Bile acids (BA)arrest colonic cell proliferation in a cell cycle-dependent fashion

Abstract

Epidemiological studies link factors that influence concentration of soluble fecal BA to etiology of colon cancer. BA promote tumor growth in vivo and increase cell transformation in vitro. HYPOTHESIS: BA alter normal cycle of colonic cell proliferation. AIM: to study cell cycle-dependent effects of BA on cell proliferation in the human colon cancer cell line, HT29. Cells were seeded at 4 x 104/cm2 . 24h later, medium was replaced with serum-free medium to render cells quiescent. GO/GI arrest was confirmed by flow cytometry. After 24h, 0.5% FBS was added to stimulate proliferation (TO), and either PBS (control), 50pM chenodeoxycholate (CDC), deoxycholate (DC), or ursodeoxycholate (UDC) were added to 112 of wells. 24h later, (T24), cells were washed to remove agents, and fresh 0.5% FBS-containing medium was added, while the same agents were freshly added to the other 112 of wells. 48h after TO (T48), DNA content was measured using a fluorescent oligonucleotide binding probe. In parallel, cells were counted with a hemocytometer, following trypan blue staining. There was no difference in number between cells exposed to either PBS or UDC, while there was a significant decrease in cell number after exposure to both CDC and DC for TO-T24. Decrease in cell number observed with DC incubation was linearly correlated with DNA content. This is consistent with either DC-induced growth arrest, or increased cell death. The latter is unlikely, since there was no change in number of either trypan-permeable (dead) cells or unattached cells after DC or CDC exposure. In contrast, the 50% decrease in cell number after TO-T24 CDC incubation was accompanied by a DNA content per cell that was 2 fold greater compared to that of control, DC or UOc. We conclude that CDC induces G2 arrest, in that DNA has been replicated without cell division. This is consistent with the observation that population doubling (PD) time of GO/GI synchronized HT29 cells is around 30-38h, as determined initially, and subsequently, 18 to 24h following addition of 0.5% FBS. Therefore by T48, I PD would have occurred. In sharp contrast to these findings, cells exposed to BA from T24-T48, showed no decrease in either cell number or DNA content. Thus the ability of DC and CDC to block cell proliferation was only observed with GO/GI synchronized cells. Results of this study show that certain BA alter normal cell cycle progression in a cell cycle-dependent fashion. Dysregulation of cell cycle control may be one mechanism by which BA induce their co-carcinogenic effects.