Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53

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.

Fig 1.

Survival of TK6 and WTK-1 cells after NO^• treatment, as determined by trypan blue exclusion. Cells were exposed to varying rates of NO^• for 2 h. Cell survival after exposure to 100 nM/s NO^• was determined only in TK6 cells. Data represent the mean of two duplicate experiments. Standard deviations were less than 15% (not shown).

Fig 2.

Mutant fractions in the HPRT and TK1 genes of TK6 and WTK-1 cells treated with NO^• at 533 nM/s for 2 h. Cells treated with argon gas or 4-NQO (140 ng/ml for 1.5 h) served as negative and positive controls, respectively. Results shown are mean ± SD of duplicate experiments.

Fig 3.

Box-and-whisker plots of Olive tail moments from neutral comet assays of TK6 and WTK-1 cells 8 h after treatment with varying rates of NO^• for 2 h. At least 40 cells were analyzed in each sample. Cells treated with argon gas or with H₂O₂ were used as negative and positive controls, respectively. Olive tail moments were significantly higher in TK6 cells than in WTK-1 cells treated with NO^• at 533 nM/s (P < 0.05) or treated with 100 μM H₂O₂ (P < 0.01), but differences between TK6 and WTK-1 cells treated with NO^• at 300 nM/s or 1 and 10 μM H₂O₂ were not significant (P > 0.05).

Fig 4.

DNA fragmentation in TK6 and WTK-1 cells treated with varying rates of NO^• for 2 h, detected by agarose gel electrophoresis.

Fig 5.

Normalized apoptosis (A) and MMP loss (B) in TK6 and WTK-1 cells treated with varying rates of NO^• for 2 h. Cells treated with argon gas were used as negative controls. Data represent the mean of two experiments, each done in duplicate. Standard deviations were 6–16% (not shown). *, P < 0.04–0.02, compared with TK6 cells.

Fig 6.

Release of mitochondrial cytochrome c into cytosol in TK6 and WTK-1 cells treated with NO^• at 533 nM/s for 2 h, detected by Western blots. Rat heart cytochrome c (25 ng) served as a positive control.

Fig 7.

Apaf-1 protein levels in TK6 and WTK-1 cells treated with NO^• at 533 nM/s for 2 h, detected by Western blots. Lysates (50 μg) of Jurkat cells treated with 4 μM staurosporine served as a positive control.

Fig 8.

XIAP protein levels in TK6 and WTK-1 cells treated with NO^• at 533 nM/s for 2 h, detected by Western blots. Lysates (25 μg) of Jurkat cells treated with 4 μM staurosporine served as a positive control.