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• W2076448066 abstract "Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells. Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells. Glucocorticoids (GCs) 1The abbreviations used are: GC, glucocorticoid; ALL, acute lymphoblastic leukemia; BH, Bcl-2 homology; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GR, glucocorticoid receptor; PBS, phosphate-buffered saline; pre-B, precursor B-cell; TA, triamcinolone acetonide; RNAi, RNA interference; RT, reverse transcription; siRNA, small-interfering RNA; shRNA, short hairpin RNA.1The abbreviations used are: GC, glucocorticoid; ALL, acute lymphoblastic leukemia; BH, Bcl-2 homology; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GR, glucocorticoid receptor; PBS, phosphate-buffered saline; pre-B, precursor B-cell; TA, triamcinolone acetonide; RNAi, RNA interference; RT, reverse transcription; siRNA, small-interfering RNA; shRNA, short hairpin RNA. are steroid hormones that maintain physiological homeostasis. Synthetic GCs such as dexamethasone and triamcinolone acetonide (TA) are widely prescribed pharmaceuticals for indications ranging from inflammation to cancer. GCs induce apoptosis in numerous lymphoid and myeloid tissues and have been successful in the treatment of childhood leukemias (1Planey S.L. Litwack G. Biochem. Biophys. Res. Commun. 2000; 279: 307-312Crossref PubMed Scopus (128) Google Scholar). The mechanism of GC-induced apoptosis involves the hallmarks of the intrinsic pathway of apoptosis, i.e. the release of cytochrome c and Smac (second mitochondria-derived activator of caspase) from the mitochondria and the activation of caspase-9 (2Planey S.L. Derfoul A. Steplewski A. Robertson N.M. Litwack G. J. Biol. Chem. 2002; 277: 42188-42196Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Determining the molecular trigger(s) that commit the cell to the activation of intrinsic apoptosis might enable strategies to combat GC-resistant leukemias. Precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) is the most common childhood cancer and a useful model to investigate the mechanism of GC-induced apoptosis (3Findley Jr., H.W. Cooper M.D. Kim T.H. Alvarado C. Ragab A.H. Blood. 1982; 60: 1305-1309Crossref PubMed Google Scholar). GC signaling occurs through the glucocorticoid receptor (GR), a nuclear receptor superfamily member that dissociates from a large Hsp70-containing complex and translocates to the nucleus upon GC binding (reviewed in Ref. 4Wright A.P. Zilliacus J. McEwan I.J. Dahlman-Wright K. Almlof T. Carlstedt-Duke J. Gustafsson J.A. J. Steroid Biochem. Mol. Biol. 1993; 47: 11-19Crossref PubMed Scopus (68) Google Scholar). The activated GR initiates a tissue-specific transcriptional program through both direct DNA binding and interaction with other transcription factors. Numerous studies with actinomycin D and cyclo-heximide have demonstrated that new transcription and protein synthesis, respectively, are required for GC-induced apoptosis (5McConkey D.J. Nicotera P. Hartzell P. Bellomo G. Wyllie A.H. Orrenius S. Arch. Biochem. Biophys. 1989; 269: 365-370Crossref PubMed Scopus (524) Google Scholar, 6Cifone M.G. Migliorati G. Parroni R. Marchetti C. Millimaggi D. Santoni A. Riccardi C. Blood. 1999; 93: 2282-2296Crossref PubMed Google Scholar). Microarray profiling has shown that a short GC exposure induces or represses the transcription of >100 genes by a factor of 3-fold or greater in multiple models of GC-induced apoptosis (7Planey S.L. Abrams M.T. Robertson N.M. Litwack G. Cancer Res. 2003; 63: 172-178PubMed Google Scholar, 8Medh R.D. Webb M.S. Miller A.L. Johnson B.H. Fofanov Y. Li T. Wood T.G. Luxon B.A. Thompson E.B. Genomics. 2003; 81: 543-555Crossref PubMed Scopus (61) Google Scholar, 9Wang Z. Malone M.H. He H. McColl K.S. Distelhorst C.W. J. Biol. Chem. 2003; 278: 23861-23867Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Among these genes are universal regulators of intrinsic apoptosis such as BCL2 and BIM, a gene encoding several splice variants of the related Bcl-2 homology (BH) region 3 domain-containing protein Bim (7Planey S.L. Abrams M.T. Robertson N.M. Litwack G. Cancer Res. 2003; 63: 172-178PubMed Google Scholar, 9Wang Z. Malone M.H. He H. McColl K.S. Distelhorst C.W. J. Biol. Chem. 2003; 278: 23861-23867Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). GC represses BCL2 transcription but induces BIM transcription. Lymphocytes containing overexpressed (7Planey S.L. Abrams M.T. Robertson N.M. Litwack G. Cancer Res. 2003; 63: 172-178PubMed Google Scholar) or high endogenous levels (10Alnemri E.S. Fernandes T.F. Haldar S. Croce C.M. Litwack G. Cancer Res. 1992; 52: 491-495PubMed Google Scholar) of Bcl-2 protein are partially GC-resistant, underscoring the significance of intrinsic pathway regulation in GC signaling. Bim protein is in the “BH3-only” subset of Bcl-2 relatives, a group that also includes Bid, Bad, Puma, and Noxa. BH3-only proteins are transcriptionally activated, post-translationally modified, or released from sequestration in response to death stimuli and promote apoptosis by interacting with Bcl-2 family members that contain multiple BH domains (BH1-BH4) (reviewed in Ref. 11Borner C. Mol. Immunol. 2003; 39: 615-647Crossref PubMed Scopus (614) Google Scholar). Bim is a critical regulator of immune cell homeostasis as well as apoptosis in several tissue types and has been recently investigated in preclinical models as a potential cancer therapeutic agent (12Yip K.W. Li A. Li J.H. Shi W. Chia M.C. Rashid S.A. Mocanu J.D. Louie A.V. Sanchez O. Huang D. Busson P. Yeh W.C. Gilbert R. O'Sullivan B. Gullane P. Liu F.F. Mol. Ther. 2004; 10: 533-544Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 13Yamaguchi T. Okada T. Takeuchi K. Tonda T. Ohtaki M. Shinoda S. Masuzawa T. Ozawa K. Inaba T. Gene Ther. 2003; 10: 375-385Crossref PubMed Scopus (18) Google Scholar). Mice lacking Bim contain strikingly high numbers of leukocytes and eventually succumb to autoimmune disease (14Bouillet P. Metcalf D. Huang D.C. Tarlinton D.M. Kay T.W. Kontgen F. Adams J.M. Strasser A. Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1290) Google Scholar). Importantly, thymocytes isolated from these mice demonstrate a delayed apoptotic response to the GC dexamethasone (14Bouillet P. Metcalf D. Huang D.C. Tarlinton D.M. Kay T.W. Kontgen F. Adams J.M. Strasser A. Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1290) Google Scholar). Loss of a single allele of BIM accelerated the rate of murine c-myc-induced lymphoma development, leading to classification of Bim as a tumor suppressor protein (15Egle A. Harris A.W. Bouillet P. Cory S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6164-6169Crossref PubMed Scopus (415) Google Scholar). Bim expression is induced by growth factor withdrawal (16Shinjyo T. Kuribara R. Inukai T. Hosoi H. Kinoshita T. Miyajima A. Houghton P.J. Look A.T. Ozawa K. Inaba T. Mol. Cell. Biol. 2001; 21: 854-864Crossref PubMed Scopus (188) Google Scholar, 17Putcha G.V. Moulder K.L. Golden J.P. Bouillet P. Adams J.A. Strasser A. Johnson E.M. Neuron. 2001; 29: 615-628Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), T-cell receptor ligation (18Villunger A. Marsden V.S. Zhan Y. Erlacher M. Lew A.M. Bouillet P. Berzins S. Godfrey D.I. Heath W.R. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7052-7057Crossref PubMed Scopus (65) Google Scholar), paclitaxel treatment (19Sunters A. Fernandez de Mattos S. Stahl M. Brosens J.J. Zoumpoulidou G. Saunders C.A. Coffer P.J. Medema R.H. Coombes R.C. Lam E.W. J. Biol. Chem. 2003; 278: 49795-49805Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar), and forced growth in suspension (20Marani M. Hancock D. Lopes R. Tenev T. Downward J. Lemoine N.R. Oncogene. 2004; 23: 2431-2441Crossref PubMed Scopus (97) Google Scholar, 21Reginato M.J. Mills K.R. Paulus J.K. Lynch D.K. Sgroi D.C. Debnath J. Muthuswamy S.K. Brugge J.S. Nat. Cell Biol. 2003; 5: 733-740Crossref PubMed Scopus (431) Google Scholar) in various cell types, suggesting that the transcriptional regulation of Bim is an upstream event in apoptosis induced by diverse stimuli. Finally, lowering BIM mRNA by RNAi causes partial protection against apoptosis induced by the drugs paclitaxel (19Sunters A. Fernandez de Mattos S. Stahl M. Brosens J.J. Zoumpoulidou G. Saunders C.A. Coffer P.J. Medema R.H. Coombes R.C. Lam E.W. J. Biol. Chem. 2003; 278: 49795-49805Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar) and imitinib (22Kuribara R. Honda H. Matsui H. Shinjyo T. Inukai T. Sugita K. Nakazawa S. Hirai H. Ozawa K. Inaba T. Mol. Cell. Biol. 2004; 24: 6172-6183Crossref PubMed Scopus (143) Google Scholar), as well as apoptosis induced by forced suspension culture (anoikis) (20Marani M. Hancock D. Lopes R. Tenev T. Downward J. Lemoine N.R. Oncogene. 2004; 23: 2431-2441Crossref PubMed Scopus (97) Google Scholar, 21Reginato M.J. Mills K.R. Paulus J.K. Lynch D.K. Sgroi D.C. Debnath J. Muthuswamy S.K. Brugge J.S. Nat. Cell Biol. 2003; 5: 733-740Crossref PubMed Scopus (431) Google Scholar). The BIM gene is transcribed as three major spice variants, BIM EL, BIM L, and BIM S, encoding the functionally distinct proteins BimEL, BimL, and BimS, respectively (23O'Connor L. Strasser A. O'Reilly L.A. Hausmann G. Adams J.M. Cory S. Huang D.C. EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (949) Google Scholar). The BimS protein has been widely reported to be the most strongly proapoptotic of the three, although there may be exceptions (16Shinjyo T. Kuribara R. Inukai T. Hosoi H. Kinoshita T. Miyajima A. Houghton P.J. Look A.T. Ozawa K. Inaba T. Mol. Cell. Biol. 2001; 21: 854-864Crossref PubMed Scopus (188) Google Scholar). Only BimEL is likely to be regulated by phosphorylation and caspase-3 cleavage (24Chen D. Zhou Q. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1235-1240Crossref PubMed Scopus (74) Google Scholar). Both BimEL and BimL (but not BimS) may be sequestered by LC8, a subunit of the dynein motor complex (25Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar), although the significance of this interaction has recently been questioned (26Zhu Y. Swanson B.J. Wang M. Hildeman D.A. Schaefer B.C. Liu X. Suzuki H. Mihara K. Kappler J. Marrack P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7681-7686Crossref PubMed Scopus (112) Google Scholar). Previous studies in our laboratory (7Planey S.L. Abrams M.T. Robertson N.M. Litwack G. Cancer Res. 2003; 63: 172-178PubMed Google Scholar) and others (9Wang Z. Malone M.H. He H. McColl K.S. Distelhorst C.W. J. Biol. Chem. 2003; 278: 23861-23867Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 27Zhang L. Insel P.A. J. Biol. Chem. 2004; 279: 20858-20865Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) have led to speculation that the transcriptional induction of Bim is required for GC-induced apoptosis. Only correlative evidence has thus far been generated in leukemic cells. To test this hypothesis directly, we used RNAi (28Scherr M. Morgan M.A. Eder M. Curr. Med. Chem. 2003; 10: 245-256Crossref PubMed Scopus (143) Google Scholar) to reduce BIM mRNA levels and found that both synthetic siRNAs and lentiviral-expressed shRNAs rendered human 697 pre-B ALL cells partially resistant to GC and inhibited GC-induced caspase-3 activity. Using splice variant-specific siRNAs, we found that all three major isoforms contribute to GC-induced apoptosis. Finally, we demonstrate that the Bim/Bcl-2 ratio is not a critical parameter in this pathway, suggesting that Bim functions primarily in a Bcl-2-independent manner in pre-B ALL cells. Cell Culture and Reagents—The pre-B ALL cells lines were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Nalm-6, Kasumi-2, and Kopn-8 cells were acquired from DSMZ (Braunschweig, Germany). TA, Polybrene, and all other reagents were purchased from Sigma unless otherwise indicated. Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. Electroporation of siRNA—siRNAs were purchased from Dharmacon, Inc. (Lafayette, CO). The sense strand sequences of the RNA duplexes used were as follows: control siRNA (firefly luciferase), 5′-CGUACGCGGAAUACUUCGA(dTdT)-3′; KIF11, 5′-AACUGAAGACCUGAAGACAAU(dTdT)-3′; BIM#1, 5′-ACCGAGAAGGUAGACAAUU(dTdT)-3′; BIM#2, 5′-CUACCUCCCUACAGACAGA(dTdT)-3′; BIM EL, 5′-CUCGAUCCUCCAGUGGGUA(dTdT)-3′; BIM EL plus BIM L (5′-CAGCACCCAUGAGUUGUGA(dTdT)-3′; BCL2#1, 5′AGAUAGUGAUGAAGUACAUUU-3′; BCL2#2, 5′-GAAGUACAUCCAUUAUAAGUU-3′; and BCL2#3, 5′-GGGAGAUAGUGAUGAAGUAUU-3′. Cells were washed twice with phosphate-buffered saline (PBS) and resuspended in serum-free RPMI 1640 medium containing 25 mm HEPES (no phenol red or antibiotics) at 107 cells/ml. A 200-μl aliquot of cells was added to a 0.2-cm gap electroporation cuvette (Bio-Rad) along with 10 μl of a 20 μm stock of siRNA and then incubated at room temperature for 20 min. Cells were then electroporated with a Bio-Rad Gene Pulser using the indicated conditions, incubated for an additional 20 min, and added to 2 ml of the above medium (excluding aggregated cell debris) supplemented with 10% fetal bovine serum. Thus, the final concentration of siRNA is 100 nm following electroporation. The standard condition of 500-microfarad capacitance (see “Results”) achieved a pulse length of 7-8 ms. Vehicle (0.05% ethanol) or TA was added after the indicated recovery times. Cell Cycle Analysis and Viability Assays—Electroporated and drug-treated 697 cells were harvested, washed with PBS, and fixed with 70% ethanol. After fixing for 18 h at 4 °C, cells were collected by centrifugation and resuspended in 1 ml of staining solution containing 50 μg/ml propidium iodide, 1 mg/ml RNase A, and 1 mg/ml glucose in PBS. Cells were analyzed for DNA content using a Beckman Coulter XL flow cytometer after 2 h of staining. 10,000 cells were counted per sample. Percentages of cells in sub-G0 or G2/M cell cycle fractions were determined using WinMDI software (Scripps Research Institute, La Jolla, CA). Trypan blue exclusion assays were performed in triplicate, counting >200 cells per sample. Immunoblot Analysis—Cells were washed once in PBS and lysed with radioimmune precipitation assay buffer containing 1× Complete protease inhibitors (Roche Applied Science). 30-50 μg of each whole cell extract was electrophoresed on Novex 12% Tris-Gly polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes. Immunoblots were performed using antibodies against Bim (1:20000 dilution; catalog number 202000, Calbiochem), Bcl-2 (1:10000; clone 100, Upstate Biotechnology, Lake Placid, NY), and GAPDH (1:20000; clone 6C5, Research Diagnostics, Flanders, NJ) using standard methods and detected using chemoluminescent horseradish peroxidase substrates (Pierce). Densitometry analyses were performed by generating net intensity values using Kodak 1D software (Rochester, NY). RT-PCR—Total RNA was purified using RNeasy columns (Qiagen, Valencia, CA). RT-PCR was performed in 50-μl reactions using 100 ng of RNA, 0.5 μm each primer, and an annealing temperature of 53 °C for 25 cycles. All other PCR conditions and reagents were supplied and recommended by the manufacturer's protocol for the Titan one-step system (Roche Applied Science). Primer sequences for BIM were 5′-GAGAAGGTAGACAATTGCAG-3′ (forward) and 5′-GACAATGTAACGTAACAGTCG-3′ (reverse); for GAPDH the primers were 5′-CACCCATGGCAAATTCCATG-3′ (forward) and 5′-TCTAGACGGCAGGTCAGGT-3′ (reverse). Caspase Analysis—For the caspase-3 substrate cleavage assay, one transfection was performed for each data point. Cells were washed with PBS, lysed, and assayed in a 96-well plate using the EnzCheck caspase-3 assay kit (Molecular Probes, Eugene, OR). Fluorescence was measured at emission and excitation settings of 485 and 530 nm, respectively, with a Bio-Tek (Winooski, VT) FL600 plate reader. For immunofluorescence, 50,000 cells were spun onto microscope slides, fixed, and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100, immunostained with an anti-active caspase-3 antibody diluted 1:50 (Cell Signaling Technology, Beverly, MA), and counterstained with 4′,6-diamidino-2-phenylindole. Fluorescent detection was performed with the Vector Laboratories (Burlingame, CA) biotin/avidin system and Alexa Fluor 488 (Molecular Probes). Lentivirus Generation and Infection—The lentiviral transfer and packaging vectors were a generous gift from Dr. Xiao-Feng Qui. Lentivirus production was conducted by co-transfection of HEK293T cells with four plasmids, i.e. a packaging defective helper construct (pMDL-g/pRRE; 3 μg), a Rev-expressing construct (pRSV-Rev; 3 μg), a construct expressing a heterologous envelope protein (pCMV-VSVg; 5 μg), and a transfer vector harboring a specific shRNA sequence under control of the H1 RNA polymerase III promoter (pH1UG/Luc-shRNA or pH1UG/Bim-shRNA; 10 μg). Briefly, 3 × 106 HEK293T cells were seeded on 10-cm plates 24 h before transfection. For each shRNA, 100 μl of FuGENE-6 (Roche Applied Science) was combined with the four plasmids in 3 ml of serum-free RPMI 1640 medium. The mixture was added dropwise onto the cells after a 30-min incubation, and the cells were analyzed for green fluorescent protein (GFP) expression by fluorescence microscopy after 24 h. At 48 h, virus-containing cell supernatants were collected and centrifuged twice to eliminate transfer of cells. shRNA-encoding pH1UG vectors were created by cloning annealed complementary oligonucleotides into BamHI and XhoI sites at the 3′ of the H1 RNA polymerase III promoter. The coding strand sequences of the shRNA-encoding oligonucleotides are 5′-GATCCCCCGTATGCGGAATACTTTGATTCAAGAGATCGAAGTATTCCGCGTACGTTTTTC-3′ for control (luciferase) and 5′-GATCCCCGACTGAGAAGGTAGATAATTTTCAAGAGAAATTGTCTACCTTCTCGGTCTTTTTC-3′ for BIM. The BIM shRNA sequence used was modeled after the BIM#1 siRNA sequence. The 697 cells were infected by adding 1 ml of viral supernatant supplemented with 4 μg/ml Polybrene to 5 × 104 cells in 24-well plates. The viral supernatant was replaced with standard growth medium after 24 h, and infection efficiency was monitored by GFP expression after 48 h. Viral titers were calculated by counting GFP-positive cells after infecting with serial dilutions of the viral supernatant. Northern Analysis—Total RNA was prepared from shRNA-expressing cell lines using Tri reagent (Sigma) according to the vendor's protocol. 30 μg of RNA were electrophoresed on a Tris borate-EDTA-urea 15% acrylamide gel (Invitrogen) and transferred to a Zeta Probe nitrocellulose membrane (Bio-Rad). The membranes were UV light-cross-linked and probed with an 32P-5′-end-labeled Bim antisense probe (5′-AATTGTCTACCTTCTCGGTC-3′) at 42 °C for 18 h. For the two hybridization control oligonucleotides (perfect match and two-base mismatch), the two complementary BIM shRNA-encoding oligonucleotides were used because wobble base-pairing in the shRNA design (29Paddison P.J. Caudy A.A. Sachidanandam R. Hannon G.J. Methods Mol. Biol. 2004; 265: 85-100PubMed Google Scholar) created two mismatches in the sense sequence of the coding strand oligonucleotide. The pro-apoptotic Bcl-2 relative Bim has been shown to be up-regulated by GC in GC-sensitive lymphocytes, suggesting that Bim may function as an early sensor for intrinsic apoptosis (7Planey S.L. Abrams M.T. Robertson N.M. Litwack G. Cancer Res. 2003; 63: 172-178PubMed Google Scholar, 9Wang Z. Malone M.H. He H. McColl K.S. Distelhorst C.W. J. Biol. Chem. 2003; 278: 23861-23867Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 27Zhang L. Insel P.A. J. Biol. Chem. 2004; 279: 20858-20865Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). To determine whether there is a correlation between Bim induction and GC sensitivity in human pre-B ALL cells, we monitored a panel of human pre-B ALL cell lines for viability in response to the potent GC triamcinolone acetonide after 48- and 72-h exposures (Fig. 1, A and B). Significant cell death was observed in the cell line 697 at low doses (5 nm) after 48 h, but the other lines tested (Kasumi-2, Kopn-8, and Nalm-6) showed no effect after 48 h and only slight (Kasumi-2, Kopn-8) or moderate (Nalm-6) cell death at the 72-h time point. We then compared the effect of TA on Bim protein levels in the sensitive and resistant cells (Fig. 1C). We observed that the three major Bim proteins (BimEL, BimL, and BimS) (23O'Connor L. Strasser A. O'Reilly L.A. Hausmann G. Adams J.M. Cory S. Huang D.C. EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (949) Google Scholar) increased after 24 h in the 697 cells but not in the other pre-B ALL cell lines. In addition, basal levels of the Bim proteins were low or undetectable in the GC-resistant lines. To further explore the kinetics of Bim induction by GC, the 697 cells were also exposed to GC for shorter time points, showing rapid up-regulation of the three Bim proteins after a 4 or 8 h exposure to 100 nm TA (Fig. 1D). Taken together, these data suggest that Bim expression and induction are factors in determining the pro-apoptotic response to GC in pre-B ALL. We then sought to reduce Bim expression in 697 cells by RNAi. The use of synthetic siRNAs has not been widely reported for lymphocytes, primarily because of the technical difficulty of nucleic acid transfection in non-adherent cultures. It has previously been shown that, for lymphocytes, electroporation is a more efficient delivery method for synthetic nucleic acids than liposome-mediated transfection (30Weil D. Garcon L. Harper M. Dumenil D. Dautry F. Kress M. BioTechniques. 2002; 33: 1244-1248Crossref PubMed Scopus (99) Google Scholar). However, the cytotoxicity caused by the cellular stress of electroporation has often been too significant to perform downstream viability measurements. It has been reported (30Weil D. Garcon L. Harper M. Dumenil D. Dautry F. Kress M. BioTechniques. 2002; 33: 1244-1248Crossref PubMed Scopus (99) Google Scholar) that an siRNA against the KIF11 gene, which encodes the mitotic kinesin KSP (also known as Eg5), is an effective tool to optimize transfection conditions because of its ability to induce mitotic block. To determine conditions for electroporating siRNAs into human 697 pre-B lymphocytes, we tested several RNA concentrations and capacitance settings using a KIF11-targeting siRNA. After 48 h, cell cycle analysis was performed using propidium iodide staining and flow cytometry. Under optimized settings (220 V/500 microfarads), the percentage of cells blocked in the G2/M stage was 2-fold higher for the KSP siRNA as compared with that for control siRNA (Fig 2A), indicating that the level of transfection was sufficient to explore biological phenotypes. We also verified that RNA uptake occurred in >90% of the cells under these transfection conditions using a nonspecific siRNA labeled with Cy3 (data not shown). Importantly, the relatively mild electroporation conditions resulted in a minimal accumulation of sub-G1/G0 cells, indicating that this RNA delivery method caused negligible cell death and is therefore suitable for performing downstream viability studies. These electroporation conditions were used for all subsequent siRNA experiments. Finally, increasing the capacitance setting or the siRNA concentration did not further increase the percentage of cells blocked in G2/M (Fig. 2B). 24 h after transfection with two BIM-targeting siRNAs, steady state levels of BimEL, BimL, and BimS were markedly reduced compared with those of a control siRNA sequence (Fig. 3A). Neither of the BIM-targeting siRNAs affected expression of the housekeeping protein GAPDH (Fig. 3A) or the other Bcl-2 family members Bax and Bad (data not shown). To determine whether the siRNA can reduce the level of Bim even after its expression is up-regulated by GC, we treated the cells with TA (100 nm) 24 h after siRNA electroporation. The 24-h recovery after electroporation allows for degradation of the existing Bim protein, the rapid turnover rate of which can be attributed in part to ubiquitin-proteasome processing (31Ley R. Balmanno K. Hadfield K. Weston C. Cook S.J. J. Biol. Chem. 2003; 278: 18811-18816Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Both siRNAs caused a marked reduction in not only basal Bim expression but also in the TA-induced levels of the three major splice variants relative to a control sequence (Fig 3A, right). The more potent Bim siRNA (sequence 2) caused a 70% reduction in basal BimEL (n = 4) and a 30% reduction in TA-induced BimEL (n = 2) (Fig. 3A, right, compare lanes 1 and 2 to lanes 5 and 6, counting from the left). To explore the RNAi effect at the mRNA level, we performed RT-PCR on total RNA isolated from siRNA-transfected 697 cells. The use of end point RT-PCR allows for visualization of all three major splice variants. Immediately following a 20-min post-electroporation recovery incubation (see “Materials and Methods”), the transfected cells were treated with vehicle or TA (100 nm) and then harvested 2.5, 5, or 7.5 h later. The mRNAs corresponding to the three major splice variants were effectively reduced by the BIM-targeting siRNA only 2.5 h after electroporation and reached near maximal silencing by that early time point, as further incubation to 7.5 h had little additional effect. Treatment of the cells with TA caused BIM mRNA induction relative to vehicle-treated controls, which was effectively abrogated by the siRNA (Fig 3B). To determine whether targeting Bim affects the apoptotic machinery in the 697 cells, we analyzed cells for caspase-3 activation (cleavage) and caspase-3 enzyme activity. Cells were electroporated as above, allowed to recover for 24 h (to allow for the degradation of pre-existing Bim protein), and then treated with TA (5 nm) for 24 h. This low, physiologically-relevant GC concentration corresponds to the approximate Kd of TA for the receptor (32Zhang S. Danielsen M. Recent Prog. Horm. Res. 1995; 50: 429-435PubMed Google Scholar). After the 24-h drug exposure, cells were immunostained with an antibody specific for the active (cleaved) form of caspase-3. Silencing of Bim reduced TA-induced caspase-3 activation, reflecting a decrease in activity of its upstream activator, caspase-9 (Fig 4A). To confirm these results, we also monitored the enzymatic activity of caspase-3 by assaying for cleavage of the fluorescent substrate, DEVD-rhodamine-110, as described (33Liu J. Bhalgat M. Zhang C. Diwu Z. Hoyland B. Klaubert" @default.
• W2076448066 created "2016-06-24" @default.
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• W2076448066 date "2004-12-01" @default.
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• W2076448066 title "Inhibition of Glucocorticoid-induced Apoptosis by Targeting the Major Splice Variants of BIM mRNA with Small Interfering RNA and Short Hairpin RNA" @default.
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