3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. of mitochondrial membrane potential and induction of ROS. Furthermore, we show thatLY573636was able to induce granulocytic/monocytic differentiation of HL60 and U937 cells.LY573636, as shown before in sound tumors, is effective in hematopoietic cell lines as well. These data suggest the use ofLY573636alone or in combination with standard chemotherapeutic regimens in hematopoietic malignancies. Keywords:small-molecule drugs, acylsulfonamides,LY573636, hematopoietic malignancies == Introduction == LY573636-sodium (hereafter referred to asLY573636), a benzamide, N-[(5-bromo-2-thienyl)sulfonyl]-2,4-dichloro-sodium salt (Lilly Research Laboratories [LRL]), was initially identified for its selective activity against solid tumors using anin vitrosoft agar disk diffusion assay. This compound has exhibited significant dose-dependent antitumor activity against xenograft models of HCT116 and SW620 human colon cancers, as well as A375 melanoma (1) (LRL, unpublished data). Other acyl sulfonamides analogues recognized during the development ofLY573636have been reported in recent years to exhibit considerable antitumor activity, particularly against human colon, lung, prostate, ovarian, and breast xenografts growing in immunodeficient mice (2,3). In addition, toxicity ofLY573636has been evaluatedin vitroandin vivoin different species including rats and beagle UNC2541 dogs (LRL, unpublished UNC2541 data). Currently, Phase 1 study ofLY573636has been completed, with bone marrow suppression, most commonly thrombocytopenia, identified as the dose-limiting toxicity. Phase 2 studies in non-small cell lung malignancy, malignant metastatic melanoma, ovarian malignancy, and soft tissue sarcoma are currently in progress (4). Importantly, National Cancer Institutes COMPARE analysis did not identify UNC2541 any similarities betweenLY573636and any known cytotoxic compound suggesting a unique mechanism of action. These findings are consistent with recent pre-clinical mechanism of action studies indicating thatLY573636-induced apoptosis occurs by a mitochondrial-targeted mechanism (LRL, unpublished data). To date, no one has evaluated the effects ofLY573636against malignant hematopoietic cells; in this study, we examined the antileukemic and antilymphoma activity of the compound using a large variety of cell lines. == Materials and methods == == Cell culture == B-cell acute lymphocytic leukemia (RCH, kindly provided by Dr. Janet Rowley, University or college of Chicago; Reh, a nice gift from Dr. Gary Gilliland, Harvard University or college; BALL-1, kindly UNC2541 provided by Dr. Sven de Vos, University or college of California, Los Angeles), Burkitts lymphoma (Daudi, American Type Culture Collection [ATCC], Manassas, VA), diffuse large B-cell lymphoma (MD901, kindly provided by Dr. Miki, Tokyo Medical and Dental care University or college, Japan; LY4, generously donated by Ari Melnick, Albert Einstein College of Medicine), and myeloid leukemia cell lines (HL60 and U937, ATCC) were managed in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10 %10 % fetal bovine serum (FBS), 10 U/ml penicillin and 10 mg/ml streptomycin (P/S) (Invitrogen) at 37 C in 5 % CO2. Mantle cell lymphoma cell lines (NCEB1, Jeko1, and SP49) (5) were maintained under comparable conditions in RPMI 1640 supplemented with 20 % FBS and P/S. == MTT proliferation assays == Cells were treated with numerous concentrations ofLY573636(kindly provided by Lilly Research Laboratories, Indianapolis, IN). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO) was performed as previously explained (6). KLRK1 Briefly, MTT was dissolved in phosphate-buffered saline (PBS) at 5 mg/mL. Approximately 1,000 cells per well were incubated in culture medium for 96 hours in 96-well plates; and then, 10 L of the MTT answer was added. After a 4-hour incubation, 100 L of solubilization answer (20 % sodium dodecyl sulfate [SDS]) was added, and the combination was incubated at 37 C for 16 hours. In this assay, MTT was cleaved to an orange formazan dye by metabolically active cells; and the absorbance of the formazan product was measured with an enzyme-linked immunoabsorbent assay (ELISA) reader at 540 nm. == Apoptosis analyses == Annexin V staining was performed as.