The Small Heat Shock Protein αB-crystallin Is a Novel Inhibitor of TRAIL-induced Apoptosis That Suppresses the Activation of Caspase-3
2005; Elsevier BV; Volume: 280; Issue: 12 Linguagem: Inglês
10.1074/jbc.m413382200
ISSN1083-351X
AutoresMerideth C. Kamradt, Meiling Lü, Michael E. Werner, Toni Kwan, Feng Chen, Anne M. Strohecker, Shayna E. Oshita, John Wilkinson, Chunjiang Yu, Patsy G. Oliver, Colin S. Duckett, Donald J. Buchsbaum, Albert F. LoBuglio, V. Craig Jordan, Vincent L. Cryns,
Tópico(s)Hepatitis B Virus Studies
ResumoTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor α family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein αB-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type αB-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of αB-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type αB-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation αB-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that αB-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of αB-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor α family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein αB-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type αB-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of αB-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type αB-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation αB-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that αB-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of αB-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 1The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Hsp, heat shock protein; RNAi, RNA interference; DISC, death-inducing signaling complex; DR, death receptor; WT, wild-type; mAb, monoclonal antibody; GFP, green fluorescent protein; TUNEL, terminal dUTP deoxynucleotidyl transferase-mediated nick end labeling; AFC, 7-amino-4-trifluoromethylcoumarin.1The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Hsp, heat shock protein; RNAi, RNA interference; DISC, death-inducing signaling complex; DR, death receptor; WT, wild-type; mAb, monoclonal antibody; GFP, green fluorescent protein; TUNEL, terminal dUTP deoxynucleotidyl transferase-mediated nick end labeling; AFC, 7-amino-4-trifluoromethylcoumarin. also known as Apo2L, is a promising antitumor agent currently in preclinical studies that preferentially induces apoptosis in cancer cells, but not normal cells (1Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Investig. 1999; 104: 155-162Crossref PubMed Scopus (1985) Google Scholar, 2LeBlanc H.N. Ashkenazi A. Cell Death Differ. 2003; 10: 66-75Crossref PubMed Scopus (750) Google Scholar). Like other members of the tumor necrosis factor family of cytokines, TRAIL is a type II transmembrane protein with an extracellular carboxyl terminus that mediates trimerization and receptor binding (3Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1632) Google Scholar, 4Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2636) Google Scholar). 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Chem. 2001; 276: 10767-10774Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, the molecular mechanisms of TRAIL resistance in the vast majority of cancers have not yet been delineated.Here we report that the small heat shock protein αB-crystallin is a novel inhibitor of TRAIL-induced apoptosis in cancer. αB-crystallin is 1 of 10 mammalian members of the conserved small heat shock protein family, which also includes Hsp27 (27Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (218) Google Scholar). Like other small heat shock proteins, αB-crystallin is a molecular chaperone that is induced by a variety of stress stimuli and confers a cytoprotective effect by suppressing aggregation of denatured proteins (28Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1735) Google Scholar, 29Klemenz R. Frohli E. Steiger R.H. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar). In addition, αB-crystallin is constitutively expressed, often at high levels, in diverse human cancers, including gliomas and breast, prostate, and renal cell carcinomas (30Aoyama A. Steiger R.H. Frohli E. Schafer R. von Deimling A. Wiestler O.D. Klemenz R. Int. J. Cancer. 1993; 55: 760-764Crossref PubMed Scopus (90) Google Scholar, 31Pinder S.E. Balsitis M. Ellis I.O. Landon M. Mayer R.J. Lowe J. J. Pathol. 1994; 174: 209-215Crossref PubMed Scopus (42) Google Scholar, 32Takashi M. Katsuno S. Sakata T. Ohshima S. Kato K. Urol. Res. 1998; 26: 395-399Crossref PubMed Scopus (56) Google Scholar, 33Chelouche-Lev D. Kluger H.M. Berger A.J. Rimm D.L. Price J.E. Cancer. 2004; 100: 2543-2548Crossref PubMed Scopus (67) Google Scholar). Within cells, αB-crystallin assembles into large ∼500-kDa homo- and hetero-oligomeric complexes (along with Hsp27) (27Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (218) Google Scholar, 34Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Importantly, the assembly of αB-crystallin into these large complexes is negatively regulated by stress-induced phosphorylation on serine residues 19, 45, and 59, and the dissociated complexes have diminished chaperone activity (35Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 36Ito H. Kamei K. Iwamoto I. Inaguma Y. Nohara D. Kato K. J. Biol. Chem. 2001; 276: 5346-5352Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Furthermore, we have recently demonstrated that αB-crystallin inhibits apoptosis induced by various stimuli, including DNA-damaging agents and growth factor deprivation, by disrupting the proteolytic activation of caspase-3 (37Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 38Kamradt M.C. Chen F. Sam S. Cryns V.L. J. Biol. Chem. 2002; 277: 38731-38736Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Here we demonstrate that TRAIL resistance in a panel of human cancer cell lines is tightly linked to the expression of αB-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) and that αB-crystallin inhibits TRAIL-induced caspase-3 activation and apoptosis. We also show that selective inhibition of αB-crystallin expression by plasmid-based RNA interference (RNAi) sensitizes cancer cells to TRAIL-induced apoptosis. These findings indicate that αB-crystallin is a novel negative regulator of TRAIL-induced apoptosis and that targeted inhibition of this small heat shock protein sensitizes cancer cells to TRAIL-induced apoptosis.EXPERIMENTAL PROCEDURESCell Culture and Reagents—Breast carcinoma cells were grown in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 4.5 g/liter glucose and 4 mm l-glutamine, 100 units/ml penicillin/streptomycin, and 10% fetal calf serum (Invitrogen). The malignant glioma cell lines were grown in Dulbecco's modified Eagle's medium/F-12 media supplemented with 10% fetal calf serum. All cell cultures were maintained in a 5% CO2 atmosphere at 37 °C.Transient and Stable Transfection Experiments—FLAG-tagged cDNAs encoding WT αB-crystallin, mutant αB-crystallin (R120G, a deletion of the carboxyl-terminal 14 amino acids (ΔC) or a triple pseudophosphorylation mutant (S19E/S45E/S59E) labeled 3XSE), or Hsp27 (all in pcDNA3-NFLAG) were described previously (37Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 38Kamradt M.C. Chen F. Sam S. Cryns V.L. J. Biol. Chem. 2002; 277: 38731-38736Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). For transient transfections, human MDA-MB-231 breast carcinoma cells were grown on glass coverslips and transfected with 1 μg of pcDNA3-NFLAG plasmids or pEGFP-N1 control vector (Clontech) using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. Cells were allowed to recover for 24 h before treatment with TRAIL. To obtain stable clones, transfected cells were grown in 800 μg/ml G418 (Invitrogen), and G418-resistant clones expressing WT αB-crystallin (made previously (37Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar)) or 3XSE αB-crystallin were identified by immunoblotting with anti-FLAG M2 mAb as described (37Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 39Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar).Expression and Purification of TRAIL—Recombinant soluble TRAIL (amino acids 95–281) was expressed in Escherichia coli by transforming BL-21 cells (Novagen) with a pET15b plasmid (Novagen) containing a partial TRAIL cDNA (40Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1546) Google Scholar). A single clone was isolated and grown to log phase, at which time 1 mm isopropyl 1-thio-β-d-galactopyranoside was added to induce protein expression. After cell growth for an additional 2 h, bacteria were lysed, and the His-tagged protein was purified under native conditions using the QIAexpress system (Qiagen). Briefly, bacteria were suspended in lysis buffer (50 mm NaH2PO4, 300 mm NaCl, and 10 mm imidazole (pH 8.0)) with 100 μg/ml phenylmethylsulfonyl fluoride and 10 μg/ml lysozyme. The lysate was then incubated on ice for 30 min, sonicated, and centrifuged at 10,000 × g for 20 min at 4 °C to pellet cellular debris. Next, the lysate was incubated with nickel-nitrilotriacetic acid resin (Qiagen) for 90 min at 4 °C with gentle shaking. The lysate-nickel-nitrilotriacetic acid mixture was then loaded onto a column and washed twice (50 mm NaH2PO4, 300 mm NaCl, and 20 mm imidazole (pH 8.0)), and His-tagged TRAIL was eluted in 250 mm imidizole. After elution, fractions that contained high concentrations of purified TRAIL (as determined by SDS-PAGE and a protein assay; Bio-Rad) were pooled and stored in aliquots containing 10% glycerol at –80 °C.Induction and Analysis of Apoptosis—All apoptosis assays were performed with TRAIL produced as detailed in the preceding paragraph, except for the transient transfection experiments. In these experiments, transiently transfected MDA-MB-231 breast cancer cells were treated with 50 ng/ml TRAIL (Calbiochem) for 30 min. After treatment, transfected cells were identified by GFP fluorescence (control vector) or indirect immunofluorescence (FLAG-tagged plasmids) with FLAG M2 mAb (Sigma) as detailed previously (41Byun Y. Chen F. Chang R. Trivedi M. Green K.J. Cryns V.L. Cell Death Differ. 2001; 8: 443-450Crossref PubMed Scopus (283) Google Scholar). Apoptotic nuclei (fragmented or condensed) were scored by staining with 10 μg/ml Hoescht 33258 as described previously (37Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 41Byun Y. Chen F. Chang R. Trivedi M. Green K.J. Cryns V.L. Cell Death Differ. 2001; 8: 443-450Crossref PubMed Scopus (283) Google Scholar). For apoptosis assays with parental or stably transfected cancer cells, apoptosis was scored by nuclear morphology (as described above) on the entire cell population. For xenograft tumors, apoptotic nuclei were detected in formalin-fixed, paraffin-embedded tissue by terminal dUTP deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay (In Situ Cell Death Detection kit, TMR Red; Roche Applied Science) according to the manufacturer's instructions. Experiments were performed at least three times; 200 or more cells were scored in each experiment. The statistical significance of results was determined by a two-tailed, paired Student's t test.Cell Proliferation Assay—The in vitro growth of MDA-MB-231 breast cancer cells stably expressing vector, WT αB-crystallin (clones A5 and D1), or mutant 3XSE αB-crystallin (clones A2 and A3) was measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega), a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. 3000 cells in 100 μl of Dulbecco's modified Eagle's medium were plated in each well of a 96-well plate. Cells were maintained at 37 °C for 0, 1, 2, or 3 days, at which time 20 μl of the CellTiter96® Aqueous One Solution were added to each well. The plate was incubated for 3 h at 37 °C, and absorbance was read using a Molecular Devices microplate reader (490 nm). Cell number was expressed as the mean percentage of viable cells present at each time point normalized to t = 0 ± S.E. (n = 3; each performed in triplicate).Immunoblotting—Cell lysates were prepared and analyzed by immunoblotting as described previously (39Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The following antibodies were used for immunoblotting: αB-crystallin, Hsp27, Hsp70, FLIP, DR4, DR5, and DcR2 (Stressgen); tubulin and FLAG (Sigma); caspase-3, caspase-8, HspB2/MKBP, Bax, and Bcl-xL (BD Transduction Laboratories); and FADD and cytochrome c (BD Pharmingen). To examine the proteolytic processing of caspases, lysates were immunoblotted with a caspase-9 polyclonal antibody (Cayman Chemical), a caspase-8 mAb (kindly provided by Marcus E. Peter, University of Chicago), or a caspase-3 mAb (BD Transduction Laboratories).Caspase-3-like Activity Assays—Caspase-3-like activity was measured by release of AFC from a synthetic fluorogenic substrate (DEVD-AFC) according to the manufacturer's instructions (ICN Biomedicals, Enzymes Systems Products) using a fluorometric microplate reader (Molecular Devices) with excitation and emission wavelengths of 400 and 505 nm, respectively.RNAi—pRNAi was constructed using methods similar to those described for the construction of pSUPER (42Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar). Briefly, the H1 RNA promoter was generated by PCR using human genomic DNA and the primers 5′-CCATGGAATTCGAACGCTGACGTC-3′ (sense) and 5′-GCAAGCTTAGATCTGTGGTCTCATACAGAACTTATAAGATTCCC-3′ (antisense). The product was then digested with EcoRI and HindIII and ligated into the pBSKII cloning vector (Stratagene). The final plasmid was verified by DNA sequencing. pRNAi-αB-crystallin was constructed by annealing the following 64-bp oligonucleotides: 5′-gatccccGGACAGGTTCTCTGTCAACttcaagagaGTTGACAGAGAACCTGTCCtttttggaaa-3′ and 5′-agcttttccaaaaaGGACAGGTTCTCTGTCAACtctcttgaaGTTGACAGAGAACCTGTCCggg-3′ (the 19-nucleotide αB-crystallin-specific target sequences are indicated in capital letters). The annealed oligonucleotides were then ligated into the BglII and HindIII cloning sites of pRNAi. pRNAi-αB-crystallin generates a hairpin RNA structure that is cleaved to form the 19-bp specific small interfering RNA sequence to αB-crystallin, 216-GGACAGGTTCTCTGTCAAC-234. The final product was verified by DNA sequencing.To determine the efficiency of pRNAi-αB-crystallin in silencing αB-crystallin expression, MDA-MB-435 breast carcinoma cells were co-transfected with 0.2 μg of pEGFP-N1 and 0.8 μg of pRNAi-αB-crystallin or pRNAi vector using Lipofectamine Plus reagent (Invitrogen). Cells were allowed to recover for 48 h, at which time cells were trypsinized and sorted for GFP expression by fluorescence-activated cell-sorting analysis. GFP-positive cells were collected and analyzed by immunoblotting. For apoptosis assays, MDA-MB 435 cells were co-transfected with pEGFP-N1 and pRNAi-αB-crystallin or pRNAi vector as described above. 48 h later, cells were treated for 6 h with vehicle (phosphate-buffered saline) or 2.5 μg/ml TRAIL, and GFP-positive cells were scored for apoptosis by nuclear morphology as indicated above.Breast Cancer Xenograft Studies—Female 4–5-week-old athymic nude mice were obtained from Harlan Sprague-Dawley. To determine the effects of αB-crystallin on xenograft tumor growth, 2.5 × 106 MDA-MB-231 cells stably expressing vector, WT, or mutant 3XSE αB-crystallin were resuspended in phosphate-buffered saline and implanted subcutaneously into both mammary fat pads (n = 5 mice/group). Tumor size was measured weekly using Vernier calipers. Tumor volume was calculated by the following equation: tumor volume (mm3) = [(length × width2)] × π /6. To examine the effect of αB-crystallin on TRAIL-induced apoptosis in vivo, 1-mm3 blocks of MDA-MB-231 tumors stably expressing vector or WT αB-crystallin were transplanted subcutaneously into the mammary fat pads of female athymic nude mice. 3 weeks after tumor implantation, nude mice were treated with TRAIL (10 mg/kg/day intraperitoneal) for 2 weeks. Apoptosis was scored in xenograft tumors by TUNEL assay (In Situ Cell Death Detection kit, TMR Red; Roche Applied Science) as described under “Induction and Analysis of Apoptosis.” All procedures involving animals were approved by the Animal Care and Use Committee of Northwestern University.RESULTSαB-crystallin Expression Correlates with Resistance to TRAIL-induced Caspase-3 Activation and Apoptosis—A panel of estrogen receptor-positive (MCF-7 and T47D) and estrogen receptor-negative (MDA-MB-231, MDA-MB-435, and MDA-MB-468) human breast carcinoma cell lines was treated with 0–5 μg/ml TRAIL for 24 h, and the percentage of apoptotic cells was determined by nuclear morphology. MDA-MB-231 cells were exquisitely sensitive to TRAIL: >50% of cells were apoptotic after treatment with the lowest concentration of TRAIL tested (200 ng/ml) (Fig. 1A). In contrast, the other four breast cancer cell lines were resistant to TRAIL: <30% of cells were apoptotic when treated with the highest dose (5 μg/ml) of TRAIL (Fig. 1A). In an effort to delineate the mechanism(s) underlying the differential sensitivity of these breast carcinoma cells to TRAIL-induced apoptosis, we examined these cells for the expression of various DISC components (Fig. 1B, left panel) and apoptosis-regulating proteins (Fig. 1B, right panels). Importantly, each of the DISC components and Bcl-2 family members examined was expressed in all of these cells. Intriguingly, MDA-MB-231 cells (which were sensitive to TRAIL) completely lacked the small heat shock protein αB-crystallin, whereas MDA-MB-435 cells (which had intermediate resistance to TRAIL; Fig. 1A) expressed low levels of αB-crystallin, and the three highly TRAIL-resistant cell lines (MDA-MB-468, MCF-7, and T47D) expressed high levels of αB-crystallin. Unlike αB-crystallin, Hsp27 and Hsp70 were expressed in all of these breast cancer cells, thereby underscoring the specificity of this observation. A similar correlation between αB-crystallin expression and TRAIL resistance was noted in human malignant glioma cell lines (Fig. 1C). Gliomas that lacked αB-crystallin (LG11 and D54MG) were sensitive to TRAIL-induced apoptosis, whereas gliomas with low (U87MG and A172) or high levels (U251MG and U373MG) of αB-crystallin expression were partly or completely resistant to TRAIL, respectively. Indeed, a survey of additional human cancer cell lines (carcinomas of the breast, lung, kidney, and pancreas) revealed that 9 of 10 cancer cell lines that expressed αB-crystallin were resistant to TRAIL (data not shown). These findings indicate that αB-crystallin expression is tightly linked to TRAIL resistance in diverse human cancer cell lines.Because we have previously demonstrated that αB-crystallin inhibits apoptosis by disrupting the activation of caspase-3 (37Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 38Kamradt M.C. Chen F. Sam S. Cryns V.L. J. Biol. Chem. 2002; 277: 38731-38736Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), we reasoned that if αB-crystallin were responsible for the observed TRAIL resistance, then TRAIL-ind
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