Supplemental Figure 1: Effect of NSC23766 on THP-1 cells. (A) Percentage of 7AAD-positive cells after exposure to 0 µM (black bars), 20 µM (hatched bars), or 40 µM (white bars) NSC23766 (n=5). (B) Cell cycle analysis of THP-1 cells was performed 48 hours after exposure to NSC23766. Graph depicts percentage of cells in G1/G0 or S phase of cell cycle 48 hours after exposure to 0 µM (black bars) or 40 µM (white bars) NSC23766, respectively (n=5). ** p<0.01
Supplemental Methods
Cell lines. ML-2 cells (AML M4, DMSZ, Braunschweig, Germany) contain a translocation t(6;11)(q27;q23) leading to the MLL-AF6 fusion gene (1). ML-2 cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Fisher Scientific, Pittsburgh, PA) supplemented with 10% fetal calf serum (FCS, Omega Scientific, Tarzana, CA) and 1% penicillin/streptomycin (P/S, Hyclone, Logan, UT). U-937 cells (histiocytic lymphoma, ATCC, Manassas, VA) were cultured in RPMI-1640, 10% FCS, 1% P/S. HL-60 cells (acute promyelocytic leukemia, ATCC) were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Fisher Scientific, Pittsburgh, PA), supplemented with 20% FCS, 1% P/S. THP-1 cells (acute monocytic leukemia, ATTC) were cultured in RPMI-1640, supplemented with 10% FCS and 0.05 mM 2-mercapto-ethanol. THP-1 cells contain an MLL-AF9 gene rearrangement (1). Human CD34+ healthy donor bone marrow cells were obtained following informed consent. CD34+ cells were positively selected using MACS cell separation columns (Miltenyi Biotec Inc., Auburn, CA). CD34+ cells were cultured in IMDM mediumsupplemented with 1xBIT serum substitute (Stem Cell technologies, Vancouver, Canada),1% P/S, 2nM L-glutamine, and the five cytokine cocktail KTF36 (10ng/mL Stem Cell Factor, Megakaryocyte Growth and Development Factor, Flt3 Ligand, interleukin-6 and interleukin-3, Amgen, Thousand Oaks, CA).
In vivo administration of NSC23766. All animals were maintained in a specific pathogen-free environment and all experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Cincinnati Children’s Research Foundation.A total of 2 X107ML-2 cells were transplanted into sublethally irradiated (350 Gy) NOD/SCID mice. Twenty-one days post-transplant, Alzet osmotic pumps (model 2002, Durect, Cupertino, CA) were subcutaneously implanted into recipient mice to administer either NSC23766 (2 pumps, 75 mM/pump) or PBS control at a flow rate of 0.5 µL/hr for 14 days. To implant the pumps, a small incision was made in the skin of the mid-scapular region of isoflurane-anesthetized mice, and curved hemostats were used to spread the subcutaneous tissue, creating a small pocket. The pumps were inserted into the subcutaneous pocket, delivery portal first. The skin incision was then closed using surgical wound clips. Fourteen days post-surgery, the pumps were exchanged in the surviving mice for pumps containing fresh NSC23766 or PBS. The second set of pumps was removed from surviving mice fourteen days later, and the wounds were sutured.
Proliferation Assay. MTS assays were performed per the manufacturer’s instructions (CellTiter 96 AqueousNon-Radioactive CellProliferation Assay, Promega, Madison, WI). Briefly, cells were plated in 96-well plates at a density of 5x103 or 10x103 cells per well in the presence of increasing concentrations of NSC23766 for 72 hours, after which time 20 µl of MTS reagent was added to each well. The absorbance of the reaction product, soluble formazan, was read at 490 nm at 1-4 hrs. Data from three independent experiments was analyzed by mixed effects model analysis. Random cell line and cell line by dose effects were included to account for “within cell line” variation from experiment to experiment and variation by dose.
Rac Activation Assay. Activation of Racwas determined by p21-activated kinase (PAK)-binding domain (PBD) pull-down on cell lysates as previously described (2). Cells were starved for 16 hours in RPMI, 1% FCS. Increasing concentrations of NSC23766 were added to the medium when indicated.
Flow cytometry. Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). All fluorochrome-labeled monoclonalantibodies were purchased from BD Pharmingen (San Jose, CA) and used according to the manufacturer’s instructions. Cell cycle analysis was performed by bromodeoxyuridine (BrdU) incorporation for one hour followed by flow cytometry using the anti-BrdU-APC labeling kit (Beckton Dickinson). Apoptosis analysis was performed by Annexin V and 7AAD staining (Annexin V kit, Beckton Dickinson). To assess the engraftment levels of ML-2(human CD45+) cells in transplanted mice, bone marrow was obtained from moribund mice. Human chimerism levels were determined following ammonium chloride red cell lysis (BD Pharm Lyse, BD Biosciences, San Jose, CA) by staining with anti-human CD45 APC and anti-mouse CD45 PE.
Supplemental Reference
1.Drexler HG, MacLeod RA. Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma. Leukemia 2004; 18(10):1569-71.
2.Benard V, Bohl BP, Bokoch GM. Characterization of Rac and Cdc42 activation in chemoattractant- stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 1999; 274(19):13198-204.