APC +/- alters colonic fibroblast proteome in FAP

Abstract

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.

Figure 1. 2D gels of FAP and control colonic fibroblasts.Three overlapping pH range 2D gels across pI 4—11 of FAP and control colonic fibroblasts

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Figure 2. RSU-1 expression in colonic fibroblasts

A. pH 6—11 range 2D gel analysis of RSU-1 protein isoforms in control colon fibroblasts. All the protein spots observed in this picture were identified with mass spectrometry. Four isoforms of RSU-1 are indicated by the arrows. The rightmost spot is unmodified RSU-1. The second spot from the left is lower in intensity in FAP fibroblasts. The right two RSU-1 spots were outside of the range of the pH 5—8 gels previously used to detect changes in RSU-1 levels. B. Western analysis of RSU1 expression in FAP colonic fibroblasts versus control colonic fibroblasts. C. Western analysis of RSU-1 expression in FAP colonic crypt cells versus those from control patients.

Figure 3. DJ-1 proteomic changes in colonic crypts

A. 1D gel Western blotting, B. 2D gel pH4—7 12% SDS-PAGE gels, and mass spectrometry protein identification, and C. 2D gel Western blotting experiments. The circled protein spots were DJ-1 identified by mass spectrometry. Corresponding spots were indicated by arrows in the 2D Western blotting images. For the 2D Western, Control was CP 203, FAP was SID 516.