Article Figures & Data
Figures
- Figure 1.
A, constitutive activation of EGFR rendered FET cells resistant to GFDS-induced apoptosis. FET vector control and FETα cells were deprived of growth factors after growth to 80% confluence. Cells were harvested at the indicated times and Western blot was done with anti-PARP antibody. B, repression of constitutive EGFR activation led to decreased activation of EGFR, Erk, and Akt. FETα cells were treated with 0.1 μg/mL of tetracycline for 3 d to repress constitutive EGFR activation. Cells were harvested and lysates were immunoprecipitated with anti-EGFR antibody. Western blot was done with anti-pTyr, anti-EGFR, anti-pErk, anti-Erk, anti-pAKT, and anti-AKT antibodies. Image Quant software was used to quantify protein levels.
- Figure 2.
A, inhibition of xenograft growth of FETα cells by tetracycline. Exponentially growing cells (5 × 106) were inoculated s.c. in athymic nude mice. Tetracycline (3 mg/mL) was added to the drinking water daily from day 30 onwards. Tumors were measured externally on the indicated days in two dimensions using calipers. Tumor volumes were determined by the equation V = (L × W2) × 0.5, where L, length; and W, width of the tumor. Values are the mean of 10 xenografts. B, GFP labeled 5 × 106 FETα cells were inoculated into athymic nude mice. Thirty days after inoculation, tetracycline (3 mg/mL) was added to the drinking water daily for 34 d.
- Figure 3.
Decreased proliferation and increased apoptosis in FETα cells treated with tetracycline. A, four xenografts were harvested on each indicated day. The CAS 200 image analyzer was used to determine the mean proliferative activity for each group. B, slides were prepared from the harvested xenografts for standard immunohistochemistry and Ki-67 staining for FETα and FETα cells treated with tetracycline (Dako Corporation). C, apoptotic rates were determined by an average of three random 75 μm2 fields at 20× magnification. D, slides were prepared from harvested xenografts for standard Apotag TUNEL assay for both FETα and FETα tetracycline–treated animals (Oncor).
- Figure 4.
A to D, immunohistochemical validation of signal transduction. FETα xenografts were harvested and placed in buffered formalin for 24 h and then paraffin embedded. Slides were prepared for standard immunohistochemistry to evaluate the effect of tetracycline on the expression of TGFα, activated EGFR, activated Erk, and activated AKT. Phosphospecific antibodies were used to generate this data. Each experiment has negative control staining (top left), FET control cells (top right), FETα-untreated cells (bottom left), and FETα tetracycline–treated cells (bottom right).
- Figure 5.
Effects of clinical EGFR antagonist on FETα cells in vivo and in vitro. A, FETα cells were treated with 5 μmol/L of CI-1033 for 3 h. Cells were harvested and lysates were immunoprecipitated with anti-EGFR antibody. Western blot was done with anti-pTyr, anti-EGFR, anti-pErk, anti-Erk, anti-pAkt, and anti-Akt antibodies. B, ten animals with FETα xenografts were treated with CI-1033. Thirteen days postinjection, animals were treated with CI-1033 at 80 mg/mL in 0.557 mol/L of isothionic acid in lactate buffer (pH 7.0) by gavage. Treatment was continued for up to 15 d. Control animals received gavage treatment with isothionic acid/lactate buffer (vehicle) only. Animals were euthanized and tumors were harvested after 15 d of treatment. Tumor volumes were determined by the equation V = (L × W2) × 0.5, where L, length; W, width of the tumor. C, FETα cells were treated with AG879, an ERBb2 antagonist, and Western blots evaluating phosphorylated ERBb2 and p-Akt. D, FETα cells were treated with the 2C4 antibody to ERBb2 and Western blots to p-ERBb2 and p-Erk, and p70S6k are shown.
- Figure 6.
A to C, CI-1033 inhibits activation of EGFR and Erk, but not AKT. Thirteen days postinoculation; xenografts were treated with CI-1033 and harvested after 10 or 14 d of treatment. Tissues were placed in buffered formalin for 24 h and paraffin embedded. Immunohistochemistry was done for p-EGFR, p-Erk, and p-AKT on negative control, vehicle control, 10 day–treated and 14 day–treated xenografts.
Volume 67, Issue 2
