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. 2005 Feb;96(2):116-9.
doi: 10.1111/j.1349-7006.2005.00018.x.

STI571 (Glivec) inhibits the interaction between c-KIT and heat shock protein 90 of the gastrointestinal stromal tumor cell line, GIST-T1

Hajime Nakatani  1 , Michiya Kobayashi, Toufeng Jin, Takahiro Taguchi, Takeki Sugimoto, Takumi Nakano, Shinichi Hamada, Keijiro Araki
Affiliations

STI571 (Glivec) inhibits the interaction between c-KIT and heat shock protein 90 of the gastrointestinal stromal tumor cell line, GIST-T1

Hajime Nakatani et al. Cancer Sci. 2005 Feb.

Abstract

The gastrointestinal stromal tumor cell line, GIST-T1, has a heterogenic 57-base pair deletion in exon 11 of the c-kit mutation, and the c-KIT protein in the GIST-T1 cells constitutively activated. We report that STI571 (Glivec; Novartis, Basel, Switzerland), a specific inhibitor of c-KIT, inhibits the clustering of c-KIT at the cell membrane of the GIST-T1 cells. Furthermore, STI571 prevents the interaction between c-KIT and the molecular chaperone, heat shock protein 90 (Hsp90). Geldanamycin, an inhibitor of Hsp90, also prevents interaction between c-KIT and Hsp90, and inhibits tyrosine phosphorylation of c-KIT. Our results indicate that c-KIT molecules are assembled on the cell surface of the GIST-T1 cells, and that the interaction between c-KIT and Hsp90 plays an important role in c-KIT activation.

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Figures

Figure 1
Figure 1
Cells, 3 × 106, were seeded on a 10‐cm diameter dish. After 24 h, cells were treated with/without STI571 (1 µg/mL) or geldanamycin (0.01, 0.1, 1 µg/mL) for 5 h. The cells were scraped and collected by centrifugation and then lyzed in radioimmunoprecipitation assay buffer as described in the Materials and Methods section. Membranes were reacted with antiphosphotyrosine (1:250) or anti‐c‐KIT (1:500) overnight at 4C°. Lanes 1–5: non‐treated, STI571 1 µg/mL, geldanamycin 0.01 µg/mL, 0.1 µg/mL and 1 µg/mL, respectively.
Figure 2
Figure 2
Cells, 3 × 106, were treated with/without STI571 (1 µg/mL) or geldanamycin (0.1 µg/mL). Immunoprecipitated with anti‐c‐KIT (1:100) overnight at 4C°, and immunoprecipitates were protein A agarose as described in the Materials and Methods section. Anti‐heat shock protein 90 antibody (1:250) was used for Western blot analysis.
Figure 3
Figure 3
Immunoelectromicroscopic analysis. Cells were treated with/without STI571 (1 µg/mL) or geldanamycin (0.1 µg/mL). Samples were prepared as described in the Materials and Methods section. (a) Non‐treated, (b) STI571 treated, (c) geldanamycin‐treated, and (d) non‐treated, but samples were reacted with anti‐c‐KIT and 15‐nm‐gold labeled secondary antibody, and then they were reacted with anti‐heat shock protein 90 antibody and 5‐nm‐gold labeled secondary antibody.
Figure 4
Figure 4
Cell viability was measured by (3,‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide assay. 1, non‐treated; 2, STI571 0.01 µg/mL; 3, STI571 0.05 µg/mL; 4, STI571 0.1 µg/mL; 5, STI571 1 µg/mL; 6, geldanamycin 0.01 µg/mL; 7, geldanamycin 0.02 µg/mL; 8, geldanamycin 0.05 µg/mL; 9, geldanamycin 0.1 µg/mL; 10, geldanamycin 1 µg/mL; 11, STI571 1 µg/mL + geldanamycin 0.1 µg/mL; 12, STI571 1 µg/mL + geldanamycin 1 µg/mL. The survival percentages are shown as the mean ± standard deviation of triplicate experiments. The y‐axis presents the percentage viability of gastrointestinal stromal tumor cell line (GIST)‐T1.
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