Cisplatin and MK-1775 Combination Therapy

HNSCC cells (50,000) were seeded in 60-mm dishes, treated the next day with 1.5 μmol/L cisplatin, 0.25 μmol/L MK-1775 either alone or in combination for 48 hours, and then harvested 0, 24, or 48 hours later. Cells were fixed in ice-cold 70% ethanol, permeabilized with 0.25% Triton X-100 in PBS, and incubated with phospho-Histone H3 (Ser10) antibody conjugated to Alexa Flour 488 (#9708; at concentration 1:100, from Cell Signaling Technology) for 2 hours at 4°C. DNA was stained with 20 μg/mL propidium iodide (PI; Sigma-Aldrich) in the presence of 100 μg/mL RNase A (Sigma-Aldrich). For apoptosis assessment, cells were treated as indicated above and DNA strand breaks detected with an APO-BrdU TUNEL assay kit (Life Technologies) according to the manufacturer’s instructions. Samples were analyzed on a Gallios Flow Cytometer (Beckman Coulter, Inc.) combined with Flo-Jo software (FloJo).

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Osman A.A., Monroe M.M., Ortega Alves M.V., Patel A.A., Katsonis P., Fitzgerald A.L., Neskey D.M., Frederick M.J., Woo S.H., Caulin C., Hsu T.K., McDonald T.O., Kimmel M., Meyn R.E., Lichtarge O, & Myers J.N. (2014). Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-Risk TP53 Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence. Molecular cancer therapeutics, 14(2), 608-619.

Publication 2014

propidium iodide (PI; RNase A APO-BrdU TUNEL assay kit Gallios Flow Cytometer

Antibody Apoptosis Assay Brdu Cells Cisplatin Cold Dishes Dna breaks Ethanol Flour Histone h3 Hnscc Mk 1775 Propidium iodide Rnase Triton x 100 Tunel

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, Huntsman Cancer Institute, University of Utah, Tufts Medical Center, Baylor College of Medicine, MUSC Hollings Cancer Center, Medical University of South Carolina, Rice University

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Variable analysis

independent variables

Cisplatin (1.5 μmol/L)
MK-1775 (0.25 μmol/L)
Combination of cisplatin and MK-1775

dependent variables

Phospho-Histone H3 (Ser10) expression (measured by flow cytometry)
DNA strand breaks (measured by APO-BrdU TUNEL assay)

control variables

HNSCC cell line (50,000 cells seeded)
Time points (0, 24, 48 hours after treatment)
Cell fixation (ice-cold 70% ethanol)
Cell permeabilization (0.25% Triton X-100 in PBS)
Phospho-Histone H3 (Ser10) antibody concentration (1:100)
DNA staining (20 μg/mL propidium iodide, 100 μg/mL RNase A)

controls

Negative control: Untreated cells

Annotations

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