cell lines Jurkat, HL 60 and IM 9 were cultured secondly in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics at 37 C in a humidified atmo sphere with 5% CO2. All cell culture reagents were from PAA, Pasching, Austria. Stromal bone marrow cells, enriched by Ficoll gradient centrifugation as described, were kindly provided by the Tumour Immunology Department of the University Hospital, Munich. Bone marrow fibroblasts were generated by allowing bone marrow cells to adhere to plastic cell culture flasks. Cells were grown for 4 weeks, and non adherent cells were regularly displaced by replacing the cell culture medium. Cells e hibited a typical fibroblast like mor phology, and fibroblasts appeared to be the only cell type from bone marrow cells that showed significant proliferation under the cell culture conditions used.

Drugs and drug treatment Nelfinavir mesylate was gener ously provided by Pfizer, Groton, CT, USA. Nelfinavir was dissolved in DMSO and stored at 20 C as a 50 mg ml stock solution. The primary concentration used in this study was 8 ug ml nelfinavir mesylate, correspond ing to a molar concentration of 12 uM. Sorafenib was stored as a 25 mg ml stock solution in DMSO. In control e periments, cells received an amount of DMSO equal to that used in the treated cells. Staurosporine was stored as a 500 uM stock solution in DMSO. Chemosensitivity assay To test the viability of the cancer cells, 5000 cells in a total volume of 200 ul were plated in flat bottomed 96 well plates and incubated with nelfinavir for 48 h at 37 C.

For cell e traction, 50 ul tumour cell e traction buffer was added to each well, mi ed thoroughly, and incubated for 20 minutes at room temperature. Using a MicroLumat LB 96P biolu minometer, Luciferin Luciferase agent was added automatically to each sample and samples were analyzed for bioluminescence. Anne in binding assay FITC labelled anne in V was added to viable cells as recommended by the sup plier in combination with propidium iodide, and cells were analyzed with a FACScan using an FL 1 setting at 575 nm and an FL 2 setting at 530 nm. FACScan analysis was performed using a Becton Dickinson FACScan analyzer. Cell cycle analysis For cell cycle analysis, leukemia cells were washed with phosphate buffered saline, fi ed with 70% metha nol, incubated with RNase, and stained with propidium iodide prior to FACScan analysis.

Mitochondrial membrane potential analysis To analyze the mitochondrial membrane potential, the MitoCapture Mitochondrial Apoptosis Detection Kit was used according to the manufacturers instructions. For FACScan analysis, an FL 1 setting at 575 nm and an FL 2 setting at 530 nm were used. Simi lar filters were used for fluorescence Carfilzomib microscopy. Western blot analysis Western blot analysis was performed as recently described. Cell e tracts were prepared with RIPA selleck chemicals Sunitinib buffer, and 20 ug of protein was subjected to SDS polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes in a B