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  • br differentiated adenocarcinoma case year old man poorly di


    272 differentiated adenocarcinoma; case 2: 51-year-old man, poorly differentiated adenocarcinoma; and case 3: 35-year-old man,
    273 poorly differentiated adenocarcinoma. Cases 4 and 5 were a 75-year-old man and a 71-year-old man, respectively, who were 274 H pylori–positive and had well-differentiated adenocarcinoma. Red indicates CAPZA1 and green indicates CD44v9. The white
    275 box contains CD44v9-positive SR11302 and is enlarged at the bottom of each image. White arrowheads indicate CD44v9-positive
    276 cells. Nuclei (blue) were stained with 40,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 mm. (B) The intensity of CAPZA1 immunostaining per cell in CD44v9-positive cells and adjacent CD44v9-negative cells. By using 19 CD44v9-positive and 277 CD44v9-negative cells detected in 10 regions of 5 SR11302 human gastric tissues (cases 1–5), the intensity of CAPZA1 staining was
    278 quantified using ImageJ. The box-and-whisker plot presents the full range of variation, interquartile range, and median values.
    279 The data points indicate the CAPZA1 staining intensity of each cell. *P < .05 by the Student t test. 
    339 281 H pylori G27 strain or H pylori G27 cytotoxin-associated caused by H pylori infection of CAPZA1-overexpressing cells 340 282 gene-pathogenicity island (cagPAI)-deleted isogenic are hypothesized to increase CD44v9 mRNA expression. To 341 283 mutant strain (H pylori G27 DcagPAI) for 5 hours and determine whether CagA accumulation helps to induce 342 284 cultured them in antibiotic-containing medium for 24 hours, CD44v9 mRNA expression, we examined CagA-expressing 343 285 and then analyzed CD44v9 mRNA expression. H pylori G27 WT-A10 cells, in which forced CagA expression was Q11344 286 infection did not increase CD44v9 mRNA expression signif- induced by the pTet-off-cagA expression vector upon cul- 345 287 icantly in AGS cells (Figure 2C). By contrast, CD44v9 mRNA ture in the absence of doxycycline for 24 hours.16 Although 346 288 expression was significantly higher in CAPZA1- the level of CD44v9 mRNA expression in WT-A10 cells 347 289 overexpressing AGS cells infected with H pylori G27 than without stable CagA expression was equivalent to that in 348 290 in noninfected CAPZA1-overexpressing AGS cells MKN-II cells, it was increased significantly in CagA- 349 291 (Figure 2C). H pylori G27 DcagPAI infection did not increase expressing WT-A10 cells (Figure 2D). We subsequently 350 292 CD44v9 mRNA expression in CAPZA1-overexpressing cells examined whether CagA accumulation increases CAPZA1 351 293 (Figure 2C). Based on these findings, accumulation of CagA expression. CAPZA1 mRNA expression was not increased in 352
    4 Tsugawa et al
    Cellular and Molecular Gastroenterology and Hepatology Vol. -, No. -
    472 indicate that accumulation of CagA resulting from H pylori overexpressing AGS cells than in AGS cells (Figure 3A, lanes 531
    473 infection in CAPZA1-overexpressing cells induces mRNA 1 and 2). CagA is reported to enhance nuclear accumulation 532
    475 CagA reportedly mediates transactivation of Sal-like nuclear accumulation of b-catenin in AGS cells (Figure 3B, 534
    477 are reprogramming factors that promote the acquisition of CAPZA1-overexpressing AGS cells was enhanced further by H 536
    478 stem cell properties in gastric epithelial cells.22 We inves- pylori G27 infection, but not by H pylori G27 DcagPAI infec- 537
    480 increased in CAPZA1-overexpressing cells infected with H degraded by autophagy in AGS cells, but escapes from auto- 539
    481 pylori. Infection of H pylori G27 increased mRNA expression phagic degradation in CAPZA1-overexpressing AGS cells and 540
    482 of SALL4 and KLF5 in CAPZA1-overexpressing AGS cells by thereby strongly induces nuclear translocation of b-catenin. 541
    483 2.4- and 3.5-fold, respectively (Figure 2E). Furthermore, To investigate whether this increase in nuclear accumulation 542
    484 mRNA expression of SALL4 and KLF5 was significantly of b-catenin was associated with the induction of CD44 543
    485 higher in CAPZA1-overexpressing AGS cells infected with H expression, we reduced the nuclear and cytosolic levels of b- 544
    486 pylori G27 than in AGS cells infected with H pylori G27 catenin in AGS cells via CCT031374 hydrobromide treat- 545
    487 (Figure 2E). On the other hand, mRNA expression of SALL4 ment.27 Treatment with CCT031374 at concentrations higher 546
    488 and KLF5 was not increased in CAPZA1-overexpressing AGS than 5 mmol/L reduced the total b-catenin level in CAPZA1- 547