Other reagents were obtained from Sigma. Cell culture The human CML cell line K562 was obtained from the American Type Culture Collection. Ba F3 wt BCR ABL cells and Ba F3 T315I 17-AAG cells were described previously. These cells were maintained in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum with 1% penicillin streptomycin in a humidified incubator at 37 C. Cell proliferation assay Cell proliferation analysis was performed as previously described. Cell signaling assays and western blot analysis Panorama Ab microarrays were analyzed according to the manufacturers instructions. The arrays were scanned using a GenePix Personal 4100A microarray scanner, and normalization was carried out using the housekeeping pro tein included with the chip.
The protein expression ratio was calculated using MS Excel. Western blot analysis was performed as previously described. DNA microarray and microarray data analysis DNA microarray analysis was performed as previously described. In brief, K562 cells were treated with 1 uM tozasertib for 16 h. Following incubation at 37 C, the cells were washed twice with ice cold phosphate buffered saline and collected immediately for RNA isolation. In this study, we used the Human Genome U133A Genechip, which contains more than 47,000 transcripts. Target prepar ation was carried out following the manufacturers ex pression analysis manual. All arrays were screened for quality by standard methods, and the mean fluorescent intensity for each probe set was determined.
Primary samples This study was approved by the Institutional Review Board of Tokyo Medical University, and informed con sent was provided by all patients in accordance with the Declaration of Helsinki. Primary samples were obtained from the peripheral blood of CML patients. Mono nuclear cells were isolated from blood samples and separated by Lymphosepar. The cells were cultured in RPMI1640 medium containing 10% fetal calf serum and analyzed as described. Flow cytometory analysis Cells were treated with the indicated concentrations of tozasertib for 48 h. Annexin V propidium iodide apop tosis assays were performed according to the manufac turers instructions. The cells were gently mixed and immediately analyzed by flow cytometry. Statistical analysis Differences between treatment groups, in terms of dose response and apoptosis, were determined using Students t test.
P values of less than 0. 05 were considered significant. Background Diffuse large B cell lymphoma is the most com mon type of non Hodgkins lymphoma. Rituximab, an anti CD20 antibody, Entinostat administered as induction or main tenance therapy in combination with CHOP significantly prolonged event free survival of DLBCL. However, contin ued use of rituximab has resulted in CD20 negative trans formation of tumor cells and failure to demonstrate benefit.