Inhibition of Invasion of Epithelial Cells by Tiam1-Rac Signaling
10.1126/science.278.5342.1464
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin–Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor–induced scattering by increasing E-cadherin–mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin–mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.

Bibliography

Hordijk, P. L., ten Klooster, J. P., van der Kammen, R. A., Michiels, F., Oomen, L. C. J. M., & Collard, J. G. (1997). Inhibition of Invasion of Epithelial Cells by Tiam1-Rac Signaling. Science, 278(5342), 1464–1466.

Authors 6
  1. Peter L. Hordijk (first)
  2. Jean Paul ten Klooster (additional)
  3. Rob A. van der Kammen (additional)
  4. Frits Michiels (additional)
  5. Lauran C. J. M. Oomen (additional)
  6. John G. Collard (additional)
References 36 Referenced 364
  1. Tapon N., Hall A., Curr. Opin. Cell Biol. 9, 86 (1997). (10.1016/S0955-0674(97)80156-1) / Curr. Opin. Cell Biol. by Tapon N. (1997)
  2. Habets G. G. M., et al., Cell 77, 537 (1994). (10.1016/0092-8674(94)90216-X) / Cell by Habets G. G. M. (1994)
  3. Michiels F., Habets G. G. M., Stam J. C., Van der Kammen R. A., Collard J. G., Nature 375, 338 (1995). (10.1038/375338a0) / Nature by Michiels F. (1995)
  4. Behrens J., Mareel M. M., Van Roy F. M., Birchmeier W., J. Cell Biol. 108, 2435 (1989). (10.1083/jcb.108.6.2435) / J. Cell Biol. by Behrens J. (1989)
  5. K. Vleminckx L.Vakaet Jr.
  6. Mareel M., Fiers W., Van Roy F., Cell 66, 107 (1991). (10.1016/0092-8674(91)90143-M) / Cell by Mareel M. (1991)
  7. Frixen U. H., et al., J. Cell Biol. 113, 173 (1991); (10.1083/jcb.113.1.173) / J. Cell Biol. by Frixen U. H. (1991)
  8. Takeichi M., Curr. Opin. Cell Biol. 5, 806 (1993); (10.1016/0955-0674(93)90029-P) / Curr. Opin. Cell Biol. by Takeichi M. (1993)
  9. Birchmeier W., Behrens J., Biochim. Biophys. Acta 1198, 11 (1994); / Biochim. Biophys. Acta by Birchmeier W. (1994)
  10. Shiozaki H., Oka H., Inoue M., Tamura S., Monden M., Cancer 77, 1605 (1996). (10.1002/(SICI)1097-0142(19960415)77:8<1605::AID-CNCR28>3.0.CO;2-2) / Cancer by Shiozaki H. (1996)
  11. 10.1126/science.275.5307.1787
  12. ; B. Rubinfeld et al. ibid. p. 1790.
  13. Hirano S., Kimoto N., Shimoyama Y., Hirohashi S., Takeichi M., Cell 70, 293 (1992); (10.1016/0092-8674(92)90103-J) / Cell by Hirano S. (1992)
  14. Becker K. F., et al., Cancer Res. 54, 3845 (1994); / Cancer Res. by Becker K. F. (1994)
  15. Berx G., et al., EMBO J. 14, 6107 (1995). (10.1002/j.1460-2075.1995.tb00301.x) / EMBO J. by Berx G. (1995)
  16. 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 Tiam1 and Rac cDNAs are described elsewhere (23).
  17. 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-aminoethyl)-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 Tiam1 (anti-C16 Santa Cruz) (1 μg per immunoprecipitate) and analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotting (12).
  18. For immunocytochemical staining cells were grown on glass coverslips for 48 hours at low density (5 × 10 4 cells per well). Endogenous Tiam1 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 (FITC)–labeled streptavidin (Zymed). The staining pattern was recorded with a charge-coupled device camera system.
  19. Michiels F., et al., J. Cell Biol. 137, 387 (1997). (10.1083/jcb.137.2.387) / J. Cell Biol. by Michiels F. (1997)
  20. Cells were grown on glass coverslips as described in (11). Cells were stained with the monoclonal antibody to the Myc tag (9E10) to detect RacV12 or RacN17 a rabbit polyclonal antiserum to Tiam1 (anti-DH) (2) or the DECMA-1 antibody to E-cadherin (Sigma) (4 5). Antibodies were visualized with FITC-labeled or trimethylrhodamine isothiocyanate (TRITC)–labeled secondary antibodies (Zymed). In most cases cells were stained simultaneously for F-actin with TRITC-labeled phalloidin (1 U/ml) (Molecular Probes). In the confocal microscopic images green and red fluorescences were recorded separately and then merged. For analysis of cell scattering medium was replaced after 48 hours and recombinant HGF (10 ng/ml) was added as indicated. After 24 hours cells were fixed in 3.7% paraformaldehyde and inspected by phase-contrast microscopy. For investigation of E-cadherin–dependent adhesion C1199Tiam1-expressing cells (2 × 10 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.
  21. 10.1146/annurev.bi.59.070190.001321
  22. Furlong R. A., BioEssays 14, 613 (1992). (10.1002/bies.950140908) / BioEssays by Furlong R. A. (1992)
  23. Ridley A. J., Comoglio P. M., Hall A., Mol. Cell. Biol. 15, 1110 (1995). (10.1128/MCB.15.2.1110) / Mol. Cell. Biol. by Ridley A. J. (1995)
  24. 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 [
  25. Takeda H., et al., J. Cell Biol. 131, 1839 (1995)]. (10.1083/jcb.131.6.1839) / J. Cell Biol. by Takeda H. (1995)
  26. Braga V. M. M., Machesky L. M., Hall A., Hotchin N. A., ibid. 137, 1421 (1997). / ibid. by Braga V. M. M. (1997)
  27. Rat tail type IV collagen (1% w/v) (Sigma) was dissolved in PBS (containing calcium and magnesium) and 1% acetic acid. This solution was diluted to 0.2% collagen in DMEM containing 10% FCS and adjusted to pH 7.4 with sodium hydroxide. Cells were resuspended in the collagen (4 × 10 3 cells per milliliter of collagen) and seeded in 12-well plates. After 7 days in culture cells were photographed [
  28. Webb C. P., Lane K., Dawson A. P., Van de Woude G. F., Warn R. M., J. Cell. Sci. 109, 2371 (1996)]. (10.1242/jcs.109.9.2371) / J. Cell. Sci. by Webb C. P. (1996)
  29. Eaton S., Auvinen P. L., Jan Y. N., Simon K., J. Cell Biol. 131, 151 (1995). (10.1083/jcb.131.1.151) / J. Cell Biol. by Eaton S. (1995)
  30. 10.1016/0092-8674(95)90370-4
  31. Van Leeuwen F. N., et al., J. Cell Biol. 139, 797 (1997); (10.1083/jcb.139.3.797) / J. Cell Biol. by Van Leeuwen F. N. (1997)
  32. ; F. Michiels R. A. van der Kammen J. G. Collard unpublished results.
  33. Qiu R. G., Chen J., Kirn D., McCormick F., Symons M., Nature 374, 457 (1995); (10.1038/374457a0) / Nature by Qiu R. G. (1995)
  34. Khosravi-Far R., Solski P. A., Clark G. J., Kinch M. S., Der C. J., Mol. Cell. Biol. 15, 6443 (1995). (10.1128/MCB.15.11.6443) / Mol. Cell. Biol. by Khosravi-Far R. (1995)
  35. F. Michiels unpublished data.
  36. 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.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:45 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 11:48 p.m.)
Indexed 2 months ago (June 26, 2025, 2:24 p.m.)
Issued 27 years, 9 months ago (Nov. 21, 1997)
Published 27 years, 9 months ago (Nov. 21, 1997)
Published Print 27 years, 9 months ago (Nov. 21, 1997)
Funders 0

None

@article{Hordijk_1997, title={Inhibition of Invasion of Epithelial Cells by Tiam1-Rac Signaling}, volume={278}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.278.5342.1464}, DOI={10.1126/science.278.5342.1464}, number={5342}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Hordijk, Peter L. and ten Klooster, Jean Paul and van der Kammen, Rob A. and Michiels, Frits and Oomen, Lauran C. J. M. and Collard, John G.}, year={1997}, month=nov, pages={1464–1466} }