Skip to main content
NCBI home page
Cancer Science logoLink to Cancer Science
. 2005 Aug 19;95(5):442–447. doi: 10.1111/j.1349-7006.2004.tb03229.x

Conditional gene silencing utilizing the lac repressor reveals a role of SHP‐2 in cagA‐positive Helicobacter pylori pathogenicity

Megumi Higuchi 1, Ryouhei Tsutsumi 1, Hideaki Higashi 1, Masanori Hatakeyama 1,✉
PMCID: PMC11160029  PMID: 15132773

Abstract

RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss‐of‐function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III‐dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP‐2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP‐2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP‐2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer‐related genes through a reversed genetic approach.

References

  • 1. Hannon GJ. RNA interference. Nature 2002; 418: 244–51. [DOI] [PubMed] [Google Scholar]
  • 2. Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics Cancer Cell 2002; 2: 17–23. [DOI] [PubMed] [Google Scholar]
  • 3. Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003; 4: 457–67. [DOI] [PubMed] [Google Scholar]
  • 4. Tijsterman M, Ketting RF, Plasterk RH. The genetics of RNA silencing. Annu Rev Genet 2002; 36: 489–519. [DOI] [PubMed] [Google Scholar]
  • 5. Carthew RW. Gene silencing by double‐stranded RNA. Curr Opin Cell Biol 2001; 13: 244–8. [DOI] [PubMed] [Google Scholar]
  • 6. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–3. [DOI] [PubMed] [Google Scholar]
  • 7. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short‐interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 6047–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Miyagishi M, Taira K. U6 promoter‐driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002; 20: 497–500. [DOI] [PubMed] [Google Scholar]
  • 9. Blaser MJ, Perez‐Perez GI, Kleanthous H, Cover TL, Peek RM, Chyou PH, Stemmermann GN, Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 1995; 55: 2111–5. [PubMed] [Google Scholar]
  • 10. Personnet J, Friedman GD, Orentreich N, Vogelman H. Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut 1997; 40: 297–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Nomura N, Lee J, Stemmermann GN, Nomura RY, Perez‐Perez GI, Blaser MJ. Helicobacter pylori CagA seropositivity and gastric carcinoma risk in a Japanese American population. J Infect Dis 2002; 186: 1138–44. [DOI] [PubMed] [Google Scholar]
  • 12. Covacci A, Censini S, Bugnoli M, Petracca R, Burroni D, Macchia G, Massone A, Papini E, Xiang Z, Figura N, Rappuoli R. Molecular characterization of the 128‐kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993; 90: 5791–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Segal ED, Cha J, Lo J, Falkow S, Tompkins LS. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci USA 1999; 96: 14559–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Asahi M, Azuma T, Ito S, Ito Y, Suto H, Nagai Y, Tsubokawa M, Tohyama Y, Maeda S, Omata M, Suzuki T, Sasakawa C. Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med 2000; 191: 593–602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Stein M, Rappuoli R, Covacci A. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag‐driven host cell translocation. Proc Natl Acad Sci USA 2000; 97: 1263–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 2000; 287: 1497–500. [DOI] [PubMed] [Google Scholar]
  • 17. Backert S, Ziska E, Brinkmann V, Zimny‐Arndt U, Fauconnier A, Jungblut PR, Naumann M, Meyer TF. Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus. Cell Microbiol 2000; 2: 155–64. [DOI] [PubMed] [Google Scholar]
  • 18. Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M. SHP‐2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 2002; 295: 683–6. [DOI] [PubMed] [Google Scholar]
  • 19. Higashi H, Tsutsumi R, Fujita A, Yamazaki S, Asaka M, Azuma T, Hatakeyama, M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites. Proc Natl Acad Sci USA 2002; 99: 14428–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Yamazaki S, Yamakawa A, Ito Y, Ohtani M, Higashi H, Hatakeyama M, Azuma T. The CagA protein of Helicobacter pylori is translocated into epithelial cells and binds to SHP‐2 in human gastric mucosa. J Infect Dis 2003; 187: 334–7. [DOI] [PubMed] [Google Scholar]
  • 21. Hu MC, Davidson N. The inducible lac operator‐repressor system is functional in mammalian cells. Cell 1987; 48: 555–66. [DOI] [PubMed] [Google Scholar]
  • 22. Brown M, Figge J, Hansen U, Wright C, Jeang KT, Khoury G, Livingston DM, Roberts TM. lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal. Cell 1987; 49: 603–12. [DOI] [PubMed] [Google Scholar]
  • 23. Hoshikawa Y, Amimoto K, Mizuguchi R, Hatakeyama M. Highly controlled heterologous gene expression through combined utilization of the tetracy‐cline‐repressible transactivator and the lac repressor. Anal Biochem 1998; 261: 211–8. [DOI] [PubMed] [Google Scholar]
  • 24. Saxton TM, Henkemeyer M, Gasca S, Shen R, Shalaby F, Feng G‐S, Pawson T. Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp‐2. EMBO J 1994; 16: 2352–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Hannon GJ, Chubb A, Maroney P, Hannon G, Altman S, Nilsen TE. Multiple cis‐acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P. J Biol Chem 1991; 266: 22796–9. [PubMed] [Google Scholar]
  • 26. Neel BG. Structure and function of SH2‐domain containing tyrosine phosphatases. Semin Cell Biol 1993; 6: 419–32. [DOI] [PubMed] [Google Scholar]
  • 27. Feng GS, Pawson T. Phosphotyrosine phosphatases with SH2 domains: regulators of signal transduction. Trend Genet 1994; 10: 54–8. [DOI] [PubMed] [Google Scholar]
  • 28. Czauderna F, Santel A, Hinz M, Fechtner M, Durieux B, Fisch G, Leenders F, Arnold W, Giese K, Klippel A, Kaufmann J. Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res 2003; 31: e127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector‐mediated druginducible RNA interference. J Virol 2003; 77: 8957–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Matsukura S, Jones PA, Takai D. Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Res 2003; 31: e77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D, Holstege FC, Brummelkamp TR, Agami R, Clevers H. Specific inhibition of gene expression using a stably integrated, inducible small‐interfering‐ RNA vector. EMBO Rep 2003; 4: 609–15. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cancer Science are provided here courtesy of Wiley

RESOURCES