Cell Culture and Reagents
The cell lines used in the study have been previously described; Follicular Lymphoma (WSU-FSCCL)  and Diffuse Large Cell Lymphoma (WSU-DLCL2) ; The WSU-FSCCL cell line has been karyotyped at least 4 times since our initial publication in 1993. The recent analysis in September of 2009 revealed the same chromosomal abnormalities as previously reported; 47,XY,+der(1)i(1)(q10)del(1)(q32),t(1;13)(p31;q12),del(6)(q21q27),t(8;11)(q24;q22),t(14;18)(q32;q21). The WSU-DLCL2 has been similarly karyotyped several times since its establishment in 1990. The cell line acquired an additional abnormality, add(8q24), that was detected for the first time in 1997. Since then the karyotype profile has remained stable with no further changes. The most recent karyotype in September of 2009 revealed: 48,XY,t(1;2)(p36;q37),der(3)t(3;7)(q13;p15),t(4;14)(q27;q32),+7,i(7)(p10),der(7)t(3;7)(q21;p11.2),+8,add(8)(q24),t(14;18)(q32;q21),del(15)(q26.1),del(16)(q22). Furthermore, fluorescent in situ hybridization (FISH) using LSI MYC dual color break-apart DNA probe (Vysis Inc.) revealed a deletion of the telomeric 3' region of CMYC gene most likely due to unbalanced translocation affecting the CMYC gene region. Cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin and incubated at 37°C in a humidified incubator with 95%/5% CO2. Primary antibody specific for Actin was obtained from Santa Cruz Biotechnology, (Santa Cruz, CA). Primary antibodies specific for Caspase-3, Caspase-9, PARP, p-IκBα and IκBα were obtained from Cell Signaling, (Danvers, MA). G3PDH was obtained from Trevigen, Inc (Gaithersburg, MD). Protein concentrations were determined using the Micro BCA protein assay (Pierce Chemical Company, Rockford, IL). Cyclophosphamide monohydrate was obtained from Mead Johnson (Evansville, IN). Doxorubicin hydrochloride was obtained from Bedford Inc (SA, Australia). Vincristine was obtained from Pharma Inc. (Bloomington, MN). ML120B was synthesized by Millennium Pharmaceuticals, Inc (Cambridge, MA) and dissolved in DMSO. Concentration of DMSO in the final culture was 0.44%.
Western Blot Analysis
Proteins obtained from cell extracts were collected 24, 48, or 72 h after single or combination treatment with the IKK-2 inhibitor (ML120B) and vincristine in lysis buffer containing protease inhibitors. Cytosolic protein extracts were prepared from control and treated cells using Nuclear/Cytosolic Fractionation Kit according to manufacturer's protocol (BioVision, Mountain View, CA). All proteins were resolved using 12% SDS-PAGE and transferred to Hybond C-extra membranes (Amersham Life Science, Arlington Heights, IL). Membranes were blocked with 5% milk in Tris buffer saline containing 0.05% Tween 20 (TBST) for 1 h at 25°C and incubated overnight at 4°C with rabbit anti-caspase 9 (H-170, Santa Cruz), rabbt anti-caspase 8 (H-134, Santa Cruz), rabbit anti-PARP (#9542, Cell Signaling), mouse anti-caspase 3 (#9668, Cell Signaling) or rabbit anti-NF-κB (H-286, Santa Cruz) in 2% Bovine serum albumin in TBST (1:1000 dilutions in BSA-TBST). Following incubation, membranes were washed with TBST and incubated with corresponding horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:5000 dilution in 5% milk-TBST) for 1 h at 25°C and then washed before proteins were visualized using picoglow HRP substrate (Michigan Diagnostics, LLC, Royal Oak, MI).
Flow Cytometric Analysis of Cell Cycle and Apoptosis
Cell cycle analysis and sub G0/G1 DNA content were determined by flow cytometry using propidium iodide (PI) staining. Cells were grown in the presence or absence of ML120B or vincristine then centrifuged and washed. The cells were then fixed with 75% ice-cold ethanol overnight and stained with 50 μg of PI and analyzed. To determine DNA fragmentation (as indication of apoptosis) induced by treatment agents, we utilized standard terminal deoxynucleotidyl transferase of dUTP nick end labeling (TUNEL) assay and propidium iodide (PI) staining. The kit used in this method (ApoDirect In Situ DNA Fragmentation Assay Kit, BioVision, Mountain View, CA; Catalog #K402-50) utilizes terminal deoxynucleotidyl transferase (TdT) to catalyze incorporation of DUTP at the 3'-hydroxyl ends of the fragmented DNA. The fluorescein-labeled DNA was detected by flow cytometry (horizontal axis in Figure 4). PI staining was simultaneously used to separate cells into G0/G1, S, G2 M and sub-G0 compartments based on DNA content (vertical axis, Figure 4). The dual staining (dUTP and PI) allowed us to assign dUTP-positive cells to a cell cycle phase. In this method, it is accepted that dUTP-positive cells are considered apoptotic . To confirm induction of apoptosis, we stained WSU-FSCCL cells with 7-AAD as previously published from our laboratory . All flow cytometry analysis of cells was done on FACScan (Becton-Dickinson, San Jose, CA).
WSU-FSCCL cells, treated and untreated, were harvested, washed once with PBS and fixed for 10 min with 3.7% formaldehyde in PBS. All procedures were carried out at room temperature. Following fixation, cells were washed 3 times with PBS, blocked for 45 min with 0.5% BSA in PBS and then incubated for 3 hr in 200 μl PBS containing 0.1% saponin (PBS-S), 1 μg/ml each of two primary antibodies, mouse anti-human NF-κBp65 and rabbit anti-tubulin. After incubation with primary antibodies, cells were carefully washed 3 times with PBS-S and then resuspended in PBS-S containing 5% goat sera and 10 μg/ml each of two fluorescently-labeled secondary antibodies and DAPI (10 μg/ml) for nuclear staining, if used. Cells were incubated for 1 hour, washed X3 with PBS-S and then fixed for 1 min with 3.7% formaldehyde. Following the final fixation, cells were washed 3 times with PBS containing no saponin. Cell suspensions were mounted on 1% gelatin-coated slide, dried, sealed with coverslips and visualized using an Olympus BX40 microscope equipped with laser light and fluorescence filter cubes for UV, green and red fluorescence. Visual recordings were captured separately using an RT-Spot Color Camera (Diagnostic Instruments, Inc, Sterling Heights, MI) and merged using Super Spot software (Diagnostic Instruments) to complete the overlay and final pictures.
All primary antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Slow Fade Light, DAPI and Alexa Fluor 488 (green) and Alexa Fluor 568 (red) fluorescently labeled secondary antibodies were purchased from Molecular Probes (a division of InVitrogen, Carlsbad, CA).
Establishment and Propagation of Xenografts
3-4 week old female ICR mice with severe combined immune deficiency (SCID) were purchased from Taconic Farms (Germantown, NY). Animals were housed in special protective environment and left to adapt for few days before beginning the experiments. To initiate the WSU-DLCL2-SCID xenografts, (5-10) × 106 WSU-DLCL2 cells in serum-free RPMI 1640 medium were injected subcutaneously (SC) in the flank areas of each animal. Palpable tumors were detected by clinical examination in about 2 weeks. When tumor weight reached 1000-1500 mg, animals were euthanized; tumors dissected out, placed in RPMI 1640 medium in sterile environment and minced into small fragments (20-30 mg each). To propagate the xenografts, tumor fragments were implanted SC, using a trocar, into flanks of 3-4 week old female ICR-SCID mice. Forty animals were implanted with WSU-DLCL2 tumors for the single agent (ML120B alone) experiment and forty for the combination study (CHOP plus ML120B). The WSU-FSCCL-SCID is a systemic model which is established by injecting 107 WSU-FSCCL cells in serum-free medium intravenously via tail vein of ICR-SCID mice. The growth pattern and assessment of response of this model to ML120B were the same as previously published from our laboratory .
Efficacy Trial Design
WSU-DLCL2 tumor-bearing animals were randomly assigned to control or one of 3 treatment dose/schedules of ML120B; 10 animals in each group. Therapy was started one week after tumor implantation. Group 1 received one dose of ML120B at 120 mg/kg. Group 2 received 60 mg/kg twice (every 12 hours). Group 3 received 60 mg/kg twice a day for 28 days. All treatments were given through oral gavage. ML120B compound was dissolved in 5% (hydroxypropyl) methylcellulose. Control group animals received vehicle alone. CHOP MTD in SCID mice was previously determined in our laboratory  for one injection (i.e.40 mg/kg, i.v. cyclophosphamide; 3.3 mg/kg,i.v. doxorubicin; 0.5 mg/kg,i.v. vincristine; and 0.2 mg/kg orally prednisone every day for 5 days). Animals were monitored 3 times per week for signs of toxicity, weight changes and tumor measurements. They were euthanized to avoid discomfort if the tumor burden reached ~2000 mg (approximately 10% of the body weight). All animal experiments were done according to protocols approved by the Animal Investigation Committee (AIC) of Wayne State University.
Statistical significance of drug-treated versus control measurements was determined by the student t-test. The interaction between ML120B and vincristine was analyzed using Calcusyn V2 software program to determine if the combinations were synergistic. Calcusyn is based on the Chou-Talalay method , which calculates a combinational index (CI) to indicate synergistic effects where CI < 0.9, is considered synergistic. Survival functions were estimated using the Kaplan-Meier method and compared by the log-rank test. P-values <0.05 were considered statistically significant.
All statistical analyses were evaluated using GraphPad Prism 4 (San Diego, CA).