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target in cancer treatment.Materials and Gossypol MethodsCell lines and reagentsDexamethasonesensitiveand Dex resistanthuman MM cell lineswere kindly provided by Dr. Steven Rosen.RPMI8226 and U266 human MM cells were obtained from American Variety CultureCollection. MelphalanresistantRPMI8266 human MM anddoxorubicinresistant RPMIDox40cell lines were provided by Dr William Dalton. OPM1 cells were provided by Dr P. LeifBergsagel. All MM cell lines were cultured as previouslydescribed. Fresh peripheral blood mononuclear cellswereobtained from four healthy volunteers. BM aspirates from MM patients were obtainedfollowing approval from the institutional review board. Immediately after mononuclear cells wereseparated, MM cells were purified by positive selection using CD138MicroBeads along with the Auto Macs magnetic cell sorter.
Bonemarrow stromal cellswere generated as Gossypol previously described.BMSCs were incubated in 96well culture platesfor 24 h, afterwashing off the medium, MM cell lines were added to the wellsandincubated with media or with growing doses of AT7519 for the specified time at 37C.AT7519 is N41Hpyrazole3carboxamide.AT7519 was obtained from Astex therapeutics Ltd, Cambridge, UK. It wasdissolved first in dimethyl sulfoxideat a concentration of 10mM,and then in culture mediumimmediately just before use. Alphaamanitin wasobtained from Axxora LLC. GSK3inhibitor was obtained fromCalbiochem.Cell viability and proliferation assaysAT7519's effects on viability of MM cell lines, primary MM cells, and PBMNCs wasassessed by measuring 32,5 diphenyl tetrasodium bromidedye Vortioxetine absorbance as previously described.
DNA PARP synthesis was measured by tritiated thymidine uptake. MMcellswere incubated in 96well culture plateswith media and different concentrations of AT7519 andor recombinant IL6or IGF1for 24 or 48 h at 37C and 3HTdR incorporation was measured aspreviously described.Detection of RNA synthesisRNA synthesis was evaluated by measuringuridineincorporation. MM.1S cellswere incubated in 96well culture plates within the presence of mediaor AT7519for 4, 6, 24 and 48h. Cells were incubated withuridinewellfor 3.5 h at 37C, harvested onto glass filters with an automatic cell harvester, and counted using the LKB Betaplatescintillation counter. 3H uptake analyses were performed intriplicate.Cell cycle analysis and detection of apoptosisMM cellswere cultured for 48h in media alone or with varying concentrations ofAT7519.
Cells were harvested, washed with icecold phosphatebuffered saline, fixedwith 70% ethanol for 20 minutes, and pretreated with10gmL RNasefor 20minutes as previously described. Apoptosis analysis was also confirmedby using Annexin VPI staining right after MM cells were cultured in media or 0.5M ofAT7519 at 37C for 6, 12, 24 hours as previously Vortioxetine described. AnnexinVPI? apoptotic cells were enumerated by using the Epics flow cytometer. The percentageof cells undergoing apoptosis was defined as the sum of early apoptosisand late apoptosis.Western blottingMM cells were cultured with AT7519 0.5M, harvested, washed, and lysed using lysisbuffer as previously described. The protein concentration of lysate wasmeasured, mixed with gel electrophoresis loading buffer, boiled for 5 min, separated bysodium dodecyl sulfatepolyacrylamide gel electrophoresis, and transferred tonitrocellulose membrane.
The membranes were blocked in TBS plus 5% non fat milkpowder and 0.1% TWEEN20 for 1 hour just before incubating with all the following antibodiesovernight at 4C: anti phosphoRNA polII serine 2 and serine 5, RNA pol II, phosphoGSK3, GSK3, phosphoAkt, Akt, phosphop4442MAPK, p4442 MAPK, phosphop70SK6, p70SK6, CDK4,CDK9, XIAP, Mcl1, caspase 3, caspase 9 and caspase 8; anticyclinD1, Gossypol cMyc; antiCDK1, CDK2, CDK5, CDK6, cyclin B1, cyclin A,Mcl1Antigenantibody complexes were detected usingsecondary antibodies conjugated to HRP and visualized using enhanced chemiluminescence. Blots were stripped and reprobed with antiαtubulin, GAPDH or αactinantibodies to ensure equal protein loading.
Quantitation of bandintensity was performed using Image J software program.Transfection and Lentivirus infectionTo determine the role of GSK3in AT7519induced apoptosis, we employed shRNA sequencesto knock down GSK3in Vortioxetine MM.1S cell line using a lentivirus transfection system. TheshRNA was kindly provided by RNAi Screening Facility of Dana Farber Cancer Institute.The sequence for with the GSK3shRNA construct was as follows: clone no.1: 5'CCACTGATTATACCTCTAGTA3'; clone no.2: 5'CCCAAACTACACAGAATTTAA3';clone no 3: 5'GCAGGACAAGAGATTTAAGAA3'; clone no 4: 5'GCTGAGCTGTTACTAGGACAA3'; clone no 5: 5'GACACTAAAGTGATTGGAAAT3'. pLKO.1 plasmidwithGSK3shRNA or pLKO.1 manage plasmid were cotransfected with pVSVG and delta 8.9plasmids into 293T cells with FuGENE 6 transfection reagent. At 48 and72 hours post transfection, superrnatant containing pseudoviral particles were collected;aliquots with 8gml polybrene were added to MM.1S cellsas previouslydescribed. Two days right after infection, cells were analyzed for GSK3andGAPDH expression by western blotting.