Helicobacter pylori CagA causes mitotic impairment and induces chromosomal instability

Abstract

Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively contribute to the progression of multistep gastric carcinogenesis.

FIGURE 1.

Establishment of CagA-inducible gastric epithelial cells that constitutively express H2B-GFP. A, schematic representation of CagA and H2B-GFP. B, fluorescence of H2B-GFP in the nucleus of A10/H2B-GFP cells. A10/H2B-GFP cells were generated by stably transfecting an H2B-GFP-expression vector into WT-A10 cells, MKN28-derived human gastric epithelial cells in which CagA is inducibly expressed by depleting Doxycycline from the culture. Fluorescence image (upper). Fluorescence image plus phase contrast image (lower). Scale bars, 20 μm. C, inducible expression of HA-tagged CagA in WT-A10 and A10/H2B-GFP cells. Cells were cultured in the presence or absence of Dox and cell lysates were subjected to immunoblotting with the indicated antibodies.

FIGURE 2.

Effect of CagA on the progression of mitosis. A10/H2B-GFP cells were cultured in the presence (CagA (−)) or absence (CagA (+)) of Dox. At 12 h after the onset of Dox treatment, cells were subjected to time-lapse fluorescence microscopic analysis. Representative images for the progression of mitosis starting from the onset of prophase (0 min) in A10/H2B-GFP cells with (lower) or without (upper) CagA expression. QuickTime videos of these experiments are included as supplemental videos 1 and 2.

FIGURE 3.

Prolonged mitotic chromosomal segregation in cells expressing CagA. The time length from the onset of chromatin condensation to the initiation of chromosomal separation was measured in A10/H2B-GFP cells with or without CagA expression. *, p < 0.05, statistically significant (Student's t test) (n = 11). Bars indicate mean.

FIGURE 4.

Delay in prophase and metaphase by CagA. A, WT-A10 cells were cultured in the presence or absence of Dox. After 12 h, cells were stained with an antibody against phosphorylated serine 10 on histone H3 (H3-pSer10) to count H3-pSer10-positive cells. Error bars indicate the mean ± S.D. *, p < 0.05, statistically significant (Student's t test) (n = 3). B, WT-A10 cells were induced to express CagA by depleting Dox in the absence or presence of the mitotic inhibitor, nocodazole. After 12, 15, and 18 h of CagA induction, cells were stained with an antibody against H3-pSer10. DMSO was used as a control of nocodazole treatment. Error bars indicate the mean ± S.D. Three hundred cells were subjected to the staining analysis in each of three independent experiments. C, WT-A10 cells were cultured in the presence or absence of Dox. After 12 h, cells were stained with 4′,6-diamidino-2-phenylindole to count anaphase cells. Error bars indicate the mean ± S.D. Two hundred cells were subjected to the staining analysis in each of three independent experiments.

FIGURE 5.

Delay in prophase and metaphase by CagA-mediated PAR1 inhibition. WT-A10 cells were induced to express CagA by depleting Dox in the presence of the indicated adenoviruses (multiplicity of infection = 200 for each virus). After 12 h, cells were stained with an antibody against phosphorylated serine 10 on histone H3 (H3-pSer10) to count H3-pSer10-positive cells. Error bars indicate the mean ± S.D. *, p < 0.05; **, p < 0.01, statistically significant (Student's t test) (n = 3) (left). Expressed protein levels are shown (right). β-Gal, β-galactosidase.

FIGURE 6.

Impaired cell division axis by CagA. A, confocal x-z plane views of spindle orientation in anaphase. A10/H2B-GFP cells were cultured in the presence or absence of Dox. After 12 h, cells were fixed, and segregation of chromosomes was visualized by confocal microscopy. B, percentages of cells with spindle misorientation in the presence or absence of CagA. In this experiment, cells in which the angles of the two daughter chromosomes (α) incline more than 10° were judged to have destabilized cell division axis (left). Fifteen anaphase cells were investigated in each of three independent experiments. Error bars indicate the mean ± S.D. *, p < 0.05, statistically significant (Student's t test) (right).

FIGURE 7.

Chromosomal instability caused by repeated exposure to CagA. WT-A10 cells were cultured for 6 days with intermittent induction of CagA on days 1, 3, and 5 by depleting Dox. WT-A10 cells without CagA induction during the culture were used as a control. After the culture, cells were harvested, stained with propidium iodide, and subjected to DNA histogram analysis using flow cytometry. The arrow shows tetraploid (8N) (upper). Percentages of cells in each cell cycle phases were determined by use of CellQuest and ModFit cell cycle analysis software. Error bars indicate the mean ± S.D. *, p < 0.001, statistically significant (Student's t test) (lower).