Cell culture
Human head and squamous cell cancer cell lines were grown in Dulbecco’s Modified Eagle’s Medium containing 10 % heat-inactive fetal bovine serum supplemented with 2 μM L-glutamine and incubated in a humidified chamber at 37 °C with 5 % CO2 as previously described [6, 7]. UM-SCC-1 and UM-SCC-5 were obtained from Dr. Thomas Carey at the University of Michigan. UM-SCC-5 cells were used to create stable transfectants of a short hairpin RNA against STAT-3 (STAT-3-2.4 cells). These cells were created by transfecting UM-SCC-5 cells with a pBABE-U6 vector containing STAT-3 short hairpin RNA (shRNA) as previously described [6]. Following transfection, the STAT-3-2.4 cells showed approximately 50 % STAT-3 knockdown as previously described [6, 7]. Control cells were also previously created by transfection with a mutated or negative STAT-3 shRNA form of the short hairpin RNA against STAT-3 (NEG 4.17 cells) and do not show significant STAT-3 knockdown [7].
Immunoblots
Immunoblots were used to analyze the protein expression levels of STAT3 as previously described [6, 7]. UM-SCC-1, −5 and transfected STAT3-2.4 and NEG4.17 cells were assessed for STAT-3, phosphorylated STAT-3 and GAPDH. Cell lysates were prepared and equal amounts of protein were loaded in each gel lane. Separation was performed by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P membrane (Millipore Corp, Bedford, MA). The immunoblots were blocked in 10 % milk–Tris-buffered saline with Tween-20 (TBS-T) (20 mmol/L Tris HCL [pH 7.5], NaCl 137 mmol/L, and 0.05 % Tween-20) for 1 h at room temperature. The primary antibodies of anti-STAT-3, anti-p-STAT-3 (Cell Signaling Technologies, Beverly, MA), anti-EGFr (Sigma-Aldrich, St. Louis, MO), anti-p-EGFr (cell signaling technologies, Beverly, MA) and anti-GAPDH (Santa Cruz Technologies, Inc., Santa Cruz, CA) were incubated overnight at 4 ° C with 2 % milk–TBS-T. The secondary antibody, anti-mouse–IgG–horseradish peroxidase antibody (Sigma Chemical Company, St. Louis, MO) was incubated at room temperature for 1 h. The blots were developed by chemiluminescence (Amersham Life Sciences, Inc., Arlington Heights, IL).
Cell proliferation
The effects of various treatments on cell proliferation were assessed by a cell proliferation assay as previously described [6, 7]. Briefly, UM-SCC-1, −5, and the transfected STAT3-2.4 and NEG 4.17 cells were plated in a manner to allow multiple days of proliferation. Following the entry of cells into an exponential growth phase, they were exposed to various combinations of cetuximab (0.5 μg/ml), 1 μM Janus Kinase inhibitor ([JAK1i] Calbiochem, LaJolla, CA) and/or radiation (2 Gy). The cells were assessed for cell counts every 24 h. The cells were removed from plates with trypsin and counted using a cell counter (Beckman Coulter, Fullerton, CA). Illustrative experiments demonstrate results following 72 h of growth.
Apoptosis
Apoptotic cell death following exposure to various treatments was quantified by using the Annexin V-FITC apoptosis detection kit (BioVision Research Products, Mountain View, CA) as previously described [8]. UM-SCC-1, −5, and the transfected STAT3-2.4 and NEG 4.17 cells were treated with various combinations of cetuximab (0.5 μg/ml), JAK1i (1 μM) and/or radiation (2 Gy) in a manner identical to the cell proliferation assay. Following 72 h of incubation, the cells were isolated and the data were collected using a Becton Dickinson FACScan system. This information was analyzed using CellQuest v3.1 software (Becton Dickinson, San Jose, CA).
Colony formation
Cell survival was assessed by colony formation as previously described [6, 7]. Briefly, cells were plated the day before the start of the experiment in order to allow time for attachment of the cells. The cells were exposed to various doses of radiation, either alone or with exposure to JAK1i, cetuximab or both agents just prior to radiation and during the post-radiation period. The cells were allowed to form colonies over 12–14 days. They were subsequently fixed, stained and counted (colonies of > 50 cells) for colony formation as previously described.
Neutral comet assay
The accumulation of DNA double strand breaks (dsbs) was assessed by the neutral comet assay as previous described [7]. Briefly, exponentially growing UM-SCC-1, −5, and the transfected UM-STAT3-2.4 and NEG 4.17 cells were plated at a concentration of 150,000 cells in 60 mm2 tissue culture dishes. After a 16 h incubation in the presence of cetuximab (5 μg/ml), JAK1i (1 μM) or the combined treatment with or without radiation, the cells were scraped and processed for Neutral Comet Assay at various times following radiation as described by Trevigen (Gaithersberg, MD). Following the staining with SYBR Green I, the slides were viewed by fluorescence microscopy (Evos f1, Advanced Microscopy Group, Bothell, WA). Tail moment and percent DNA in the tail were analyzed using CometScore Freeware.
Statistical analysis
All experiments were performed with at least three independent experiments. Representative immunoblots were selected for presentation. For experiments other than the immunoblots, results were expressed as the mean ± standard error (SE). For the cell proliferation and apoptosis assays, the statistical significance between treatment groups was determined using a two-way ANOVA assessment with software from GraphPad Prism, San Diego, California. Overall comparisons of certain conditions were made using comparisons of all four cell lines in a simultaneous two-way ANOVA assessment.
Ethics
This research did not involve human subject.