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Two-Dimensional Complementary Deoxyribonucleic Acid Electrophoresis Revealing Up-Regulated Human Epididymal Protein-1 and Down-Regulated CL-100 in Thyroid Papillary Carcinoma

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.

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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