Background and aims: C-myc over expression is implicated in malignancy although to date this has not been studied in Barrett's metaplasia. We sought to determine c-myc expression in the malignant progression of Barrett's metaplasia and whether it may be induced by bile acids seen in gastro-oesophageal refluxate. Methods: C-myc protein and mRNA levels were assessed in 20 Barrett's metaplasia and 20 oesophageal adenocarcinoma samples by western blotting and real time polymerase chain reaction. Levels of c-myc and proliferation were also assessed in cell lines OE21, OE33, SW-480, and TE-7 stimulated with pulses or continuous exposure to the bile acids deoxycholic acid and chenodeoxycholic acid. Results: C-myc protein was upregulated in 50% of Barrett's metaplasia and 90% of oesophageal adenocarcinoma samples compared with squamous, gastric, and duodenal controls. C-myc immunolocalisation in Barrett's metaplasia revealed discrete nuclear localisation, becoming more diffuse with progression from low to high grade dysplasia to adenocarcinoma. Both continual and pulsed bile acid induced c-myc at pH 4, with no effect at pH 7 or with acidified media alone. Pulsed bile acid treatment induced proliferation (p<0.05); in contrast, continuous exposure led to suppression of proliferation (p<0.05). Conclusions:We have shown upregulation of c-myc with malignant progression of Barrett's metaplasia and suggest that acidified bile may be a novel agent responsible for induction of this oncogene.

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Tadpole collagenase (EC 3.4.24.3) cleaved chick cranial bone procollagen into two triple-stranded fragments, PCA and PCB. Only pCB, with an estimated molecular weight of about 60,000 for each component chain after reduction, was found to contain interchain disulfide bonds. The analogous cleavage of collagen is known to produce a large NH2-terminal fragment with a molecular weight of 70 Since the initial identification of a biosynthetic precursor to collagen, it has become apparent that the nontriple-helical portion of the molecule is important for several functions, which may include molecular assembly, maintenance of intracellular solubility, transmembrane transport, and regulation of fibril formation (see ref. 1 for a review). Early structural studies of collagen precursors suggested, on the basis of electron micrographs of segment-long-spacing crystallites, that there was a single "noncollagenous" domain at the NH-terminus of each precursor chain (2, 3). Subsequent chemical studies of these precursors and of those procollagen-derived proteins which accumulated in the tissues of animals affected with dermatosparaxis tended to confirm the structural studies (4, 5).More recent studies of (a) peptides derived from procollagen secreted into culture medium by cells, (b) precursor chains protected from proteolysis during purification, and (c) peptides secreted into culture medium by cranial bone have suggested that the nonhelical, noncollagenous domain is considerably larger than was previously thought (6-9). Tanzer and his colleagues (10) have recently indicated that there is a noncollagenous peptide extension at the COOHterminus of the procollagen molecule, in addition to that at the NH2-terminus. In this report, we present data to show that the COOH-terminal domain of procollagen is larger than the NH2-terminal extensions, that the composition is different, and that interchain disulfide bonds occur exclusively in the COOH-terminal region. MATERIALS AND METHODSProcollagen, pulse labeled with L-[2,3-3H]proline (43.1 Ci/ mmol, New England Nuclear) for 18 min or with L-[G-3H]tryptophan (7.9 Ci/mmol, New England Nuclear) for 2 hr (7, 36) was extracted from 17-day-old chick embryo cranial bones with 1 M NaCl, 50 mM Trip-HCl, pH 7.5, to which 25 mM EDTA, 10 mM N-ethylmaleimide, 1 mM diisopropylfluorophosphate or phenylmethylsulfonylfluoride, and 1 mM benzamidine-HCl had been added to inhibit proteolysis. The bones were homogenized in that solvent, centrifuged at 39,000 X g for 15 min, and the labeled procollagen together with coextracted collagen was precipitated from the supernatant with 20% NaCl. The precipitate was collected by centrifugation, redissolved in extraction buffer, precipitated with 5% cold trichloroacetic acid and then dissolved in extraction buffer containing 0.1 M Tris-HCl, pH 7.5. Acid-extracted procollagen was obtained as previously described (4), except that extraction was limited to 1 hr, and was further purified by chromatography on DEAE-cellulose by minor modifications of the method of S...