Release of decay-accelerating factor into stools of patients with colorectal cancer by means of cleavage at the site of glycosylphosphatidylinositol anchor.

The expression of decay-accelerating factor (DAF), a cell-membrane-complement regulator, is enhanced in colorectal cancer, and DAF is detected in the stools of patients with colorectal cancer. In this study, to elucidate mechanisms whereby DAF is released into the colonic lumen, we analyzed and compared the properties of DAF in stools and colorectal-cancer tissues. Stool specimens taken before surgery and tissue samples from surgically resected colorectal cancers were obtained from 21 patients. We analyzed DAF in stool and tissue specimens using immunoblotting, ultracentrifugation, and phase separation with Triton X-114. We analyzed the expression profile of DAF mRNA in cancer tissues using reverse transcription-polymerase chain reaction to determine whether DAF transcripts for a secretory form of DAF were present. With the use of immunoblotting, stool DAF was detected as a broad band with a molecular weight of around 70,000 kDa that migrated slightly more slowly than cancer-tissue DAF. About 90% of stool DAF was present as a soluble form that remained in the 100,000 g supernatant after ultracentrifugation. On phase separation with Triton X-114, the soluble stool DAF was partitioned mainly into the aqueous phase, indicating its hydrophilic nature and lack of the fatty-acid glycosylphosphatidylinositol anchor component. In colorectal cancer tissues, reverse transcription-polymerase chain reaction experiments revealed a nonspliced DAF messenger RNA that encodes a secretory form of DAF in just 2 of the 21 specimens examined. These data suggest that DAF is released from colorectal cancer cells by way of cleavage of membrane-bound DAF at the site of the glycosylphosphatidylinositol anchor.

Improvements in the measurement of stool decay-accelerating factor in the detection of colorectal cancer.

We have previously developed an enzyme-linked immunosorbent assay (ELISA) to measure stool decay-accelerating factor (DAF) and found that stool DAF concentrations were significantly elevated in patients with colorectal cancer, suggesting that the measurement of stool DAF may be valuable for the detection of colorectal cancer. In order to refine the assay for the measurement of stool DAF, we investigated 1) effects of centrifugation of stool samples, 2) effects of detergents, and 3) adequate combination of various anti-DAF monoclonal antibodies for the ELISA system using only monoclonal antibodies. We found that high-speed centrifugation could be omitted and that only the removal of large undigested food residues by centrifugation of short duration in a low-speed benchtop microcentrifuge sufficed to adequately prepare the stool samples. Addition of 2 detergents, octyl beta-glucoside and sodium deoxycholate, known to solubilize glycosyl-phosphatidylinositol-anchored proteins such as DAF, did not influence stool DAF values. By using 2 mouse anti-DAF monoclonal antibodies (clone 4F11 and 1C6), we were able to achieve a stable ELISA for the measurement of stool DAF using a uniform source of antibodies. The results should allow us to consistently apply the DAF assay for routine use in the detection of colorectal cancer.