Threshold effects of nitric oxide-induced toxicity and cellular responses in wild-type and p53-null human lymphoblastoid cells

Abstract

Toxicity induced by nitric oxide (NO(*)) has been extensively investigated in many in vitro and in vivo experimental models. Recently, our laboratories found that both concentration and cumulative total dose are critical determinants of cell death caused by NO(*). Here, we report results of studies designed to define total dose thresholds and threshold effects for several NO(*)-induced toxicity and cellular responses and to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53-null NH32 cells to NO(*) delivered by a membrane delivery system. Cells were exposed at a steady state concentration of 0.6 microM for varying lengths of time to deliver increasing cumulative doses (expressed in units of microM min), and several end points of cytotoxicity and mutagenesis were quantified. Threshold doses for NO(*)-induced cytotoxicity were 150 microM min in TK6 cells and 300 microM min in NH32 cells, respectively. Threshold doses for NO(*)-induced apoptosis were identical to those for cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity and apoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposed to sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicity threshold) doses of NO(*). In TK6 cells (p53), the sublethal threshold dose induced DNA double-strand breaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increased only modestly (<50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TK1) locus was observed only at the toxic dose of NO(*). Treatment of NH32 cells with NO(*) at the threshold or toxic dose elevated mutagenesis of the TK1 gene, but did not cause detectable levels of DNA double-strand breaks. At similar levels of cell viability, the frequency of DNA recombinational repair was higher in p53-null NH32 cells than in wild-type TK6 cells. NO(*) treatment induced p53-independent cell cycle arrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in the modulation of toxic responses of NO(*). These findings indicate that exposure to doses of NO(*) at or above the cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellular responses to NO(*) damage. Furthermore, recombinational repair of DNA may contribute to resistance to NO(*) toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responses in human lymphoblastoid cells.

Figure 1

Cell viability in TK6 and NH32 cells 48 h after NO^• treatment, as determined by MTT assay. Data represent the mean of two to four duplicate experiments. Standard deviations were less than 15% (not shown).

Figure 2

NO^• dose-dependent mitochondrial membrane potential loss (MMP loss) and apoptosis in TK6 and NH32 cells 48 h after exposure. The threshold doses for NO-induced MMP loss and apoptosis were approximately 35 and 150 μM min in TK6 cells, and 225 and 300 μM min in NH32 cells. Data represent the mean ± SD of three independent experiments, each was done in duplicate.

Figure 3

Box-and-whisker plots of Olive tail moments from neutral comet assays of TK6 and NH32 cells right after NO^• treatment. At least 40 cells were analyzed in each sample. Cells treated with argon (Ar) gas or with H₂O₂ were used as negative and positive controls, respectively. Data from H₂O₂ treatment were not shown. * p < 0.01, compared with argon-treated control.

Figure 4

Formation of dU, dX, and dI in DNA of TK6 (solid) and NH32 (open) cells exposed to NO^• at the sublethal, threshold and toxic doses (50, 150 and 300 μM min for TK6 cells; 150, 300 and 600 μM min for NH32 cells), respectively. The only statistically significant increase in a DNA deamination product over control occurred with dU in NH32 cells treated with 600 μM min NO^• (p <0..05). Data represent the mean ± SD for N = 4.

Figure 5

Immunoblot analysis of levels of SOD1, catalase and glutathione peroxidase (GPX) proteins in TK6 and NH32 cells following various doses of NO^• treatment. Jurkat cells treated with 4 μM staurosporine were used as positive controls for SOD1 and catalase, and HL60 cells for GPX. Lower panels show changes from argon-treated negative controls. Values are mean quantitative densitometric values and 95% confidence intervals from two to three independent experiments. * p < 0.05, compared with argon-treated controls.

Figure 6

Immunoblot analysis of Akt and NF-κB p50 and p65 proteins in TK6 and NH32 cells following various doses of NO^• treatment. Jurkat cells treated with 4 μM staurosporine were used as positive controls and densitometric analyses were as indicated in Figure 5. Values are mean quantitative densitometric values and 95% confidence intervals from two to three independent experiments. * p < 0.05 and ** p < 0.01, compared with argon-treated controls. Changes in levels of NF-κB p50 protein were not statistically significant (p > 0.05) and the densitometric values not shown.

Figure 7

Immunoblot analysis of p53 (A), PTEN (B), PI3 kinase (PI3'K) (C) and phosphorylated Akt (p-Akt) (D) in TK6 (300 μM min) and NH32 cells (600 μM min) following treatment of toxic doses of NO^•. Jurkat cells treated with 4 μM staurosporine were used as positive controls and densitometric analysis were as indicated in Figure 5. Values are mean quantitative densitometric values and 95% confidence intervals from two to three independent experiments. + p53 protein was not detectable in NH32 cells. * p < 0.05, compared with argon-treated controls.

Figure 8

Schematic summary of NO^•-induced toxicity pathways in human lymphoblastoid cells, in which p53 plays a central role.