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Human Colorectal Carcinomas Specifically Accumulate M_r 42,000 Ubiquitinconjugated Cytokeratin 8 Fragments¹

Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104 [H. N., Y. M., K. I., T. O., S. H.]; Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160 [H. N., M. K.]; Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236 [N. K., A. I.]; and Fuji Research Laboratories, Kyowa Medex Co., Ltd., 600-1 Minami-ishiki, Nagaizumi-cho, Sunto-gun, Shizuoka 411 [H. K., S. S.], Japan

Recent studies have shown that various tumor cells accumulate ubiquitin (Ub)-conjugated proteins, the profiles of which differ from those of normal cells. To identify the Ub-conjugated proteins accumulated specifically by human carcinoma cells, a two-dimensional immunoblot analysis of 31 surgically resected human primary colorectal carcinoma tissues was performed using an anti-Ub monoclonal antibody, KM691. Two distinct M_r 42,000 and 45,000 proteins in the Triton X-insoluble fractions of carcinoma tissues reacted with this antibody, whereas only one M_r 45,000 protein reacted in normal tissues. The M_r 42,000 Ub-conjugated proteins were specific to carcinoma tissues from 25 patients (80.6%). One of the purified M_r 42,000 proteins was digested with Achromobacter protease I. This protein was identified as a cytokeratin 8 (CK 8) fragment based on both molecular mass determination and molecular mass searching of Achromobacter protease I-digested fragments of proteins registered in a protein sequence data base. Two-dimensional immunoblot analysis with an anti-CK 8 antibody confirmed that all of the M_r 42,000 proteins were CK 8 degradation products. These results demonstrate that human colorectal carcinomas specifically accumulate M_r 42,000 Ub-conjugated CK 8 fragments. This accumulation was observed frequently not only in advanced (18/22, 81.8%), but also in early stage cases (7/9, 77.8%), suggesting that it occurs even in the early stages of colorectal carcinoma progression.

¹ This work was supported by a Grant-in-Aid for the Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.

² Awardee of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.

³ To whom requests for reprints should be addressed.

Received 11/ 8/95. Accepted 4/11/96.

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