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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing
Chun-Qi Li1, Laura J Trudel, Gerald N Wogan
1Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
Nitric oxide (NO(*)) is mutagenic and, under appropriate conditions of exposure, also induces apoptosis in many in vitro and in vivo experimental models. Biochemical and cellular mechanisms through which NO(*) induces apoptosis are incompletely understood, but involve p53/mitochondria-dependent signaling pathways. In this study, we exposed human lymphoblastoid cells harboring either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) to NO(*), delivered by diffusion through Silastic tubing. Cells were exposed for 2 h at constant rates of 100-533 nM/s, similar to levels estimated to occur in vivo in inflamed tissues. DNA double-strand breaks and fragmentation detected 8-48 h after NO(*) treatment were more extensive in TK6 cells than in WTK-1 cells, whereas NO(*)-induced mutant fractions in both HPRT and TK1 genes were significantly lower in TK6 cells than in WTK-1 cells (P < 0.01-0.05). Treatment of TK6 cells with NO(*) caused extensive apoptosis, but this response was delayed and greatly reduced in magnitude in WTK-1 cells. Mitochondrial membrane depolarization and cytochrome c release were induced in both cell types. However, elevation of apoptotic protease-activating factor-1 (Apaf-1) protein and reduction of X-chromosome-linked inhibitor of apoptosis (XIAP) protein were observed only in TK6 cells. These results indicate that p53 status is an important modulator of NO(*)-induced mutagenesis and apoptosis, and suggest that levels of the Apaf-1 and XIAP proteins, but not mitochondrial depolarization and cytochrome c release, are regulated by p53 in these human lymphoblastoid cells. Thus, Apaf-1 and XIAP may play important roles in the regulation of p53-mediated apoptotic responses.
Insights
Nitric oxide (NO) induces mutations and apoptosis. The p53 protein
Area of Science:
- Cellular and Molecular Biology
- Toxicology
- Cancer Research
Background:
- Nitric oxide (NO) is a signaling molecule with known mutagenic and apoptotic effects.
- The precise mechanisms by which NO induces apoptosis, particularly involving the p53 protein and mitochondria, are not fully elucidated.
- Understanding these pathways is crucial for comprehending cellular responses to NO in inflammatory conditions.
Purpose of the Study:
- To investigate the role of p53 status in modulating NO-induced mutagenesis and apoptosis in human lymphoblastoid cells.
- To elucidate the specific cellular and biochemical pathways affected by NO in a p53-dependent manner.
- To compare the effects of NO on DNA damage, gene mutation, and apoptosis in cells with wild-type versus mutant p53.
Main Methods:
- Exposure of human lymphoblastoid TK6 (wild-type p53) and WTK-1 (mutant p53) cells to NO via Silastic tubing at controlled rates.
- Assessment of DNA double-strand breaks and fragmentation.
- Quantification of gene mutations in HPRT and TK1 genes.
- Analysis of apoptosis, mitochondrial membrane potential, cytochrome c release, and levels of Apaf-1 and XIAP proteins.
Main Results:
- NO induced more extensive DNA damage in wild-type p53 cells (TK6) than in mutant p53 cells (WTK-1).
- Mutagenesis was significantly lower in wild-type p53 cells compared to mutant p53 cells.
- Apoptosis was pronounced in wild-type p53 cells but reduced and delayed in mutant p53 cells.
- Mitochondrial depolarization and cytochrome c release occurred in both cell types, but Apaf-1 elevation and XIAP reduction were specific to wild-type p53 cells.
Conclusions:
- p53 status is a critical regulator of NO-induced mutagenesis and apoptosis.
- p53 influences NO-induced apoptosis through pathways involving Apaf-1 and XIAP protein levels, not solely mitochondrial events.
- These findings highlight the importance of Apaf-1 and XIAP in p53-mediated apoptotic responses to NO.
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