Pivotal role of inosine triphosphate pyrophosphatase in maintaining genome stability and the prevention of apoptosis in human cells

Abstract

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.

Figure 1. HAP treatment leads to the appearance of EndoV sensitive sites in HeLa DNA.

We extracted genomic DNA from HeLa cells grown with or without HAP. Treatment of this DNA with bacterial EndoV creates 3′ nicks, which are substrates for nick-translation (BioProbe® Nick translation kit with bio-16-dUTP (Enzo Life Sciences)) as described in Materials and Methods. A. Agarose gel electrophoresis of nick-translated DNA from HeLa cells. 1- from untreated cells; 1a – from untreated cells digested with DNase; 2 – from cells grown in 2.64 mM HAP; 3- from untreated cells, DNA incubated with Endo V; and 4 - from cells grown in 2.64 mM HAP, DNA incubated with Endo V. B. Detection of newly synthesized biotinylated DNA separated by alkaline agarose electrophoresis. 1- from untreated cells; 2 – from cells grown in 2.64 mM HAP; 3- from untreated cells, DNA incubated with Endo V; and 4 - from cells grown in 2.64 mM HAP, DNA incubated with Endo V.

Figure 2. Effects of HAP treatment on apoptosis and ITPA levels in HeLa cells.

(A) HAP treatment causes apoptosis in HeLa cells after treatment for 24 or 48 hours. By two-way ANOVA, p_time = 0.001 and p_{concentration} = 0.0046. **p<0.01 by Bonferroni multiple comparison post test for column analysis comparing means for 24 hours vs. 48 hours. (B) HAP treatment leads to the increase of ITPA protein levels in HeLa extracts following treatment for both 24 hours and 48 hours. Western blots were performed as described in Materials and Methods. P- pure ITPA protein, C- untreated control, DMSO – solvent only. (C) HAP treatment does not increase levels of ITPA transcripts. The analysis was performed as described in Materials and Methods. HAP treatment was for 24 hours. Mr – 100 bp ladder.

Figure 3. HAP-induced apoptosis occurs through the intrinsic pathway.

(A) Protection from HAP-induced apoptosis by overexpression of Bcl-xL. Both HeLa and HeLa-xL cell lines were treated with increasing doses of HAP for 48 hours and the percentage of apoptotic cells was determined by Hoechst staining. By two-way ANOVA, p_{cell line}<0.0001 and p_{concentration} = 0.009. **p<0.01,***p<0.001 by Bonferroni multiple comparison post test for column analysis comparing means for HeLa vs. HeLa-xL hours. (B) Confirmation of protection from HAP-induced apoptosis by Bcl-xL overexpression by immunoblot for PARP cleavage. HAP induced dose-dependent cleavage of PARP in regular HeLa cells but not in HeLa cells overexpressing Bcl-xL. PAPR cleavage product is marked as cl₈₉. (C) HAP treatment results in similar levels of DNA breaks in HeLa and HeLa+Bcl-xL cell lines (p>0.05).

Figure 4. ITPA protects against HAP-induced apoptosis.

ITPA knockdown sensitizes cells to HAP-induced apoptosis. As compared to the control and non-targeting shRNA transfected cells, ITPA knockdown cells undergo approximately 30–50% apoptosis upon HAP treatment for 24 hours. Hydrogen peroxide treatment (0.1 mM, four hours) was used as a positive control. The difference between control cells and cells with the ITPA knockdown is highly significant (***p<0.001, ****p<0.0001). There was no difference between the control versus the non-targeting cell lines in all HAP doses tested. No significant difference in hydrogen peroxide-induced apoptosis was observed for all three cell lines.

Figure 5. ITPase overexpression suppresses HAP-induced cytotoxicity.

(A) Effect of overexpression of the ITPA and the gene encoding yeast ITPase, HAM1, on HAP-induced apoptosis in HeLa cells. Differences are significant (*p<0.05, **p<0.01). (B) Overexpression of the yeast HAM1 could rescue ITPA knockdown cells from hypersensitivity to HAP-induced apoptosis (****p<0.0001, n.s., not significant).

Figure 6. ITPA protects against HAP-induced DNA breaks.

Alkaline comet assay data reveals that as compared to the control and non-targeting shRNA-expressing cell lines, ITPA knockdown cells accumulated elevated levels of DNA breaks after 24 hours of treatment with HAP. At high doses of HAP, the sizes of the comet tails in the ITPA knockdown cells were too large to be quantified. The assay was performed at lower doses of HAP treatment in order to obtain measurable comet tails. As compared to the cells with vector, the overexpression of HAM1 suppressed the accumulation of HAP-induced DNA breaks in the ITPA knockdown cells. Statistical differences were measured by one-way ANOVA followed by Dunns multiple comparisons for column analysis. By two-way ANOVA, p = 0.008. Post-test analysis revealed differences (p<0.05) for knockdown versus knockdown+HAM1 and knockdown+vector versus knockdown+HAM1. No difference in levels of DNA breaks was observed for hydrogen peroxide treatment for all three cell lines.

Figure 7. ITPA protects against HAP-induced mutagenesis.

The data represent the induced HPRT mutant frequency for the HAP-treated control and ITPA knockdown cells. At a low dose (0.1 mM) of HAP treatment for 24 hours the difference between cell lines was not significant but at dose 1 mM ITPA knockdown cells were more sensitive to HAP mutagenesis. (**p<0.01, n.s., not significant).

Figure 8. Model for the protective role of ITPA against HAP-induced genotoxicity and mutagenesis.

In the presence of functional ITPA, the accumulation of non-canonical nucleotides like dHAPTP is abrogated by the ITPase, thereby preventing their incorporation into DNA. In the absence of functional ITPase, dHAPTP accumulates in the precursor pool and is incorporated into DNA by the replicative DNA polymerases. Grey circles represent HAP accumulation in DNA. Slow excision of base analogs by an unknown nuclease/glycosylase results in the accumulation of single-strand DNA breaks, which triggers apoptosis. Increased levels of apoptosis contribute to the onset of degenerative diseases. In the absence of repair, HAP persists in DNA causing incorrect pairing with T or C, thus leading to the accumulation of mutations, which predisposes individuals to the development of cancer.