Hydrophobic Bile Acid-Induced Micronuclei Formation, Mitotic Perturbations, and Decreases in Spindle Checkpoint Proteins: Relevance to Genomic Instability in Colon Carcinogenesis

Abstract

We show, for the first time, that hydrophobic bile acids cause aberrations of the mitotic machinery of colon cells that can give rise to aneuploidy, the chromosomal perturbations common in colon tumors. First, we show that DOC induces a statistically significant fourfold increase in the number of micronuclei in NCM-460 cells (a noncancerous colon cell line) and a threefold increase in the number of micronuclei in binucleated HT-29 colon cancer cells using the cytokinesis block micronucleus assay. Second, we observed mitotic aberrations after DOC treatment, including improper alignment of chromosomes at the metaphase plate, lagging chromosomes during anaphase, anaphase/telophase chromatin bridges, multipolar divisions, and formation of polynucleated cells. It was determined that there was a statistically significant threefold increase in the number of aberrant metaphases after short-term and long-term exposure of HT-29 and HCT-116 cells, respectively. Third, we showed with Western blots and immunohistochemistry that a likely basis for these mitosis-related perturbations included decreased expression of the spindle checkpoint proteins, Mad2, BubR1, and securin. Fourth, results of DOC treatment on nocodazole-challenged cells further indicated deficiencies in activation of the spindle assembly checkpoint. This study provides mechanisms by which hydrophobic bile acids can induce genomic instability in colon epithelial cells.

ACKNOWLEDGMENTS

This work was supported in part by National Institute of Health 5 R01 CA119087, Arizona Biomedical Research Commission Grant No. 0803, VA Merit Review Grant 0142 of the Southern Arizona Veterans Affairs Health Care System, and Biomedical Diagnostics & Research, Inc., Tucson, Arizona. The work was performed at both the University of Arizona (Department of Cell Biology and Anatomy) and at Biomedical Diagnostics and Research, Inc. (Tucson, Arizona).

Notes

^a Cells incubated for 24 h with DOC.

^b Long-passage control cells incubated in vitro in the absence of DOC for approximately 40 wk (HCT-116SA cell line); mean and SD of 3 separate cell cultures.

^c Apoptosis-resistant cells persistently exposed to DOC for approximately 40 wk (HCT-116RB, HCT-116RC, and HCT-116RD cell lines); mean value of all 3 cell lines combined.