Inhibition of invasion of epithelial cells by Tiam1-Rac signaling

Science

Volumn 278, Issue 5342, 1997, Pages 1464-1466

  Hordijk, Peter L ^a,b   Ten Klooster, Jean Paul ^a   Van Der Kammen, Rob A ^a   Michiels, Frits ^a   Oomen, Lauran C J M ^a   Collard, John G ^a  

^a NETHERLANDS CANCER INSTITUTE   (Netherlands)

^b CENTRAL LABORATORY   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

GUANINE NUCLEOTIDE EXCHANGE FACTOR; GUANOSINE TRIPHOSPHATASE; RAC PROTEIN; RAS PROTEIN; TIAM 1 PROTEIN; UNCLASSIFIED DRUG;

ACTIN POLYMERIZATION; ANIMAL CELL; ARTICLE; CELL ADHESION; CELL CONTACT; CELL INVASION; CELL JUNCTION; CONTROLLED STUDY; DOG; EPITHELIUM CELL; NONHUMAN; PRIORITY JOURNAL; PROTEIN LOCALIZATION; SIGNAL TRANSDUCTION; T LYMPHOCYTE;

ACTINS; ANIMALS; CADHERINS; CELL ADHESION; CELL LINE; CELL LINE, TRANSFORMED; CELL MOVEMENT; CELL TRANSFORMATION, NEOPLASTIC; CYTOPLASM; EPITHELIAL CELLS; GTP-BINDING PROTEINS; HEPATOCYTE GROWTH FACTOR; INTERCELLULAR JUNCTIONS; NEOPLASM INVASIVENESS; PHENOTYPE; PROTEINS; RAC GTP-BINDING PROTEINS; SIGNAL TRANSDUCTION;

EID: 0030698191     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5342.1464     Document Type: Article

References (33)

1. N. Tapon and A. Hall, Curr. Opin. Cell Biol. 9, 86 (1997).
2. G. G. M. Habets et al., Cell 77, 537 (1994).
3. F. Michiels, G. G. M. Habets, J. C. Stam, R. A. Van der Kammen, J. G. Collard, Nature 375, 338 (1995).
4. J. Behrens, M. M. Mareel, F. M. Van Roy, W. Birchmeier, J. Cell Biol. 108, 2435 (1989).
5. K. Vleminckx, L.Vakaet Jr., M. Mareel, W. Fiers, F. Van Roy, Cell 66, 107 (1991).
6. U. H. Frixen et al., J. Cell Biol. 113, 173 (1991); M. Takeichi, Curr. Opin. Cell Biol. 5, 806 (1993); W. Birchmeier and J. Behrens, Biochim. Biophys. Acta 1198, 11 (1994); H. Shiozaki, H. Oka, M. Inoue, S. Tamura, M. Monden, Cancer 77, 1605 (1996).
7. U. H. Frixen et al., J. Cell Biol. 113, 173 (1991); M. Takeichi, Curr. Opin. Cell Biol. 5, 806 (1993); W. Birchmeier and J. Behrens, Biochim. Biophys. Acta 1198, 11 (1994); H. Shiozaki, H. Oka, M. Inoue, S. Tamura, M. Monden, Cancer 77, 1605 (1996).
8. U. H. Frixen et al., J. Cell Biol. 113, 173 (1991); M. Takeichi, Curr. Opin. Cell Biol. 5, 806 (1993); W. Birchmeier and J. Behrens, Biochim. Biophys. Acta 1198, 11 (1994); H. Shiozaki, H. Oka, M. Inoue, S. Tamura, M. Monden, Cancer 77, 1605 (1996).
9. U. H. Frixen et al., J. Cell Biol. 113, 173 (1991); M. Takeichi, Curr. Opin. Cell Biol. 5, 806 (1993); W. Birchmeier and J. Behrens, Biochim. Biophys. Acta 1198, 11 (1994); H. Shiozaki, H. Oka, M. Inoue, S. Tamura, M. Monden, Cancer 77, 1605 (1996).
10. P. J. Morin et al., Science 275, 1787 (1997); B. Rubinfeld et al., ibid., p. 1790.
11. P. J. Morin et al., Science 275, 1787 (1997); B. Rubinfeld et al., ibid., p. 1790.
12. S. Hirano, N. Kimoto, Y. Shimoyama, S. Hirohashi, M. Takeichi, Cell 70, 293 (1992); K. F. Becker et al., Cancer Res. 54, 3845 (1994); G. Berx et al., EMBO J. 14, 6107 (1995).
13. S. Hirano, N. Kimoto, Y. Shimoyama, S. Hirohashi, M. Takeichi, Cell 70, 293 (1992); K. F. Becker et al., Cancer Res. 54, 3845 (1994); G. Berx et al., EMBO J. 14, 6107 (1995).
14. S. Hirano, N. Kimoto, Y. Shimoyama, S. Hirohashi, M. Takeichi, Cell 70, 293 (1992); K. F. Becker et al., Cancer Res. 54, 3845 (1994); G. Berx et al., EMBO J. 14, 6107 (1995).
15. MDCK2 and MDCKf3 cells (5) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL) with 10% fetal calf serum (FCS). Stable cell lines expressing hemagglutinin (HA)-epitope-tagged C1199Tiam1 or C580Tiam1 constructs (see Fig. 1C), or Myc-epitope-tagged RacV12 or RacN17, were generated by retroviral transduction. Construction, transfection, and production of viral vectors and amphotropic retroviruses containing the Tiaml and Rac cDNAs are described elsewhere (23).
16. Cells were grown to confluency in 10-cm dishes, washed with ice-cold phosphate-buffered saline (PBS), and lysed in 0.5 ml of lysis buffer [50 mM tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 20 μM leupeptin, 20 μM aprotinin, and 1 mM 4-(2-amino-ethyl)-benzenesulfonyl fluoride hydrochloride (Boehringer)]. After 10 min on ice, cell lysates (Triton-soluble fraction) were collected, and the remaining cell fraction was collected in the same buffer containing 1 % sodium deoxycholate and 0.1% SDS (RIPA fraction).Tiam1 was immunoprecipitated with the antiserum to Tiam 1 (anti-C16, Santa Cruz) (1 μg per immunoprecipitate) and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting (12).
17. 4 cells per well). Endogenous Tiam 1 was visualized with the antibody to the DH domain (1:100 in PBS) (2) followed by incubation with biotin-labeled mouse antiserum to rabbit immunoglobulin and fluorescein isothiocyanate (FITQ-labeled streptavidin (Zymed). The staining pattern was recorded with a charge-coupled device camera system.
18. F. Michiels et al., J. Cell Biol. 137, 387 (1997).
19. 4 cells per well in a 12-well plate) were grown for 72 hours in the presence of control antibody or the DECMA-1 antibody (12 μg/ml). Medium and antibodies were replaced every 24 hours. HGF (10 ng/ml) was added for the last 24 hours; then the cells were fixed and processed for immunocytochemistry.
20. M. Takeichi, Annu. Rev. Biochem. 59, 237 (1990).
21. R. A. Furlong, BioEssays 14, 613 (1992).
22. A. J. Ridley, P. M. Comoglio, A. Hall, Mol. Cell. Biol. 15, 1110 (1995).
23. Cells were grown to confluency and collected in PBS with calcium and magnesium. Cells were pipetted 30 times with a 5-ml pipette and then photographed. The extent of cell dissociation was quantified by counting the number of cell clumps (particles) (Np) and the total number of cells (Nc) (1000 to 2000 cells per different cell line) on the photographs and is represented by the ratio Np/Nc [H. Takeda et al., J. Cell Biol. 131, 1839 (1995)].
24. V. M. M. Braga, L. M. Machesky, A. Hall, N. A. Hotchin, ibid. 137, 1421 (1997).
25. 3 cells per milliliter of collagen) and seeded in 12-well plates. After 7 days in culture, cells were photographed [C. P. Webb, K. Lane, A. P. Dawson, G. F. Van de Woude, R. M. Warn, J. Cell. Sci. 109, 2371 (1996)].
26. S. Eaton, P. L. Auvinen, Y. N. Jan, K. Simon, J. Cell Biol. 131, 151 (1995).
27. C. D. Nobes and A. Hall, Cell 81, 53 (1995); F. N. Van Leeuwen et al., J. Cell Biol. 139, 797 (1997); F. Michiels, R. A. van der Kammen, J. G. Collard, unpublished results.
28. C. D. Nobes and A. Hall, Cell 81, 53 (1995); F. N. Van Leeuwen et al., J. Cell Biol. 139, 797 (1997); F. Michiels, R. A. van der Kammen, J. G. Collard, unpublished results.
29. C. D. Nobes and A. Hall, Cell 81, 53 (1995); F. N. Van Leeuwen et al., J. Cell Biol. 139, 797 (1997); F. Michiels, R. A. van der Kammen, J. G. Collard, unpublished results.
30. R. G. Qiu, J. Chen, D. Kim, F. McCormick, M. Symons. Nature 374, 457 (1995); R. Khosravi-Far, P. A. Solski, G. J. Clark, M. S. Kinch, C. J. Der, Mol. Cell. Biol. 15, 6443 (1995).
31. R. G. Qiu, J. Chen, D. Kim, F. McCormick, M. Symons. Nature 374, 457 (1995); R. Khosravi-Far, P. A. Solski, G. J. Clark, M. S. Kinch, C. J. Der, Mol. Cell. Biol. 15, 6443 (1995).
32. F. Michiels, unpublished data.
33. We thank M. Mareel for providing MDCK2 and MDCKf3 cells, G. Nolan for the LZRS vector and packaging cells, A. Hall for the RacV12 construct, I. Weimar for recombinant HGF, N. Ong for photographs, and F. van Leeuwen and E. Sander for discussions. Supported by grants from the Dutch Cancer Society to J.G.C.