JCI - Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins

Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins

Marta Guix, Anthony C. Faber, Shizhen Emily Wang, Maria Graciela Olivares, Youngchul Song, Sherman Qu, Cammie Rinehart, Brenda Seidel, Douglas Yee, Carlos L. Arteaga, Jeffrey A. Engelman

Published in Volume 118, Issue 7

J Clin Invest. 2008; 118(7):2609–2619 doi:10.1172/JCI34588

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Figure 4

IGFBPs are downregulated in GR cells.

(A) Parental and A431 GR cells (10⁶ cells) were incubated for 24 hours in serum-free medium. The conditioned medium (C.M.) was concentrated by ultrafiltration, and IGF-I and IGF-II levels were determined by immunoassay as indicated in Methods. Bars represent the mean ± SD of 3 experiments. (B) Left panel: conditioned medium from 2 × 10⁶ parental and A431 GR cells was collected after 24 hours incubation and concentrated 20-fold by ultrafiltration. Medium was subjected to electrophoresis under nonreducing conditions, transferred to a nitrocellulose membrane, and incubated with ¹²⁵I–IGF-I overnight at 4°C. Signal was captured with a phosphorimager. Human serum and MCF-7 cells were used as positive controls for ¹²⁵I–IGF-I binding and IGFBP-4. Right panel: conditioned medium or 50 μg total protein from whole-cell lysates was subjected to immunoblot analysis with IGFBP-3, IGFBP-4, or Akt antibodies. (C) A431 GR cells were grown in 12-well plates in 0.5% FBS-containing medium for 72 hours with or without gefitinib (1 μM) and/or IGFBP-3 (1 μg/ml) and harvested by trypsinization. Cell numbers were determined with a Coulter Counter. Error bars represent the mean ± SD of 3 wells. Student’s t test was used for statistical comparisons. (D) A431 GR cells were treated with vehicle or gefitinib (1 μM) ± IGFBP-3 at the indicated concentrations for 6 hours. Cell lysates were prepared and analyzed with Western blots using the indicated antibodies.