HUMAN COLORECTAL CARCINOMAS SPECIFICALLY ACCUMULATE M(R)42,000 UBIQUITIN-CONJUGATED CYTOKERATIN-8 FRAGMENTS

Recent studies have shown that various tumor cells accumulate ubiquiti n (Ub)-conjugated proteins, the profiles of which differ from those of normal cells. Tn identify the UB-conjugated proteins accumulated spec ifically by human carcinoma sells, a two-dimensional immunoblot analys is of 31 surgically resected human primary colorectal carcinoma tissue s ti as performed using an anti-Ub monoclonal antibody. KM691. Two dis tinct M(r) 42,000 and 45,000 proteins in tile Triton X-insoluble fract ions 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-con jugated proteins were specific to carcinoma tissues from 25 patients ( 80.6%). One of the purified 42,000 proteins was digested with Achromob acter protease I. This protein wa identified as a cytokeratin 8 (CK 8) fragment based oil both molecular mass determination and molecular ma ss searching of Achromobacter protease I-digested fragments of protein s registered in a a protein sequence data base, Two-dimensional immuno blot analysis with an anti-CK 8 antibody confirmed that all cf tire M( r) 42,000 proteins were CK 8 degradation products. These results demon strate that human colo- rectal carcinomas specifically accumulate M(r) 42,000 Ub-conjugated CK 8 fragments. This accumulation was observed f requently not only in advanced (18/22, 81.8%), but also in early stage casts (7/9, 77.8%), suggesting that it occurs even in the early stage s of colorectal carcinoma progression.