Animals censored before day 20 were excluded from analysis. p-values were calculated using the Mann-Whitney U-test. FaDu cells were prepared from monolayer culture (log-phase growth) by trypsinization. The antiproliferative effect of BIBW 2669 and BIBW 2992 on FaDu cells was examined following replicate plating of 2.5 × 104 cells in 25-cm2 tissue flasks (Nunc, Roskilde, Denmark) containing 5 ml Dulbecco’s modified Eagle’s medium supplemented with 2 mM stabile glutamine, 10% fetal calf serum, 1 mM sodium pyruvate, 1% (v/v) non-essential amino acids, 20 mM HEPES, and 1% (v/v) PD184352 penicillin- streptomycin (all from Biochrom, Berlin, Germany). 24 h later, the medium was replaced by fresh medium containing 3, 30, or 300 nM BIBW 2669 or BIBW 2992. Controls received DMSO in the same concentration as present in the highest drug group. For each determination duplicates were prepared.

After incubation at 37 °C (5% CO2, 95% humidity), cells were trypsinized and centrifuged (250 g; 5 min). ber was determined using a hemocytometer. Each of the three independent experiments was evaluated separately by fitting the curve under the Docetaxel Microtubule inhibitor assumption of exponential growth according to N = N0*e(K*t), where N0 is the cell number at the start and K the constant for exponential increase in cell number with time (t). The doubling time (CDT) equals CDT = ln2/K. Treatment groups were compared using the CDT values obtained from each experiment and the paired t-test. To determine the impact of BIBW 2669 or BIBW 2992 on clonogenic survival, FaDu cells were seeded in 25-cm2 tissue culture flasks. After 24 h, control medium or 300 nM BIBW 2669 or BIBW 2992 was added for 3 days. After irradiation with 0, 2, 4, 6, or 8 Gy (200-kV X-rays, 0.5 mm Cu, ~1 Gy min–1), cells were trypsinized and counted. Appropriately diluted single-cell suspensions were incubated in Petri dishes for 14 days, fixed and stained with crystal violet. Colonies with ≥ 50 cells were scored as survivors. The medians of the surviving fraction and their standard errors (SE) were determined for each treatment group. Cell survival curves were fitted according to the linear- quadratic model. Exponentially growing

FaDu cells were incubated in 25-cm2 tissue culture flasks for 4, 7, or 9 days with 3, 30, or 300 nM BIBW 2669, BIBW 2992 or control medium and harvested by buy Docetaxel trypsinization. After centrifugation, the cells were washed, suspended in PBS/EDTA, fixed on ice with 96% ethanol and stored at –20 °C. For flow cytometry, cells were washed with PBS by centrifugation and stained with propidium iodide (10 μg ml–1; Molecular Probes; Eugen, OR, USA) supplemented with RNase (1 mg ml–1; Roche Diagnostics, Mannheim, Germany). Cells were measured using a FACScan (Becton Dickinson, San Jose, CA, USA) and analyzed using ModFit LT 2.0 software (Verity Software House, Topsham, ME, USA). Comparison of means and their standard errors (SEM) were performed using t-test (GraphPad Prism 4.03 Software, Inc., San Diego, CA, USA). Absolute EGFR and ErbB2 levels in cultured cells and in tumor xenografts were measured using sandwich ELISA assays (DuoSet™ IC Human Total EGFR ELISA, R&D Systems, Wiesbaden, Germany, and ERBB2/HER2/neu ELISA, Oncogene, Cambridge, MA, USA). Approximately 100 mg of solid tumor tissue was disrupted with an Ultra-Turrax™ disperser (IKA, Staufen, Germany) in a tenfold volume of homogenization buffer (10 mM Tris/HCl, pH 7.4; 1.5 mM

EDTA; 10% (v/v) glycerol; 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM purchase Docetaxel benzamidine, 1 mM PMSF). Cultured cells were scraped in 50 mM Tris/HCl pH 7.4, 5 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM benzamidine, 1 mM PMSF and disrupted by passaging ten times through a 20-gauge needle. Receptor protein was solubilized from the homogenates by adding antigen extraction buffer from the ELISA kit (Oncogene) according to the manufacturer’s instructions. Cell debris was removed by centrifugation (14,000 g, 10 min, 4 °C). The optical density of the samples was measured with a Thermomax microplate reader (Molecular Devices, Menlo Park, CA, USA) at 450 nm using wavelength correction at 540 nm. Total receptor concentrations were calculated from standard curves using the Softmax software from the same supplier. FaDu is positive for EGFR, ErbB2, and ErbB3, but not for ErbB4 [6]. The number of EGFR molecules per cell as determined by Eicheler et al. [11] was higher in cultured cells compared to xenograft tumors: 2.4 × 105 EGFR molecules per cell in vitro and 3.4 × 104 EGFR molecules per cell in vivo. The ErbB2 expression was 2.0 × 104 ErbB2 molecules per cell in BIBW 2669 or BIBW 2992 showed a significant inhibitory effect on tumor cell proliferation. This effect increased with increasing concentration of the drugs (Figure 1, Table 1). No significant differences could be shown between BIBW 2669 and BIBW 2992, when the same drug concentrations were used. Cell-cycle distribution was investigated by flow cytometry after 4, 7, and 9 days of incubation with BIBW 2669 or BIBW 2992.

Compared to control cells, incubation with BIBW 2669 or BIBW 2992 revealed a significant and dose-dependent increase of the G0/G1 fraction (Figure 2). The proportion of cells in S- and G2/Mphase was significantly lower in BIBW 2669- and BIBW 2992-incubated cells compared to control cells (Figure 2). No significant difference was detectable between cells incubated with BIBW 2669 or BIBW 2992 at the same concentrations.