Project Description

Toronto Preclinical Laboratory needed for preclinical study testing: Cell Culture
Human pulmonary carcinoid cancer cell lines (H727 and H720) will be maintained in RPMI 1640 and supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 100ug/ml streptomycin, sodium pyruvate, D-glucose, 10mM HEPES. The cell lines will be maintained at 37C in 5% CO2 and 95% air.

Cellular Proliferation
Cellular proliferation will be assessed with 3-[4.5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. 3 X 104 H727 cells and 3 X 104 H720 will be seeded in quadruplicates in 24 well plates and incubated overnight for cell adhesion. Following incubation, cells will be treated with 0-100uM decitabine (10, 30 and 100 um decitabine). Every 48 hours treatment medium will be replaced with fresh drug treatment medium. The MTT assay will be performed over 6 day period and cells will be incubated in 250ul of phenol red-free RPMI 1640 containing 0.5 mg/ML MTT solution for 3.5 hours. Following incubation 750 ul of dimethyl sulfoxide will be added to each well and mixed thoroughly. Absorbance will be measured at 540nm with a spectrophotometer. Readings will be plotted as average and standard error of mean.

Immunoblotting
H727 and H720 cells will be plated in 10 cm plates and incubated overnight tor cellular adhesion. Following incubation cells will be treated 2 days with 0-100uM of decitabine (10, 30 and 100 uM of ) decitabine. Protein will be harvested and quantified BCA protein assay kit. Denatured cell extracts will be resolved onto 10% SDS-PAGE gel, transferred onto nitrocellulose membranes and blocked in milk. Membranes will be incubated overnight with the antibodies at the dilutions: 1:1000 for chromogranin A (CgA); 1:1000 for Neuron Specific Enolase (NSE); 1:1000 for Cyclin B1; and 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Membranes will be incubated in horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody. Protein signals will be detected using either Immunstar (CgA, NSE, and GAPDH) or Supersignal West Femto (Cyclin B1) kits, according to the manufacturer's instructions.

Flow cytometry
Cells will be incubated in 10 cm2 cell culture plates overnight to enable cell adhension. Following 48 hours of incubation cells were treated with decitabine (10 uM, 30 uM and 100 uM). Following treatment period, cells will be trypsinized, washed twice with PBS, centrifuged twice at 1000rpm for five minutes and fixed with Ethanol. Cells will be stained with 50µg/mL Propidium Iodide (PI) solution that contains 100 µg/mL RNAse A and stored overnight in the dark at 4°C. The second day, cells in solution will be filtered through 40µm silk membranes and subject to Flow Cytometric analysis. Samples will be loaded into a BD FACSCalibur multi-color two-dimensional flow cytometer and absorbance was measured with 488 nM and 633 nM laser beams to detect both fluorescence and propidum iodide. DNA cell cycle analysis of flow cytometric data will be carried out using the software Modfit LT.