor protein, which had minimal variation in all normal and neoplastic gastric samples that we tested. Fold overexpression was calculated according to the formula 2(Rt - Et)/2(Rn - En) as described previously (17)
, where Rt is the threshold cycle number for the reference gene observed in the tumor, Et is the threshold cycle number for the experimental gene observed in the tumor, Rn is the threshold cycle number for the reference gene observed in the normal sample, and Rt is the threshold cycle number for the reference gene observed in the tumor sample. Rn and En values were an average for the 13 normal samples that were analyzed. Samples that had expression levels
5-fold were considered overexpressed.
| Results |
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Among the 20 most up-regulated genes (P < 0.01), 5 calcium-binding proteins (S100A2, S100A7, S100A8, S100A9, and S100A10) were overexpressed (Table 1)
. Other genes such as keratins (keratin 6A, 13, and 17), small praline-rich protein 3, and annexin A1 were also overexpressed (Table 1)
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| Discussion |
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In this analysis, we report, for the first time, the use of SAGE technology to compare the GC cell transcriptome with that of normal gastric mucosa. The normal gastric expression was evaluated on a pool of normal gastric mucosal samples prepared from endoscopic biopsies. The quality of normal samples to compare findings in the cancers is important to ensure accurate analysis. The use of endoscopic biopsies to obtain the normal samples has the unique ability to enrich for epithelial cells. Endoscopic biopsy samples are relatively free of stromal elements, with epithelial cells comprising the overwhelming majority of cells.
We have found that several members of the S100A protein family are overexpressed in GC. By RT-PCR, S100A2 was overexpressed in almost all primary gastric tumors that we studied. Other S100A proteins such as S100A7, S100A8, S100A9, and S100A10 were also overexpressed at varying levels in our tumors. To date, 20 members of this protein family have been identified that are known to regulate intracellular processes such as cell growth and motility, cell cycle regulation, transcription, and differentiation (18) .
S100A2 has been described as a potential tumor suppressor gene that is down-regulated in a number of tumors such as lung, breast, and head and neck carcinomas (19, 20, 21) . On the other hand, tumors such as ovarian cancer melanoma and epithelial tumors of the skin have shown overexpression of S100A2 (22, 23, 24) . In melanoma, S100A2 was moderately to highly expressed in early lesions, whereas none of the metastases expressed S100A2 mRNA (22) . The notion that S100A2 was overexpressed in >90% of our gastric tumors of various stages suggests that S100A2 overexpression may be an early tumorigenic event rather than a tumor progression marker in gastric carcinogenesis.
S100A7, S100A8, S100A9, and S100A10 were differentially expressed in 2545% of our gastric tumors. S100A7 has been identified in association with the transition from preinvasive to invasive breast cancer (25) . In addition, S100A7 is considered as a potential tumor biomarker for the noninvasive follow-up of patients with urinary bladder squamous cell carcinoma. There is sparse information regarding the role of S100A8, S100A9, and S100A10 in cancer. Typically, S100A8 and S100A9 are known to be differentially expressed at sites of acute and chronic inflammation (18) . However, recent reports indicate that S100A8 and S100A9 are overexpressed during skin carcinogenesis and at the invasive margin of colorectal carcinoma (26 , 27) . S100A10 is an annexin 2 protein ligand (28) . Few data are available in literature about this protein. To our knowledge, there is only one report about this gene in human cancer, in which S100A10 is overexpressed in human renal cell carcinoma (29) .
Using SAGE analysis, we have identified transcripts likely to be important in gastric tumorigenesis. Moreover, our findings add to the developing literature about the possible role of S100A calcium-binding proteins in cancer. The fact that several members of the S100A protein family were differentially overexpressed suggests, for the first time, that these proteins may be critical for the development and/or progression of GC. Additional analyses are needed to elucidate the exact role of S100A proteins in gastric tumorigenesis.
| FOOTNOTES |
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1 Supported by National Cancer Institute Grants 1R01CA93999-01 (to W. E-R.) and CA/DK 67900-06A1 (to S. M. P.) and by the Cancer Center at the University of Virginia (W. E-R.). ![]()
2 To whom requests for reprints should be addressed, at Digestive Health Center of Excellence, University of Virginia Health System, P. O. Box 800708, Charlottesville, VA 22908-0708. Phone: (434) 243-6158; Fax: (434) 243-9645; E-mail: wme8n{at}virginia.edu ![]()
3 The abbreviations used are: GC, gastric cancer; SAGE, serial analysis of gene expression; RT-PCR, reverse transcription-PCR. ![]()
4 http://www.sagenet.org/sage_protocol.htm. ![]()
5 ftp://ftp.ncbi.nih.gov/pub/sage/map/Hs/Nla3/. ![]()
6 http://www.ncbi.nlm.nih.gov/SAGE/. ![]()
Received 8/ 8/02. Accepted 10/18/02.
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