Resistance of Human Ovarian Cancer Cells to Tumor Necrosis Factor Is a Consequence of Nuclear Factor B-Mediated Induction of Fas-Associated Death Dom
Abstract'k, http://www.100md.com
The purpose of the present studies was to examine the role and regulation of Fas-associated death domain-like IL-1- converting enzyme-like inhibitory protein [FLIP; long (FLIPL) and short (FLIPS) forms] in human ovarian epithelial cancer cells by TNF{alpha} and their significance in the resistance of the cells to the proapoptotic action of the cytokine. OV2008, A2780-s, and OVCAR-3 cells were cultured in serum-free media with or without cycloheximide (CHX, 10 µg/ml) ± TNF{alpha} (5, 10, 20 ng/ml) or transfected with a mammalian expression vector containing either a dominant negative inhibitor {kappa} B (I{kappa} B), FLIPS sense or antisense cDNA and cultured with or without TNF{alpha} . In the presence of CHX, TNF{alpha} increased caspase-8 and -3 cleavage and apoptosis. It also induced I{kappa} B phosphorylation, nuclear factor {kappa} B activation, and the expression of FLIPS but not of FLIPL. Overexpression of dominant negative I{kappa} B attenuated TNF{alpha} -induced FLIPS expression and enhanced TNF{alpha} -induced apoptosis. Apoptosis induced by TNF{alpha} and CHX was facilitated by FLIPS antisense expression but attenuated by sense transfection. This study demonstrates that TNF{alpha} up-regulates FLIPS expression, and this effect is mediated by the activation of nuclear factor {kappa} B. The induction of FLIPS expression by TNF{alpha} might contribute to the resistance of ovarian epithelial cancer cells to the proapoptotic action of the cytokine.
Introductionq5, http://www.100md.com
HUMAN OVARIAN SURFACE epithelial cancer is the leading cause of death from gynecologic malignancy in the Western world. Although clinical and histological prognostic factors are well understood, the biologic process leading to uncontrolled cellular growth is unclear. It is well established that the control of cellular homeostasis involves a balance between cell proliferation and apoptosis and the uncontrolled cell proliferation and/or suppressed apoptosis lead to excessive tissue growth.q5, http://www.100md.com
TNF{alpha} is a pleiotropic cytokine that can induce differentiation, proliferation, and apoptosis in many cell types (1, 2) and has been suggested to play an important role in the biology of ovarian cancer and tumorigenesis. Ovarian tumor cells produce a macrophage colony-stimulating factor, a potent chemoattractant for monocytes that secretes TNF{alpha} . TNF{alpha} concentrations are significantly increased in ovarian cancer patients (3), and the levels of TNF{alpha} expression are positively correlated with tumor grade (4). TNF{alpha} has selective cytolytic activity against some but not all tumor cells (5). The resistance of human epithelial tumor cells to TNF{alpha} appears to be associated with the expression of this cytokine (5, 6, 7, 8) and controlled by a protein synthesis-dependent mechanism (9). However, the intracellular mechanism(s) involved in the resistance of ovarian cancer cells to TNF{alpha} is not clear.
The actions of TNF{alpha} are mediated by its two receptors, TNFR1 and TNFR2 (10, 11, 12). TNFR1 contains an intracellular death domain required for induction of apoptosis and is coupled to a nuclear factor {kappa} B (NF{kappa} B) activation pathway. Binding of TNF{alpha} to its receptors activates caspase-8 and caspase-3 (13, 14, 15, 16) as well as induces I{kappa} B phosphorylation and degradation and activates NF{kappa} B (17, 18, 19, 20, 21, 22). NF{kappa} B activation regulates the expression of a number of genes involved in the modulation of TNF{alpha} -induced apoptosis, including zinc finger protein A20 (23, 24, 25), members of the Bcl-2 family (26), Bcl-2 homolog Bfl-1/A1 (27), inhibitor of apoptosis proteins (IAP) (28, 29), and Fas-associated death domain (FADD)-like IL-1ß-converting enzyme-like inhibitory protein (FLIP) (30, 31, 32).6w-2^, http://www.100md.com
FLIP is a FADD-binding suppressor of apoptosis. FLIP is present in two spliced isoforms, long (FLIPL) and short (FLIPS) (33). Both isoforms contain two death effector domains, a structure resembling the N-terminal half of caspase-8 (34, 35, 36). In addition, FLIPL isoform has an inactive caspase-like domain. FLIP is recruited to the death-inducing signaling complex through the adaptor molecule, FADD, thereby preventing the recruitment of caspase-8 into the complex and subsequent caspase-8 activation and then suppresses apoptosis (33, 35, 37). However, the role of FLIP is controversial in some cell types because its overexpression has been reported to induce apoptosis (36, 38, 39, 40). Although recent data have shown that FLIP plays an important role in TNF{alpha} -induced, NF{kappa} B-mediated antiapoptotic response, the expression and role of the two FLIP splice variants appears to be cell type specific (30, 31). Furthermore, whether FLIP overexpression is related to the resistance of human ovarian epithelial cancer cells to TNF{alpha} is unknown.
In the present studies, we examined the role and regulation of FLIP expression by TNF{alpha} in a human ovarian cancer cell lines in vitro and demonstrated that TNF{alpha} induces NF{kappa} B-mediated FLIPS expression, which protects the cells from cytotoxic action of the cytokine.k63{ie, 百拇医药
Materials and Methodsk63{ie, 百拇医药
Chemicals and reagentsk63{ie, 百拇医药
Agarose, Tris, phenylmethylsulfonyl fluoride (PMSF), and EGTA were from Sigma (St. Louis, MO). Enhanced chemiluminescence Western blotting detection kit and [{gamma} 32P]-dATP (30 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). RPMI 1640, DMEM/F-12 media, and fetal bovine serum (FBS) were from Life Technologies, Inc. (Burlington, Ontario, Canada). Nitrocellulose membrane, acrylamide (electrophoresis grade), N,N’-methylene-bis-acrylamide, ammonium persulfate, dithiothreitol (DTT), glycine, and a protein assay kit were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). X-ray films were from Eastman Kodak Co. (Rochester, NY). Recombinant human TNF{alpha} was from R&D Systems Inc. (Minneapolis, MN). CHX was from BDH Laboratory Supplies (Poole, UK). NF{kappa} B probe and T4 polynucleotide kinase were from Promega Corp. (Madison, WI). Polyclonal rabbit caspase-3 antibody was from PharMingen (Mississauga, Canada). Rabbit polyclonal antihuman phosphorylated and total I{kappa} B-{alpha} antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody recognizing full-length caspase-8, the cleavage intermediates p43 and p41, and the p18 active subunits were generously provided by Dr. M. Peter (German Cancer Research Center, Heidelberg, Germany). Rabbit polyclonal antihuman X-linked IAP (XIAP) antibody was a generous gift from Dr. Eric LaCasse, ApoptoGen Inc. (Ottawa, Canada). Rabbit polyclonal antihuman FLIP antibody was from Alexis Biochemicals (San Diego, CA). The pCMV-I{kappa} B construct containing serine-to-alanine mutation at residue 32 on I{kappa} B{alpha} that cannot be degraded because of mutated phosphorylation sites was from Shrikanth Reddy (M. D. Anderson Cancer Center, Houston, TX).
Cell culture%!/?, http://www.100md.com
Human ovarian epithelial cancer cells were cultured in RPMI 1640 (for OV2008 and OVCAR-3) or DMEM/F-12 (for A2780-s) supplemented with FBS (10%, vol/vol), nonessential amino acids (0.1 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C under 5% CO2 and 95% air. After a 24-h plating period, the culture medium was changed and the treatments were added as described hereafter. At the end of the culture period, cells were trypsinized and aliquoted for the assessment of nuclear morphology and protein extraction. Cell number in each treatment group was determined by hemocytometry. Cell viability was determined by the trypan blue dye exclusion test as previously described (41).%!/?, http://www.100md.com
Preparation of plasmid DNA%!/?, http://www.100md.com
The cDNA fragment encoding the open reading frame of human FLIPS (nucleotides 294–956) was prepared by RT-PCR using a set of primers: 5'-ATGTCTGCTGAAGTCATCCA-3' (294–313) and 5'-CATGGAACAATTTCCAAGAA-3' (937–956). The primers were designed based on the human FLIPS sequences (GenBank accession no. U97075) obtained from the GenBank database and the PCR products were subcloned into pcDNA3.1/CT-GFP-TOPO expression vector (Invitrogen, Carlsbad, CA). The sense and antisense hFLIPS-pcDNA3.1/CT-GFP constructs were verified by automated sequence analysis.
Transient transfection+]?r), http://www.100md.com
OV2008, A2780-s, and OVCAR-3 cells were seeded in 6-well plates (1 x 106 cell/well) and transfected the following day with 4 µg of the vectors pcDNA3.1/CT-GFP, pCMV alone, or pcDNA3.1/CT-GFP containing hFLIPS and pCMV containing mutated I{kappa} B, using the Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with TNF{alpha} (20 ng/ml) for 6 h and then harvested for further analyses. The overall transfection efficiency assessed by the presence of green fluorescent protein (GFP) expression under a fluorescent microscope is about 40–50%.+]?r), http://www.100md.com
Quantitation of FLIP mRNA by semiquantitative RT-PCR+]?r), http://www.100md.com
Total RNA was isolated from cultured cells with TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Two micrograms total RNA were reversely transcribed for cDNA synthesis, using oligo-deoxythymidine as primer. One tenth of the cDNA synthesized was then amplified with the following primers: human FLIPL [forward: 5'-GACAGCTGAGACAACAAGGACC-3' (58–79), reverse: 5'-GTCTCCACAGCTTTTCTGTCCA-3' (624–602)]; human FLIPS [forward: 5'-GAGACCACCCAGAAGGAAAGAG-3' (66–87), reverse: 5'-G GGTCTCCACAGCTTTTCTGTC-3' (537–516)]; ß-actin [forward: 5'-GAAACTACCTTCAACTCCATC-3', reverse: 5'-CGAGGCCAGGATGGAGCCGCC-3']. Human FLIPL and FLIPS PCR cycle conditions were 95 C for 15 min, 94 C for 45 sec, 60 C for 1 min, and 72 C for 1 min for 35 cycles, 72 C for 10 min. Human ß-actin conditions were 95 C for 15 min, 94 C for 45 sec, 55 C for 1 min, and 72 C for 1 min for 25 cycles, 72 C for 10 min. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide. FLIPS mRNA levels were normalized with its respective ß-actin contents.
Protein extraction and Western blot analysis)x9lljp, http://www.100md.com
Cells were sonicated in a lysis buffer (pH 7.4) containing NaCl (150 mM), sodium dodecyl sulfate (0.1%), sodium deoxycholate (0.5%), Nonidet P-40 (1%) in PBS and protease inhibitors [PMSF (1 mM), aprotinin (10 µg/ml), sodium orthovanadate (1 mM)]. The sonicates were pelleted (15,000 x g, 20 min) and supernatant was retained and stored at -20 C. Protein content of the extracts was determined with the DC protein assay reagent (Bio-Rad Laboratories, Inc.). Samples were mixed with loading buffer, resolved by 12% SDS-PAGE and electrotransferred (30 V, overnight) onto nitrocellulose membranes. The total protein on the nitrocellulose membranes was stained with ponceau S solution and scanned. After blocking for 1 h with nonfat milk powder (5%) in Tris-buffered saline [Tris (10 mM), NaCl (150 mM)] and Tween-20 (0.05%; TBS-T), membranes were incubated for 3 h with primary antibodies in TBS-T containing 5% nonfat milk powder, and subsequently with horseradish peroxidase-conjugated secondary antibody (1:5,000 to 10,000) in TBS-T with milk powder (reverse transcription, 45 min). Immunoreactivity was detected by chemiluminescence autoradiography (enhanced chemiluminescence kit) in accordance with the manufacturer’s instructions. The intensity of protein bands of interest was densitometrically determined and normalized by the respective stained total protein.
EMSA*, 百拇医药
Nuclear extracts of OV2008 cells were prepared as previously described (42) with minor modifications. Briefly, 3 x 106 cells were pelleted (200 x g, 5 min) and resuspended in 30 µl buffer A [HEPES (10 mM, pH 7.9), KCl (10 mM), MgCl (1.5 mM), DTT (0.5 mM), PMSF (0.5 mM), Nonidet P-40 (0.67%)]. Cells were allowed to swell (0 C, 15 min), and centrifuged (10,000 x g, 4 C). The supernatant was collected and stored at -80 C. The cell pellet (containing cell nuclei) was resuspended in 30 µl buffer B [HEPES (20 mM, pH 7.9), NaCl (0.4 M), EDTA (0.2 mM), MgCl (1.5 mM), DTT (0.5 mM), PMSF (0.5 mM)] and rocked vigorously (4 C, 15 min). The nuclear extract was centrifuged (10,000 x g, 30 min) and stored at -80 C. Double-stranded DNA oligonucleotides containing consensus sequences (5'-AGTTGAGGGGACTTTCCCAGGC-3') for NF{kappa} B was 32P-labeled with [32P]-ATP and T4 polynucleotide kinase. Nuclear proteins (8 µg) were incubated with radiolabeled DNA probes (reverse transcription, 20 min) in the binding buffer (20 mM HEPES, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5% glycerol, 2 mM DTT, pH 7.9). Nuclear acid-protein complexes were resolved on a native 5% polyacrylamide gel in Tris-buffered EDTA (1x, pH 8.0) and detected by autoradiography.
Assessment of apoptosiskn, http://www.100md.com
Cells were fixed in 4% neutral buffered formalin and then resuspended in Hoechst 33248 staining solution (0.1 µg/ml, overnight), as previously described (43, 44). Cells with typical apoptotic nuclear morphology were identified and counted.kn, http://www.100md.com
Statistical analyseskn, http://www.100md.com
Results are expressed as the mean ± SEM of three independent experiments. Statistical analyses were carried out by one- or two-way ANOVA. Significant differences between treatment groups were determined by the Tukey’s test. Statistical significance was inferred at P < 0.05.kn, http://www.100md.com
Resultskn, http://www.100md.com
TNF induced apoptosis in the presence of CHXkn, http://www.100md.com
OV2008 cells were cultured in serum-free RPMI medium for 0, 3, and 6 h in the absence or presence of TNF (20 ng/ml), the protein synthesis inhibitor CHX (10 µg/ml), or CHX (10 µg/ml) plus TNF (20 ng/ml). Neither TNF nor CHX alone could induce cell death, but in the presence of CHX, TNF significantly increased the number of apoptotic cells in a time-dependent manner (P < 0.001; Fig. 1A). The proapoptotic effects of TNF{alpha} during a 6-h culture in the presence of CHX were concentration dependent (P < 0.001), and a significant increase in apoptosis was evident at concentrations as low as 5 ng/ml (P < 0.001; Fig. 1B). To examine whether the above proapoptotic effects of TNF were specific to the OV2008 cell line, twoadditional ovarian epithelial cancer cell lines (A2780-s and OVCAR-3) were treated with TNF{alpha} in the absence or presence of CHX for 6 or 12 h. In the presence of CHX, TNF significantly induced apoptosis in all three cell lines (Fig. 2, A–C), although the response of OVCAR-3 cells to TNF was much lower than those of OV2008 and A2780-s. Nonetheless, these findings suggest that a cell survival factor(s) might have been induced by TNF{alpha} and suppressed the apoptotic process elicited by the cytokine.
fig.ommitteed:xcf#ch, 百拇医药
Figure 1. Time- and dose-dependent effect of TNF and cycloheximide (CHX) on apoptosis in OV2008. Cells were cultured (A) in RPMI alone (CTL) or in the presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX + TNF for 0, 3, or 6 h or (B) in the presence of TNF (5, 10, 20 ng/ml) or TNF+CHX for 6 h. The cells were collected for apoptosis analysis. Mean ± SEM (n = 3).:xcf#ch, 百拇医药
fig.ommitteed:xcf#ch, 百拇医药
Figure 2. CHX decreased TNF{alpha} -induced FLIPS content and induced apoptosis in ovarian epithelial cancer cells. OV2008 (A and E), A2780s (B and F), and OVCAR-3 (C and G) cells were cultured in media alone (CTL) or in the presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX + TNF{alpha} for 6 or 12 h. The cells were collected for apoptosis (A–C) and Western blot (D–G) analyses. D, Representative images of three replicate experiments of each cell line. The images for each experiment were scanned, quantified, and normalized by respective stained total protein (E–G). Mean ± SEM (n = 3).:xcf#ch, 百拇医药
TNF{alpha} induced the cleavages of caspase-8, caspase-3, and XIAP in the presence of CHX
To identify the potential survival factor(s) induced by TNF{alpha} in the ovarian cancer cells and determine where on the TNF{alpha} death pathway this factor could be acting, we examined by Western analysis the influence of the cytokine on the cleavage of procaspase-8 and procaspase-3 as well as XIAP in the absence and presence of CHX in vitro. Neither TNF{alpha} (20 ng/ml) nor CHX (10 µg/ml) alone had any apparent effects on the processing of these intracellular proteins. However, in the presence of CHX, TNF{alpha} induced cleavage of the caspases and XIAP, a phenomenon suppressed by the presence of caspase inhibitors [ZVAD (50 µM) and DEVD (20 µM); Fig. 3]. Moreover, TNF{alpha} alone had no effect on XIAP content in OV2008 cells. These observations suggest that, in the presence but not absence of the protein synthesis inhibitor, TNF{alpha} is capable of activating caspase-8 and -3 and inducing XIAP cleavage during the induction of apoptosis. Moreover, the putative survival factor is likely acting upstream of caspase-8.o9, 百拇医药
fig.ommitteedo9, 百拇医药
Figure 3. Effect of TNF and CHX on caspase-8, -3, and XIAP cleavages in OV2008. Cells were pretreated with or without either ZVAD (50 µM) or DEVD (20 µM) for 2 h and then cultured in RPMI alone (CTL) or in the presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX + TNF for 6 h. The cells were collected for Western blot analysis of caspase-8, -3, and XIAP. The panels show representative images of three repeated experiments.(Chao Wu Xiao Xiaojuan Yan Yulian Li Shrikanth A. G. Reddy and Benjamin K. Tsang)
The purpose of the present studies was to examine the role and regulation of Fas-associated death domain-like IL-1- converting enzyme-like inhibitory protein [FLIP; long (FLIPL) and short (FLIPS) forms] in human ovarian epithelial cancer cells by TNF{alpha} and their significance in the resistance of the cells to the proapoptotic action of the cytokine. OV2008, A2780-s, and OVCAR-3 cells were cultured in serum-free media with or without cycloheximide (CHX, 10 µg/ml) ± TNF{alpha} (5, 10, 20 ng/ml) or transfected with a mammalian expression vector containing either a dominant negative inhibitor {kappa} B (I{kappa} B), FLIPS sense or antisense cDNA and cultured with or without TNF{alpha} . In the presence of CHX, TNF{alpha} increased caspase-8 and -3 cleavage and apoptosis. It also induced I{kappa} B phosphorylation, nuclear factor {kappa} B activation, and the expression of FLIPS but not of FLIPL. Overexpression of dominant negative I{kappa} B attenuated TNF{alpha} -induced FLIPS expression and enhanced TNF{alpha} -induced apoptosis. Apoptosis induced by TNF{alpha} and CHX was facilitated by FLIPS antisense expression but attenuated by sense transfection. This study demonstrates that TNF{alpha} up-regulates FLIPS expression, and this effect is mediated by the activation of nuclear factor {kappa} B. The induction of FLIPS expression by TNF{alpha} might contribute to the resistance of ovarian epithelial cancer cells to the proapoptotic action of the cytokine.
Introductionq5, http://www.100md.com
HUMAN OVARIAN SURFACE epithelial cancer is the leading cause of death from gynecologic malignancy in the Western world. Although clinical and histological prognostic factors are well understood, the biologic process leading to uncontrolled cellular growth is unclear. It is well established that the control of cellular homeostasis involves a balance between cell proliferation and apoptosis and the uncontrolled cell proliferation and/or suppressed apoptosis lead to excessive tissue growth.q5, http://www.100md.com
TNF{alpha} is a pleiotropic cytokine that can induce differentiation, proliferation, and apoptosis in many cell types (1, 2) and has been suggested to play an important role in the biology of ovarian cancer and tumorigenesis. Ovarian tumor cells produce a macrophage colony-stimulating factor, a potent chemoattractant for monocytes that secretes TNF{alpha} . TNF{alpha} concentrations are significantly increased in ovarian cancer patients (3), and the levels of TNF{alpha} expression are positively correlated with tumor grade (4). TNF{alpha} has selective cytolytic activity against some but not all tumor cells (5). The resistance of human epithelial tumor cells to TNF{alpha} appears to be associated with the expression of this cytokine (5, 6, 7, 8) and controlled by a protein synthesis-dependent mechanism (9). However, the intracellular mechanism(s) involved in the resistance of ovarian cancer cells to TNF{alpha} is not clear.
The actions of TNF{alpha} are mediated by its two receptors, TNFR1 and TNFR2 (10, 11, 12). TNFR1 contains an intracellular death domain required for induction of apoptosis and is coupled to a nuclear factor {kappa} B (NF{kappa} B) activation pathway. Binding of TNF{alpha} to its receptors activates caspase-8 and caspase-3 (13, 14, 15, 16) as well as induces I{kappa} B phosphorylation and degradation and activates NF{kappa} B (17, 18, 19, 20, 21, 22). NF{kappa} B activation regulates the expression of a number of genes involved in the modulation of TNF{alpha} -induced apoptosis, including zinc finger protein A20 (23, 24, 25), members of the Bcl-2 family (26), Bcl-2 homolog Bfl-1/A1 (27), inhibitor of apoptosis proteins (IAP) (28, 29), and Fas-associated death domain (FADD)-like IL-1ß-converting enzyme-like inhibitory protein (FLIP) (30, 31, 32).6w-2^, http://www.100md.com
FLIP is a FADD-binding suppressor of apoptosis. FLIP is present in two spliced isoforms, long (FLIPL) and short (FLIPS) (33). Both isoforms contain two death effector domains, a structure resembling the N-terminal half of caspase-8 (34, 35, 36). In addition, FLIPL isoform has an inactive caspase-like domain. FLIP is recruited to the death-inducing signaling complex through the adaptor molecule, FADD, thereby preventing the recruitment of caspase-8 into the complex and subsequent caspase-8 activation and then suppresses apoptosis (33, 35, 37). However, the role of FLIP is controversial in some cell types because its overexpression has been reported to induce apoptosis (36, 38, 39, 40). Although recent data have shown that FLIP plays an important role in TNF{alpha} -induced, NF{kappa} B-mediated antiapoptotic response, the expression and role of the two FLIP splice variants appears to be cell type specific (30, 31). Furthermore, whether FLIP overexpression is related to the resistance of human ovarian epithelial cancer cells to TNF{alpha} is unknown.
In the present studies, we examined the role and regulation of FLIP expression by TNF{alpha} in a human ovarian cancer cell lines in vitro and demonstrated that TNF{alpha} induces NF{kappa} B-mediated FLIPS expression, which protects the cells from cytotoxic action of the cytokine.k63{ie, 百拇医药
Materials and Methodsk63{ie, 百拇医药
Chemicals and reagentsk63{ie, 百拇医药
Agarose, Tris, phenylmethylsulfonyl fluoride (PMSF), and EGTA were from Sigma (St. Louis, MO). Enhanced chemiluminescence Western blotting detection kit and [{gamma} 32P]-dATP (30 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). RPMI 1640, DMEM/F-12 media, and fetal bovine serum (FBS) were from Life Technologies, Inc. (Burlington, Ontario, Canada). Nitrocellulose membrane, acrylamide (electrophoresis grade), N,N’-methylene-bis-acrylamide, ammonium persulfate, dithiothreitol (DTT), glycine, and a protein assay kit were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). X-ray films were from Eastman Kodak Co. (Rochester, NY). Recombinant human TNF{alpha} was from R&D Systems Inc. (Minneapolis, MN). CHX was from BDH Laboratory Supplies (Poole, UK). NF{kappa} B probe and T4 polynucleotide kinase were from Promega Corp. (Madison, WI). Polyclonal rabbit caspase-3 antibody was from PharMingen (Mississauga, Canada). Rabbit polyclonal antihuman phosphorylated and total I{kappa} B-{alpha} antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody recognizing full-length caspase-8, the cleavage intermediates p43 and p41, and the p18 active subunits were generously provided by Dr. M. Peter (German Cancer Research Center, Heidelberg, Germany). Rabbit polyclonal antihuman X-linked IAP (XIAP) antibody was a generous gift from Dr. Eric LaCasse, ApoptoGen Inc. (Ottawa, Canada). Rabbit polyclonal antihuman FLIP antibody was from Alexis Biochemicals (San Diego, CA). The pCMV-I{kappa} B construct containing serine-to-alanine mutation at residue 32 on I{kappa} B{alpha} that cannot be degraded because of mutated phosphorylation sites was from Shrikanth Reddy (M. D. Anderson Cancer Center, Houston, TX).
Cell culture%!/?, http://www.100md.com
Human ovarian epithelial cancer cells were cultured in RPMI 1640 (for OV2008 and OVCAR-3) or DMEM/F-12 (for A2780-s) supplemented with FBS (10%, vol/vol), nonessential amino acids (0.1 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C under 5% CO2 and 95% air. After a 24-h plating period, the culture medium was changed and the treatments were added as described hereafter. At the end of the culture period, cells were trypsinized and aliquoted for the assessment of nuclear morphology and protein extraction. Cell number in each treatment group was determined by hemocytometry. Cell viability was determined by the trypan blue dye exclusion test as previously described (41).%!/?, http://www.100md.com
Preparation of plasmid DNA%!/?, http://www.100md.com
The cDNA fragment encoding the open reading frame of human FLIPS (nucleotides 294–956) was prepared by RT-PCR using a set of primers: 5'-ATGTCTGCTGAAGTCATCCA-3' (294–313) and 5'-CATGGAACAATTTCCAAGAA-3' (937–956). The primers were designed based on the human FLIPS sequences (GenBank accession no. U97075) obtained from the GenBank database and the PCR products were subcloned into pcDNA3.1/CT-GFP-TOPO expression vector (Invitrogen, Carlsbad, CA). The sense and antisense hFLIPS-pcDNA3.1/CT-GFP constructs were verified by automated sequence analysis.
Transient transfection+]?r), http://www.100md.com
OV2008, A2780-s, and OVCAR-3 cells were seeded in 6-well plates (1 x 106 cell/well) and transfected the following day with 4 µg of the vectors pcDNA3.1/CT-GFP, pCMV alone, or pcDNA3.1/CT-GFP containing hFLIPS and pCMV containing mutated I{kappa} B, using the Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with TNF{alpha} (20 ng/ml) for 6 h and then harvested for further analyses. The overall transfection efficiency assessed by the presence of green fluorescent protein (GFP) expression under a fluorescent microscope is about 40–50%.+]?r), http://www.100md.com
Quantitation of FLIP mRNA by semiquantitative RT-PCR+]?r), http://www.100md.com
Total RNA was isolated from cultured cells with TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Two micrograms total RNA were reversely transcribed for cDNA synthesis, using oligo-deoxythymidine as primer. One tenth of the cDNA synthesized was then amplified with the following primers: human FLIPL [forward: 5'-GACAGCTGAGACAACAAGGACC-3' (58–79), reverse: 5'-GTCTCCACAGCTTTTCTGTCCA-3' (624–602)]; human FLIPS [forward: 5'-GAGACCACCCAGAAGGAAAGAG-3' (66–87), reverse: 5'-G GGTCTCCACAGCTTTTCTGTC-3' (537–516)]; ß-actin [forward: 5'-GAAACTACCTTCAACTCCATC-3', reverse: 5'-CGAGGCCAGGATGGAGCCGCC-3']. Human FLIPL and FLIPS PCR cycle conditions were 95 C for 15 min, 94 C for 45 sec, 60 C for 1 min, and 72 C for 1 min for 35 cycles, 72 C for 10 min. Human ß-actin conditions were 95 C for 15 min, 94 C for 45 sec, 55 C for 1 min, and 72 C for 1 min for 25 cycles, 72 C for 10 min. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide. FLIPS mRNA levels were normalized with its respective ß-actin contents.
Protein extraction and Western blot analysis)x9lljp, http://www.100md.com
Cells were sonicated in a lysis buffer (pH 7.4) containing NaCl (150 mM), sodium dodecyl sulfate (0.1%), sodium deoxycholate (0.5%), Nonidet P-40 (1%) in PBS and protease inhibitors [PMSF (1 mM), aprotinin (10 µg/ml), sodium orthovanadate (1 mM)]. The sonicates were pelleted (15,000 x g, 20 min) and supernatant was retained and stored at -20 C. Protein content of the extracts was determined with the DC protein assay reagent (Bio-Rad Laboratories, Inc.). Samples were mixed with loading buffer, resolved by 12% SDS-PAGE and electrotransferred (30 V, overnight) onto nitrocellulose membranes. The total protein on the nitrocellulose membranes was stained with ponceau S solution and scanned. After blocking for 1 h with nonfat milk powder (5%) in Tris-buffered saline [Tris (10 mM), NaCl (150 mM)] and Tween-20 (0.05%; TBS-T), membranes were incubated for 3 h with primary antibodies in TBS-T containing 5% nonfat milk powder, and subsequently with horseradish peroxidase-conjugated secondary antibody (1:5,000 to 10,000) in TBS-T with milk powder (reverse transcription, 45 min). Immunoreactivity was detected by chemiluminescence autoradiography (enhanced chemiluminescence kit) in accordance with the manufacturer’s instructions. The intensity of protein bands of interest was densitometrically determined and normalized by the respective stained total protein.
EMSA*, 百拇医药
Nuclear extracts of OV2008 cells were prepared as previously described (42) with minor modifications. Briefly, 3 x 106 cells were pelleted (200 x g, 5 min) and resuspended in 30 µl buffer A [HEPES (10 mM, pH 7.9), KCl (10 mM), MgCl (1.5 mM), DTT (0.5 mM), PMSF (0.5 mM), Nonidet P-40 (0.67%)]. Cells were allowed to swell (0 C, 15 min), and centrifuged (10,000 x g, 4 C). The supernatant was collected and stored at -80 C. The cell pellet (containing cell nuclei) was resuspended in 30 µl buffer B [HEPES (20 mM, pH 7.9), NaCl (0.4 M), EDTA (0.2 mM), MgCl (1.5 mM), DTT (0.5 mM), PMSF (0.5 mM)] and rocked vigorously (4 C, 15 min). The nuclear extract was centrifuged (10,000 x g, 30 min) and stored at -80 C. Double-stranded DNA oligonucleotides containing consensus sequences (5'-AGTTGAGGGGACTTTCCCAGGC-3') for NF{kappa} B was 32P-labeled with [32P]-ATP and T4 polynucleotide kinase. Nuclear proteins (8 µg) were incubated with radiolabeled DNA probes (reverse transcription, 20 min) in the binding buffer (20 mM HEPES, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5% glycerol, 2 mM DTT, pH 7.9). Nuclear acid-protein complexes were resolved on a native 5% polyacrylamide gel in Tris-buffered EDTA (1x, pH 8.0) and detected by autoradiography.
Assessment of apoptosiskn, http://www.100md.com
Cells were fixed in 4% neutral buffered formalin and then resuspended in Hoechst 33248 staining solution (0.1 µg/ml, overnight), as previously described (43, 44). Cells with typical apoptotic nuclear morphology were identified and counted.kn, http://www.100md.com
Statistical analyseskn, http://www.100md.com
Results are expressed as the mean ± SEM of three independent experiments. Statistical analyses were carried out by one- or two-way ANOVA. Significant differences between treatment groups were determined by the Tukey’s test. Statistical significance was inferred at P < 0.05.kn, http://www.100md.com
Resultskn, http://www.100md.com
TNF induced apoptosis in the presence of CHXkn, http://www.100md.com
OV2008 cells were cultured in serum-free RPMI medium for 0, 3, and 6 h in the absence or presence of TNF (20 ng/ml), the protein synthesis inhibitor CHX (10 µg/ml), or CHX (10 µg/ml) plus TNF (20 ng/ml). Neither TNF nor CHX alone could induce cell death, but in the presence of CHX, TNF significantly increased the number of apoptotic cells in a time-dependent manner (P < 0.001; Fig. 1A). The proapoptotic effects of TNF{alpha} during a 6-h culture in the presence of CHX were concentration dependent (P < 0.001), and a significant increase in apoptosis was evident at concentrations as low as 5 ng/ml (P < 0.001; Fig. 1B). To examine whether the above proapoptotic effects of TNF were specific to the OV2008 cell line, twoadditional ovarian epithelial cancer cell lines (A2780-s and OVCAR-3) were treated with TNF{alpha} in the absence or presence of CHX for 6 or 12 h. In the presence of CHX, TNF significantly induced apoptosis in all three cell lines (Fig. 2, A–C), although the response of OVCAR-3 cells to TNF was much lower than those of OV2008 and A2780-s. Nonetheless, these findings suggest that a cell survival factor(s) might have been induced by TNF{alpha} and suppressed the apoptotic process elicited by the cytokine.
fig.ommitteed:xcf#ch, 百拇医药
Figure 1. Time- and dose-dependent effect of TNF and cycloheximide (CHX) on apoptosis in OV2008. Cells were cultured (A) in RPMI alone (CTL) or in the presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX + TNF for 0, 3, or 6 h or (B) in the presence of TNF (5, 10, 20 ng/ml) or TNF+CHX for 6 h. The cells were collected for apoptosis analysis. Mean ± SEM (n = 3).:xcf#ch, 百拇医药
fig.ommitteed:xcf#ch, 百拇医药
Figure 2. CHX decreased TNF{alpha} -induced FLIPS content and induced apoptosis in ovarian epithelial cancer cells. OV2008 (A and E), A2780s (B and F), and OVCAR-3 (C and G) cells were cultured in media alone (CTL) or in the presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX + TNF{alpha} for 6 or 12 h. The cells were collected for apoptosis (A–C) and Western blot (D–G) analyses. D, Representative images of three replicate experiments of each cell line. The images for each experiment were scanned, quantified, and normalized by respective stained total protein (E–G). Mean ± SEM (n = 3).:xcf#ch, 百拇医药
TNF{alpha} induced the cleavages of caspase-8, caspase-3, and XIAP in the presence of CHX
To identify the potential survival factor(s) induced by TNF{alpha} in the ovarian cancer cells and determine where on the TNF{alpha} death pathway this factor could be acting, we examined by Western analysis the influence of the cytokine on the cleavage of procaspase-8 and procaspase-3 as well as XIAP in the absence and presence of CHX in vitro. Neither TNF{alpha} (20 ng/ml) nor CHX (10 µg/ml) alone had any apparent effects on the processing of these intracellular proteins. However, in the presence of CHX, TNF{alpha} induced cleavage of the caspases and XIAP, a phenomenon suppressed by the presence of caspase inhibitors [ZVAD (50 µM) and DEVD (20 µM); Fig. 3]. Moreover, TNF{alpha} alone had no effect on XIAP content in OV2008 cells. These observations suggest that, in the presence but not absence of the protein synthesis inhibitor, TNF{alpha} is capable of activating caspase-8 and -3 and inducing XIAP cleavage during the induction of apoptosis. Moreover, the putative survival factor is likely acting upstream of caspase-8.o9, 百拇医药
fig.ommitteedo9, 百拇医药
Figure 3. Effect of TNF and CHX on caspase-8, -3, and XIAP cleavages in OV2008. Cells were pretreated with or without either ZVAD (50 µM) or DEVD (20 µM) for 2 h and then cultured in RPMI alone (CTL) or in the presence of TNF (20 ng/ml), CHX (10 µg/ml), or CHX + TNF for 6 h. The cells were collected for Western blot analysis of caspase-8, -3, and XIAP. The panels show representative images of three repeated experiments.(Chao Wu Xiao Xiaojuan Yan Yulian Li Shrikanth A. G. Reddy and Benjamin K. Tsang)