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Accumulation of abasic sites induces genomic instability in normal human gastric epithelial cells during Helicobacter pylori infection.

Kidane D, Murphy DL, Sweasy JB - Oncogenesis (2014)

Bottom Line: Here, we show that upon H. pylori infection, abasic (AP) sites accumulate and lead to increased levels of double-stranded DNA breaks (DSBs).In contrast, downregulation of the OGG1 DNA glycosylase decreases the levels of both AP sites and DSBs during H. pylori infection.Processing of AP sites during different phases of the cell cycle leads to an elevation in the levels of DSBs.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology and Toxicology, College of Pharmacy, Dell Pediatric Research Institute, The University of Texas at Austin, Austin, TX, USA.

ABSTRACT
Helicobacter pylori infection of the human stomach is associated with inflammation that leads to the release of reactive oxygen and nitrogen species (RONs), eliciting DNA damage in host cells. Unrepaired DNA damage leads to genomic instability that is associated with cancer. Base excision repair (BER) is critical to maintain genomic stability during RONs-induced DNA damage, but little is known about its role in processing DNA damage associated with H. pylori infection of normal gastric epithelial cells. Here, we show that upon H. pylori infection, abasic (AP) sites accumulate and lead to increased levels of double-stranded DNA breaks (DSBs). In contrast, downregulation of the OGG1 DNA glycosylase decreases the levels of both AP sites and DSBs during H. pylori infection. Processing of AP sites during different phases of the cell cycle leads to an elevation in the levels of DSBs. Therefore, the induction of oxidative DNA damage by H. pylori and subsequent processing by BER in normal gastric epithelial cells has the potential to lead to genomic instability that may have a role in the development of gastric cancer. Our results are consistent with the interpretation that precise coordination of BER processing of DNA damage is critical for the maintenance of genomic stability.

No MeSH data available.


Related in: MedlinePlus

(a–e) Infection with H. pylori induces chromosomal aberrations and cellular transformation. (a) Representative images of metaphase spreads from non-infected and GES-1 cells infected with H. pylori for 24 h. Note that arrowheads show fusion, black arrows show chromatid breaks and gray arrows show chromosome breaks. (b) Estimated percentage of different types aberrations. (c) Representative image of transformed cells after H. pylori infection for 35 days coculture. (d) Estimated number of foci/field for transformed cells that were infected with H. pylori for 35 days. All statistical analysis was performed using the paired t-test on GraphPad prism software. (e) Proposed model of DNA damage response and repair in H. pylori-infected gastric epithelial cells. H. pylori induces RONS that could result in 8oxodG lesions (shown as orange circles) that are likely removed with OGG1, resulting in the formation of AP sites (represented by blue circles). AP sites are clustered in close proximity on the opposite strands of the DNA, and processed with AP lyase activity that cleaves the 3′-side of AP site40, 45, 88, 89 or with APE1 that cleaves the phosphate backbone and generates DSBs (left side of the model) in G1 phase of cell cycle. In contrast, OGG1 downregulation reduces the numbers of AP sites that accumulate during G1 phase of the cell cycle and reduces the levels of DSBs during infection. The second alternative is if AP sites are localized in the vicinity of active replication forks, then cleavage of the AP site at the replication fork could lead to replication fork collapse at DSBs (right side of the model) during the S/G2 phase of cell cycle. We propose that repair at G1 phase of DSBs may lead to chromosomal aberrations that are manifested at the metaphase stage of cell division and eventually repaired via non-homologous end-joining pathways that likely induce cellular transformation. In contrast repair at S/G2 phase is processed by homologous recombination-dependent repair.
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fig7: (a–e) Infection with H. pylori induces chromosomal aberrations and cellular transformation. (a) Representative images of metaphase spreads from non-infected and GES-1 cells infected with H. pylori for 24 h. Note that arrowheads show fusion, black arrows show chromatid breaks and gray arrows show chromosome breaks. (b) Estimated percentage of different types aberrations. (c) Representative image of transformed cells after H. pylori infection for 35 days coculture. (d) Estimated number of foci/field for transformed cells that were infected with H. pylori for 35 days. All statistical analysis was performed using the paired t-test on GraphPad prism software. (e) Proposed model of DNA damage response and repair in H. pylori-infected gastric epithelial cells. H. pylori induces RONS that could result in 8oxodG lesions (shown as orange circles) that are likely removed with OGG1, resulting in the formation of AP sites (represented by blue circles). AP sites are clustered in close proximity on the opposite strands of the DNA, and processed with AP lyase activity that cleaves the 3′-side of AP site40, 45, 88, 89 or with APE1 that cleaves the phosphate backbone and generates DSBs (left side of the model) in G1 phase of cell cycle. In contrast, OGG1 downregulation reduces the numbers of AP sites that accumulate during G1 phase of the cell cycle and reduces the levels of DSBs during infection. The second alternative is if AP sites are localized in the vicinity of active replication forks, then cleavage of the AP site at the replication fork could lead to replication fork collapse at DSBs (right side of the model) during the S/G2 phase of cell cycle. We propose that repair at G1 phase of DSBs may lead to chromosomal aberrations that are manifested at the metaphase stage of cell division and eventually repaired via non-homologous end-joining pathways that likely induce cellular transformation. In contrast repair at S/G2 phase is processed by homologous recombination-dependent repair.

Mentions: DSBs can lead to chromosomal fragmentation and genomic rearrangements if not repaired in an accurate and timely manner.61 Previous work has documented the existence of chromosomal aberrations but only in gastric cancer cells infected with H. pylori. However, it is not known whether H. pylori infection induces chromosomal aberrations in normal gastric epithelial GES-1 cells, which are the cells that are initially infected with this pathogen. Therefore, we asked whether H. pylori induces chromosomal aberrations in normal human gastric epithelial cells. Infection of GES-1 cells with H. pylori for 24 h results in significantly increased levels of chromosomal fusions (64%), chromosomal fragments (48%), chromatid breaks (36%) and chromosome breaks (n=21%) versus non-infected cells (P=0.0001; Figures 7a and b). Our observation is significant because the levels of chromosomal aberrations are more pronounced in normal gastric epithelial cells than in previously reported gastric cancer cell lines infected with H. pylori.53 We tested the hypothesis that the genomic instability resulting from H. pylori infection induces cellular transformation. We infected normal gastric epithelial cells with H. pylori for 35 days and monitored focus formation. In the focus formation assay, non-transformed cells will grow to confluence forming a monolayer (Figure 7c), whereas transformed cells will continue to grow after reaching confluence, thereby forming foci (Figure 7c). We find that the number of foci/field are significantly increased in infected cells versus non-infected control cells (Figure 7d; P=0.0001).


Accumulation of abasic sites induces genomic instability in normal human gastric epithelial cells during Helicobacter pylori infection.

Kidane D, Murphy DL, Sweasy JB - Oncogenesis (2014)

(a–e) Infection with H. pylori induces chromosomal aberrations and cellular transformation. (a) Representative images of metaphase spreads from non-infected and GES-1 cells infected with H. pylori for 24 h. Note that arrowheads show fusion, black arrows show chromatid breaks and gray arrows show chromosome breaks. (b) Estimated percentage of different types aberrations. (c) Representative image of transformed cells after H. pylori infection for 35 days coculture. (d) Estimated number of foci/field for transformed cells that were infected with H. pylori for 35 days. All statistical analysis was performed using the paired t-test on GraphPad prism software. (e) Proposed model of DNA damage response and repair in H. pylori-infected gastric epithelial cells. H. pylori induces RONS that could result in 8oxodG lesions (shown as orange circles) that are likely removed with OGG1, resulting in the formation of AP sites (represented by blue circles). AP sites are clustered in close proximity on the opposite strands of the DNA, and processed with AP lyase activity that cleaves the 3′-side of AP site40, 45, 88, 89 or with APE1 that cleaves the phosphate backbone and generates DSBs (left side of the model) in G1 phase of cell cycle. In contrast, OGG1 downregulation reduces the numbers of AP sites that accumulate during G1 phase of the cell cycle and reduces the levels of DSBs during infection. The second alternative is if AP sites are localized in the vicinity of active replication forks, then cleavage of the AP site at the replication fork could lead to replication fork collapse at DSBs (right side of the model) during the S/G2 phase of cell cycle. We propose that repair at G1 phase of DSBs may lead to chromosomal aberrations that are manifested at the metaphase stage of cell division and eventually repaired via non-homologous end-joining pathways that likely induce cellular transformation. In contrast repair at S/G2 phase is processed by homologous recombination-dependent repair.
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Related In: Results  -  Collection

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fig7: (a–e) Infection with H. pylori induces chromosomal aberrations and cellular transformation. (a) Representative images of metaphase spreads from non-infected and GES-1 cells infected with H. pylori for 24 h. Note that arrowheads show fusion, black arrows show chromatid breaks and gray arrows show chromosome breaks. (b) Estimated percentage of different types aberrations. (c) Representative image of transformed cells after H. pylori infection for 35 days coculture. (d) Estimated number of foci/field for transformed cells that were infected with H. pylori for 35 days. All statistical analysis was performed using the paired t-test on GraphPad prism software. (e) Proposed model of DNA damage response and repair in H. pylori-infected gastric epithelial cells. H. pylori induces RONS that could result in 8oxodG lesions (shown as orange circles) that are likely removed with OGG1, resulting in the formation of AP sites (represented by blue circles). AP sites are clustered in close proximity on the opposite strands of the DNA, and processed with AP lyase activity that cleaves the 3′-side of AP site40, 45, 88, 89 or with APE1 that cleaves the phosphate backbone and generates DSBs (left side of the model) in G1 phase of cell cycle. In contrast, OGG1 downregulation reduces the numbers of AP sites that accumulate during G1 phase of the cell cycle and reduces the levels of DSBs during infection. The second alternative is if AP sites are localized in the vicinity of active replication forks, then cleavage of the AP site at the replication fork could lead to replication fork collapse at DSBs (right side of the model) during the S/G2 phase of cell cycle. We propose that repair at G1 phase of DSBs may lead to chromosomal aberrations that are manifested at the metaphase stage of cell division and eventually repaired via non-homologous end-joining pathways that likely induce cellular transformation. In contrast repair at S/G2 phase is processed by homologous recombination-dependent repair.
Mentions: DSBs can lead to chromosomal fragmentation and genomic rearrangements if not repaired in an accurate and timely manner.61 Previous work has documented the existence of chromosomal aberrations but only in gastric cancer cells infected with H. pylori. However, it is not known whether H. pylori infection induces chromosomal aberrations in normal gastric epithelial GES-1 cells, which are the cells that are initially infected with this pathogen. Therefore, we asked whether H. pylori induces chromosomal aberrations in normal human gastric epithelial cells. Infection of GES-1 cells with H. pylori for 24 h results in significantly increased levels of chromosomal fusions (64%), chromosomal fragments (48%), chromatid breaks (36%) and chromosome breaks (n=21%) versus non-infected cells (P=0.0001; Figures 7a and b). Our observation is significant because the levels of chromosomal aberrations are more pronounced in normal gastric epithelial cells than in previously reported gastric cancer cell lines infected with H. pylori.53 We tested the hypothesis that the genomic instability resulting from H. pylori infection induces cellular transformation. We infected normal gastric epithelial cells with H. pylori for 35 days and monitored focus formation. In the focus formation assay, non-transformed cells will grow to confluence forming a monolayer (Figure 7c), whereas transformed cells will continue to grow after reaching confluence, thereby forming foci (Figure 7c). We find that the number of foci/field are significantly increased in infected cells versus non-infected control cells (Figure 7d; P=0.0001).

Bottom Line: Here, we show that upon H. pylori infection, abasic (AP) sites accumulate and lead to increased levels of double-stranded DNA breaks (DSBs).In contrast, downregulation of the OGG1 DNA glycosylase decreases the levels of both AP sites and DSBs during H. pylori infection.Processing of AP sites during different phases of the cell cycle leads to an elevation in the levels of DSBs.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology and Toxicology, College of Pharmacy, Dell Pediatric Research Institute, The University of Texas at Austin, Austin, TX, USA.

ABSTRACT
Helicobacter pylori infection of the human stomach is associated with inflammation that leads to the release of reactive oxygen and nitrogen species (RONs), eliciting DNA damage in host cells. Unrepaired DNA damage leads to genomic instability that is associated with cancer. Base excision repair (BER) is critical to maintain genomic stability during RONs-induced DNA damage, but little is known about its role in processing DNA damage associated with H. pylori infection of normal gastric epithelial cells. Here, we show that upon H. pylori infection, abasic (AP) sites accumulate and lead to increased levels of double-stranded DNA breaks (DSBs). In contrast, downregulation of the OGG1 DNA glycosylase decreases the levels of both AP sites and DSBs during H. pylori infection. Processing of AP sites during different phases of the cell cycle leads to an elevation in the levels of DSBs. Therefore, the induction of oxidative DNA damage by H. pylori and subsequent processing by BER in normal gastric epithelial cells has the potential to lead to genomic instability that may have a role in the development of gastric cancer. Our results are consistent with the interpretation that precise coordination of BER processing of DNA damage is critical for the maintenance of genomic stability.

No MeSH data available.


Related in: MedlinePlus