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Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila.

Wandler AM, Guillemin K - PLoS Pathog. (2012)

Bottom Line: This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells.We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis.In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT
Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.

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CagA expression causes apoptosis and epithelial disruption.(A) Schematic illustrating the fate of different domains within the wing imaginal disc. Each color-coded region of the larval structure on the left corresponds to the specified region of the adult wing on the right (modified from [24]). (B–G) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression and stained with an antibody against active caspase-3 to mark apoptotic cells. A control wing disc epithelium expressing only mGFP with the bx-GAL4 dorsal wing driver (B) lacks apoptotic cells. Ubiquitous expression of CagA in the wing disc with the 765-GAL4 driver (C) does not cause apoptosis, while expressing CagA with bx-GAL4 (D) triggers formation of apoptotic clusters within the expression domain. Expressing two copies of CagA with bx-GAL4 (E) causes a dose-dependent enhancement of the apoptosis phenotype. Expressing CagAEPISA with bx-GAL4 (F) does not cause a phenotype, while expressing two copies of CagAEPISA (G) produces small apoptotic clusters. Scale bars, 50 µm. (H) XZ confocal plane of a male wing imaginal disc epithelium expressing mGFP and CagA with bx-GAL4 stained with antibodies against active caspase-3 to show basal extrusion of apoptotic cells and matrix metalloproteinase 1 (Mmp1) to show evidence of basement membrane breakdown. Scale bar, 20 µm. (I–N) Adult wing images from male flies of each indicated genotype. Neither expression of mGFP alone with bx-GAL4 (I) nor expression of CagA with 765-GAL4 (J) causes a phenotype in the adult wing. Dorsal wing expression of CagA with bx-GAL4 (K) disrupts epithelial integrity in a dose-dependent manner (L). Expressing CagAEPISA with bx-GAL4 (M) does not cause an adult wing phenotype, while expressing two copies of CagAEPISA (N) causes epithelial disruption. Scale bar, 500 µm.
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ppat-1002939-g001: CagA expression causes apoptosis and epithelial disruption.(A) Schematic illustrating the fate of different domains within the wing imaginal disc. Each color-coded region of the larval structure on the left corresponds to the specified region of the adult wing on the right (modified from [24]). (B–G) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression and stained with an antibody against active caspase-3 to mark apoptotic cells. A control wing disc epithelium expressing only mGFP with the bx-GAL4 dorsal wing driver (B) lacks apoptotic cells. Ubiquitous expression of CagA in the wing disc with the 765-GAL4 driver (C) does not cause apoptosis, while expressing CagA with bx-GAL4 (D) triggers formation of apoptotic clusters within the expression domain. Expressing two copies of CagA with bx-GAL4 (E) causes a dose-dependent enhancement of the apoptosis phenotype. Expressing CagAEPISA with bx-GAL4 (F) does not cause a phenotype, while expressing two copies of CagAEPISA (G) produces small apoptotic clusters. Scale bars, 50 µm. (H) XZ confocal plane of a male wing imaginal disc epithelium expressing mGFP and CagA with bx-GAL4 stained with antibodies against active caspase-3 to show basal extrusion of apoptotic cells and matrix metalloproteinase 1 (Mmp1) to show evidence of basement membrane breakdown. Scale bar, 20 µm. (I–N) Adult wing images from male flies of each indicated genotype. Neither expression of mGFP alone with bx-GAL4 (I) nor expression of CagA with 765-GAL4 (J) causes a phenotype in the adult wing. Dorsal wing expression of CagA with bx-GAL4 (K) disrupts epithelial integrity in a dose-dependent manner (L). Expressing CagAEPISA with bx-GAL4 (M) does not cause an adult wing phenotype, while expressing two copies of CagAEPISA (N) causes epithelial disruption. Scale bar, 500 µm.

Mentions: In order to examine the effects of expressing the H. pylori effector protein CagA on an intact epithelium, we used the GAL4/UAS system to drive its expression in the wing imaginal disc. The Drosophila wing begins to form during early larval life when it exists as a primordial sac which contains both a simple columnar epithelium and the squamous epithelium of the peripodial membrane [23]. Cells within the wing imaginal disc proliferate extensively in larval stages followed by disc evagination during pupation, resulting in the adult wing structure. This developmental process is distinct from that of the eye imaginal disc used to model CagA pathogenesis previously [7]–[10], which undergoes systematic differentiation during larval stages. In addition, the fate of imaginal disc cells is specified early in development [24] which allowed us to express CagA in distinct regions of the wing disc (Figure 1A).


Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila.

Wandler AM, Guillemin K - PLoS Pathog. (2012)

CagA expression causes apoptosis and epithelial disruption.(A) Schematic illustrating the fate of different domains within the wing imaginal disc. Each color-coded region of the larval structure on the left corresponds to the specified region of the adult wing on the right (modified from [24]). (B–G) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression and stained with an antibody against active caspase-3 to mark apoptotic cells. A control wing disc epithelium expressing only mGFP with the bx-GAL4 dorsal wing driver (B) lacks apoptotic cells. Ubiquitous expression of CagA in the wing disc with the 765-GAL4 driver (C) does not cause apoptosis, while expressing CagA with bx-GAL4 (D) triggers formation of apoptotic clusters within the expression domain. Expressing two copies of CagA with bx-GAL4 (E) causes a dose-dependent enhancement of the apoptosis phenotype. Expressing CagAEPISA with bx-GAL4 (F) does not cause a phenotype, while expressing two copies of CagAEPISA (G) produces small apoptotic clusters. Scale bars, 50 µm. (H) XZ confocal plane of a male wing imaginal disc epithelium expressing mGFP and CagA with bx-GAL4 stained with antibodies against active caspase-3 to show basal extrusion of apoptotic cells and matrix metalloproteinase 1 (Mmp1) to show evidence of basement membrane breakdown. Scale bar, 20 µm. (I–N) Adult wing images from male flies of each indicated genotype. Neither expression of mGFP alone with bx-GAL4 (I) nor expression of CagA with 765-GAL4 (J) causes a phenotype in the adult wing. Dorsal wing expression of CagA with bx-GAL4 (K) disrupts epithelial integrity in a dose-dependent manner (L). Expressing CagAEPISA with bx-GAL4 (M) does not cause an adult wing phenotype, while expressing two copies of CagAEPISA (N) causes epithelial disruption. Scale bar, 500 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475654&req=5

ppat-1002939-g001: CagA expression causes apoptosis and epithelial disruption.(A) Schematic illustrating the fate of different domains within the wing imaginal disc. Each color-coded region of the larval structure on the left corresponds to the specified region of the adult wing on the right (modified from [24]). (B–G) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression and stained with an antibody against active caspase-3 to mark apoptotic cells. A control wing disc epithelium expressing only mGFP with the bx-GAL4 dorsal wing driver (B) lacks apoptotic cells. Ubiquitous expression of CagA in the wing disc with the 765-GAL4 driver (C) does not cause apoptosis, while expressing CagA with bx-GAL4 (D) triggers formation of apoptotic clusters within the expression domain. Expressing two copies of CagA with bx-GAL4 (E) causes a dose-dependent enhancement of the apoptosis phenotype. Expressing CagAEPISA with bx-GAL4 (F) does not cause a phenotype, while expressing two copies of CagAEPISA (G) produces small apoptotic clusters. Scale bars, 50 µm. (H) XZ confocal plane of a male wing imaginal disc epithelium expressing mGFP and CagA with bx-GAL4 stained with antibodies against active caspase-3 to show basal extrusion of apoptotic cells and matrix metalloproteinase 1 (Mmp1) to show evidence of basement membrane breakdown. Scale bar, 20 µm. (I–N) Adult wing images from male flies of each indicated genotype. Neither expression of mGFP alone with bx-GAL4 (I) nor expression of CagA with 765-GAL4 (J) causes a phenotype in the adult wing. Dorsal wing expression of CagA with bx-GAL4 (K) disrupts epithelial integrity in a dose-dependent manner (L). Expressing CagAEPISA with bx-GAL4 (M) does not cause an adult wing phenotype, while expressing two copies of CagAEPISA (N) causes epithelial disruption. Scale bar, 500 µm.
Mentions: In order to examine the effects of expressing the H. pylori effector protein CagA on an intact epithelium, we used the GAL4/UAS system to drive its expression in the wing imaginal disc. The Drosophila wing begins to form during early larval life when it exists as a primordial sac which contains both a simple columnar epithelium and the squamous epithelium of the peripodial membrane [23]. Cells within the wing imaginal disc proliferate extensively in larval stages followed by disc evagination during pupation, resulting in the adult wing structure. This developmental process is distinct from that of the eye imaginal disc used to model CagA pathogenesis previously [7]–[10], which undergoes systematic differentiation during larval stages. In addition, the fate of imaginal disc cells is specified early in development [24] which allowed us to express CagA in distinct regions of the wing disc (Figure 1A).

Bottom Line: This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells.We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis.In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT
Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.

Show MeSH
Related in: MedlinePlus