ABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of autophagy in MnCl2-induced apoptosis in human bronchial epithelial 16HBE cells.</p><p><b>METHODS</b>Cell proliferation was measured by MTT assay. Mitochondrial membrane potential (MMP) and apoptosis were measured by flow cytometry. Autophagic vacuoles were detected by fluorescence microscopy. Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.</p><p><b>RESULTS</b>16HBE cell proliferation was inhibited by MnCl2 in a dose- and time-dependent manner. MnCl2-induced 16HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis. Our data revealed that MnCl2-induced apoptosis in 16HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3. It was observed that when we exposed 16HBE cells to MnCl2 in a dose-dependent manner, the formation of autophagic vacuoles and the levels of LC-3B-II were elevated. RNA interference of LC3B in these MnCl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced. Additionally, the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis, but did not affect the cellular levels of LC3B in MnCl2-treated 16HBE cells.</p><p><b>CONCLUSION</b>MnCl2 dose- and time-dependently inhibits 16HBE cell proliferation and induces MMP loss and apoptosis. Autophagy acts in a protective role against MnCl2-induced apoptosis in 16HBE cells.</p>
Subject(s)
Humans , Amino Acid Chloromethyl Ketones , Pharmacology , Apoptosis , Autophagy , Physiology , Bronchi , Cell Line , Chlorides , Pharmacology , Down-Regulation , Epithelial Cells , Gene Expression Regulation , Manganese Compounds , PharmacologyABSTRACT
In this study, a new parameter, S phase cell percentage (S fraction) normalized BrdU (SFN-BrdU) incorporation rate, was introduced to detect S arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, SFN-BrdU incorporation rate can be used to detecting S arrest.
Subject(s)
Humans , Bromodeoxyuridine , Pharmacokinetics , Cell Proliferation , DNA Damage , Epithelial Cells , Cell Biology , S Phase , S Phase Cell Cycle CheckpointsABSTRACT
<p><b>BACKGROUND</b>Mutations in mitotic checkpoint genes have been detected in several human cancers, which exhibit chromosome instability. We wanted to know whether mutation of hBub1 could occur in transformed human embryo lung fibroblasts (HELF) cells induced by a chemical carcinogen.</p><p><b>METHODS</b>HELF cells were transformed by N-methyl-N'-nitro-N-nitrosoguaridine (MNNG), and three flasks of transformed HELF cells (named as T1, T2, and T3) were selected as amplifiers, and mutations of hBub1 in these transformed cells were analyzed by PCR-SSCP and sequencing.</p><p><b>RESULTS</b>It was found that any one of three transformed cell lines exhibited aneuploidy with a low mitotic checkpoint function. Subsequent PCR-SSCP and sequence analysis showed an AGT to CGT or ATT mutation at codon 80 in hBub1 gene in T1 cells with a resultant change in amino acid sequence.</p><p><b>CONCLUSION</b>Our study demonstrated that the mitotic checkpoint genes could be targets of MNNG.</p>