A stable isotope GC/MS method of measuring colon epithelial cell turnover in vivo and its applications on curcumin and on long-term label-retaining cells with stemness.

摘要

Currently available methods for measuring proliferation rates in colon have technical as well as practical limitations. We recently developed a stable-isotope mass spectrometric method for measuring DNA synthesis and thus, cell proliferation, by metabolically labeling the deoxyribose moiety of purine deoxyribonucleotides using deuterated water (2H ₂O). Here we apply the method to measure proliferation rates of colon epithelial cells (CECs) in rats. Initial studies revealed a turnover time of 6 days for complete replacement of the proliferative fraction of the crypt and 8 days to replace cells in the entire crypt. These kinetic results were consistent with cell cycle analysis and also correlated well with 5 ′-bromodeoxyuridine (BrdU) labeling. This 2H ₂O method was then shown to be sensitive to detect dose-dependent expansion of the proliferative fraction of the crypt and increase in cell proliferation rates in response to dietary cholic acid, a known stimulator of CEC proliferation.; Curcumin is a naturally occurring anti-carcinogenic, an anti-inflammatory agent and a cyclooxygenase 2 inhibitor present in food. From in vitro and in vivo carcinogenesis studies, anti-carcinogenic properties of curcumin have been reported and presumed to be due to anti-proliferative actions. The purpose of the study was to investigate if curcumin has anti-proliferative effects on normal CECs. We applied the 2H₂O method for measuring CEC proliferation rates after feeding curcumin to normal rats. Curcumin induced dose-dependent increases in CEC proliferation in CEC from both mature and proliferative fractions. The lengths of the colonic crypts and the fraction of cells in S-phase increased in high dietary curcumin groups implying the expansion in both CEC pool size and new cell production.; Adult and epithelial stem cells remain poorly characterized due to the difficulty in isolating them from tissue and their scarcity in tissues. Since there are many similarities between adult stem cells and long term label retaining cells (LRCs), we used a double-labeling approach to physically isolate LRCs based on their kinetic behavior. BrdU and 2H₂O were used in combination to isolate and measure proliferation rates of LRCs from rat colon. Flow cytometry was used to sort, purify and collect LRCs. The percent of LRCs after a 2 week BrdU delabeling period was consistent with the data obtained after 2 weeks of 2H₂O delabeling (9.9%). Based on argentaffin staining, the contamination from other long-lived cells (enteroendocrine cells) was negligible. (Abstract shortened by UMI.)