Uterine Leiomyosarcoma Tumorigenesis in Lmp2-deficient Mice: Involvement of Impaired Anti-oncogenic Factor IRF1

Materials and Methods

Chemicals. The following reagents were purchased from Gibco/BRL Life Technologies (Carlsbad, CA, USA): Hank's balanced salt solution (HBSS) with and without Mg²⁺ or Ca²⁺, Dulbecco's modified Eagle's medium: F12 nutrient mixture (DMEM:F12), antibiotics and FBS. NG-Mono-methyl-L-arginine (L-NMMA), Nw-nitro-L-arginine methyl ester (L-NAME), trypsin (type II), DNase (type IV), and mouse recombinant IFN-γ were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). PromoCell Smooth Muscle Cell Growth Medium 2 and supplement solution (0.5 ng/ml epidermal growth factor, 2 ng/ml basic fibroblast growth factor, 5 μg/ml insulin) as the smooth muscle cell medium were purchased from PromoCell, Heidelberg, Germany. Mouse recombinant TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). Collagenase (type IV) was purchased from Life Technologies (Carlsbad, CA, USA). Monoclonal antibodies against STAT1, IRF1, IRF2, IκBα E2F1, DP1, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against LMP2, LMP7, LMP3, and LMP10 were purchased from Sigma-Aldrich Co. Antibodies to CDK2, CDK4, CDK6, RB, and cyclin D were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Animals and cells. C57BL/6 mice and BALB/c Nude (nu/nu) mice were purchased from CLEA Japan, Inc. (Meguro, Tokyo, Japan). Trp53-deficient mice (B6.129S2-Trp53^tm1Tyj/J) were purchased from Charles River Laboratories International, Inc. (Wilmington MA, USA). Lmp2-deficient mice were a generous donation from Dr. Susumu Tonegawa (Massachusetts Institute of Technology, Cambridge, MA, USA). All wild-type, homozygous mice deficient in Lmp2 were littermates of F10 heterozygous mice generated by backcrossing with C57BL/6J mice.

The same 2 original male Trp53^−/− mice were bred with 2 female Lmp2^−/− mice, to generate F1 Lmp2^+/−Trp53^+/− mice. F1 mice were bred with each other, and the resulting Lmp2^−/−Trp53^−/− males were bred with Lmp2^−/−Trp53^+/− or Lmp2^−/−Trp53^−/− females to generate the Lmp2^−/−Trp53^−/− mice used in this study. Occasionally, Lmp2^+/− mice were included in breeding, to maintain the colony. Every mouse was screened by genomic PCR, to determine its genotype.

Genomic PCR. A 1-cm sample from the tip of each tail was digested in 0.5 ml of autoclaved digestion buffer [50 mM Tris, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100 (pH 8.0)] with 0.2 mg/ml proteinase K (Life Technologies, Grand Island, NY, USA) at 55°C for 16 h. The DNA sample was then boiled for 5 min and diluted 1:5 in sterile water. PCR reaction mixture was combined with 2.5 μl of Ex Taq PCR buffer (Takara Bio Inc., Otsu, Shiga, Japan), 1.0 μl of 50 mM MgCl₂, 1.0 μl of each 100 mM dNTP, 0.5 μl of Ex Taq DNA polymerase (all from Takara Bio Inc., Otsu, Shiga, Japan), 1.0 μl of each 25 mM primer and water to achieve a final volume of 25 μl. PCR samples were incubated in a Mastercycler (Eppendorf AG, Barkhausenweg, Hamburg, Germany) at 94°C for 1 min, followed by 25 of the following cycles: 30 sec at 94°C, 30 sec at 60°C and 1.5 min at 72°C. After these cycles, samples were incubated at 72°C for 5 min. PCR products were electrophoresed at 90 V in 1.5% agarose gels with 0.5% Tris-Acetate-EDTA buffer solution (TAE). Primers were as follows: wild-type Trp53 5’-GTGTTTCATTAGTTCCCCACCTTGAC-3’, 5’-ATGGGAGGCTGCCAGTCCTAACCC-3’; mutant Trp53 5’-GTGGGAGGGACAAAAGTTCG AGGCC-3’, 5’-TT TACGGAGCCCTGGCGCTCGATGT-3’, wild-type Lmp2 5’-GGGTGTCAGCGGGGTGA GTAG-3’, 5’-CAT GAATGCCATGGATTCAG-3’.

All animal-related procedures were performed in compliance with Guide for the animal care, which is prescribed by The Japanese association of Laboratory Animal Facilities of National University Corporation the National Institute of Health Guide for the Care. These mice were kept in a specific pathogen-free environment at Shinshu University's animal facilities, in accordance with institutional guidelines (approval no. 03–28–008).

Fresh mouse embryonic fibroblasts (MEFs) were obtained from C57BL/6 or Lmp2-deficient embryos at 13.5 days by standard methods. Stat1-deficient MEFs were a generous donation from Dr. David E Levy (New York University, New York, NY, USA). Ifn-γR-deficient MEFs were a generous donation from Dr. Tagawa (The University of Tokyo, Minato-ku, Tokyo, Japan). Cytosolic and nuclear extracts were prepared from 1×10⁷ cells and treated with or without 250 U/ml of mouse IFN-γ (Pepro Tech, Inc., Rocky Hill, NJ, USA) for 1 to 2 h for the periods indicated in each figure, essentially as described previously. (14). The cells were collected by centrifuging for 10 min at 1200 r.p.m., 130 × g washed in 5 ml of ice-cold PBS, and centrifuged again for 5 min at 12000 r.p.m. 13,000 × g at 4°C. The cells were pelleted and washed once in 400 μl of buffer A [10mM HEPES, pH 7.8; 10 mM KCl; 2 mM MgCl₂; 1 mM DTT; 0.1 mM EDTA; complete protease inhibitor cocktail (Hoffmann-La Roche Ltd., Basel, Switzerland)] and were vigorously mixed for 1 h at 4°C and centrifuged for 5 min at 12,000 r.p.m. 13,000 × g. The supernatant was collected as cytosolic extract and stored at −80°C. Pelleted nuclei were resuspended in 40 μl of buffer C (50 mM HEPES, pH 7.8; 50 mM KCl; 300 mM NaCl; 0.1 mM EDTA; 1 mM DTT; 10% (v/v) glycerol), mixed for 2 h at 4°C, and centrifuged for 5 min at 12000 r.p.m. 13000 × g at 4°C. The supernatant containing the nuclear proteins was harvested and stored at −80°C.

Cell culture. The myometria of eight 10-week-old Lmp2-deficient mice were removed, cleaned of fat and blood vessels, and rinsed thoroughly in HBSS containing 1% antibiotics (penicillin and streptomycin) and 20 mM HEPES. The mouse Ut-LMS tissues of eight 8-month-old Lmp2-deficient mice were removed, cleaned of fat and blood vessels, and rinsed thoroughly in HBSS containing 1% antibiotics (penicillin and streptomycin) and 20 mM HEPES. We scraped off the endometrial tissue, and the remaining myometrial tissues or mUt-LMS tissues were subjected to the enzymatic dispersion of cells. Briefly, we washed the tissues three times with HBSS and incubated them in enzyme solution in HBSS containing collagenase (0.1%), DNase I (0.02%), protease (0.01%), and trypsin (0.01%) for 10 min at 37°C with shaking. The supernatant was discarded, replaced with fresh enzyme solution, and incubated for another 10 min. During this incubation period, the tissue pieces were gently broken up by drawing the mixture slowly through a large-bore siliconized pipette. This procedure was continued for 30 s every 3 min. The mixture was then centrifuged at 20 × g for 5 min and supernatant was removed and saved. Fresh enzyme solution was added to the remaining pieces, and the above process was repeated three times. After enzyme digestion, the combined supernatant solution containing cells was filtered through a 100-mm nylon mesh and centrifuged at 430 × g for 10 min. The cell pellet was washed in 1:1 DMEM:F12 nutrient medium (GIBCO, Carlsbad, CA, USA) containing 10% FCS, 1% antibiotics, and sodium bicarbonate. These cells were resuspended in culture medium, PromoCell Smooth Muscle Cell Growth Medium 2 (PromoCell) with FCS (final concentration, 5%) plus SmGM-2 SingleQuots (0.5 ng/ml epidermal growth factor, 2 ng/ml basic fibroblast growth factor, 5 μg/ml insulin; CAMBREX, MD, USA), and plated on 10-cm dishes. We removed the medium 24 h later, and the myometrial cells were washed and incubated in fresh medium containing 0.1% BSA and 1% antibiotic solution. We identified 95% of the cells in culture as smooth muscle cells by the detection of α-smooth muscle actin (α-SMA) as a smooth muscle cell marker using immunohistochemical methods.

Isolation of mouse embryonic fibroblasts (MEFs). The following two steps were performed under aseptic conditions. Pregnant mice were anesthetized by the intraperitoneal administration of a mixture of xylazine (10 mg/kg) and ketamine (70 mg/kg), and were sacrificed at 13 or 14 day post-coitum. The uterine horns were quickly dissected, rinsed in 70% (v/v) ethanol, and placed into a falcon tube containing PBS without Ca²⁺Mg²⁺ (Gibco). The following steps were carried out in a tissue culture hood under aseptic conditions using sterile instruments. Each embryo was separated from its placenta and embryonic sac, and the head and red organ were dissected. The tissue was finely minced using a sterile razor blade until it could be pipetted. One milliliter of solution S [0.05% trypsin/EDTA (Gibco, Invitrogen), including 100 units Kunitz units of DNase I], was added per embryo, which were then incubated for 15 min at 37°C. To inactivate trypsin, one volume of MEF culture medium (components to make 500 ml of media, all components mixed and filtered): 450 ml of DMEM, 50 ml of FBS (10% v/v), 5 ml of 200 mM L-glutamine (1/100 v/v), and 5 ml of penicillin-streptomycin (1/100 v/v) were added to the cell suspending solution. The cells were centrifuged at a low speed (300 × g) for 5 min, and the supernatant was then carefully removed. MEFs were placed in flasks. Ideally, cells were 80-90% confluent after 48 h and, at this stage, the majority of P0 cells were frozen for future usage.

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Figure 1.

The p53 protein is crucial in multi-cellular organisms, in which it regulates the cell cycle and, thus, functions as a tumor suppressor, preventing cancer. More than 50% of human tumors contain a mutation or deletion in the TP53 gene. To increase the tumor incidence and better-assess the role of the systemic expression of Trp53 in response to initiation of mouse Ut-LMS tumorigenesis, Lmp2-deficient mice were bred with Trp53-deficient mice to create Lmp2^−/−Trp53^−/− mice and closely matched control Lmp2^−/−Trp53^+/+ mice. However, no significant differences were observed in the tumor incidence between these three genetically-modified mouse groups.

Treatment with proteasome inhibitors. Influence of proteasome inhibitors on the expression of IRF1 and IRF2 were examined with MEFs treated with proteasome inhibitors. MG 115 (N-Cbz-Leu-Leu-Nva-CHO) and MG 132 (N-Cbz-Leu-Leu-Nle-CHO) were purchased from Calbiochem (San Diego, CA, USA). MG 115 or MG 132 were added in the culture media at the concentration of 50 mM and incubated with MEFs for 6 h, and then incubated further with 5 mM MG 115 or MG 132 for another 18 h at 37°C. After the 24 h treatment with MG115 or MG132, MEFs were stimulated with TNFα (20 ng/ml) or IFN-γ (250 U/ml) for varying period indicated in Figure 5, and then the expression of appropriated proteins were analyzed by western blotting experiments.

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Figure 2.

Defective IRF1 expression in the IFN-γ signal cascade in mouse embryonic fibroblasts (MEFs) derived from IFN-γ receptor-deficient mice. A: Key role of the IFN-γ signaling pathway on LMP2 expression in human myometrium. After the binding of IFN-γ to the type II IFN receptor, Janus activated kinase 1 (JAK1) and JAK2 are activated and phosphorylate signal transducer and activator of transcription 1 (STAT1) on the tyrosine residue at position 701 (Tyr701). The tyrosine-phosphorylated form of STAT1 forms homodimers that translocate to the nucleus and bind to IFN-γ-activated site (GAS) elements that are present in the promoters of IFN-γ-regulated genes. The phosphorylation of Ser727 is not essential for the translocation of STAT1 to the nucleus or for the binding of STAT1 to DNA, but is required for full transcriptional activation. IfnγR1, Ifn-γ receptor subunit 1; IfnγR2, Ifn-γ receptor subunit 2. B: Defect in IFN-γ-induced IRF1 expression in MEFs, which were isolated from homozygous or heterozygous mice deficient in IfnγR, and wild-type mice. Cytosolic extracts were prepared from MEFs treated with IFN-γ (250 U/ml) for the individual times indicated, and 50 μg of cytosolic extracts was resolved using a 10% sodium dodecyl sulfate-polyacrylamide gel. The expression levels of p-STAT1, STAT1, LMP2, LMP7, LMP3, LMP10, IRF1, IRF2, and β-actin were examined by western blotting analysis with appropriate antibodies. The DNA-binding activities of IRF1 and IRF2 were examined using an electrophoretic mobility shift assay (EMSA).

Western blotting. Equal amounts of proteins (20 mg) were size-fractionated using 7.5% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene difluoride membrane (PVDF). The blots were allowed to air dry and then placed in blocking buffer (1% BSA in 10 mM Tris buffer with 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h at room temperature. The blots were then incubated with specific primary antibodies for 1 h at room temperature. All primary antibodies were mouse monoclonal or rabbit polyclonal antibodies, obtained from several suppliers, and used at different final dilutions (1:500-1:1000) in the blocking buffer. These antibodies were raised using the following proteins as immunogens: IRF1 (37.3 kDa), IRF2 (39.5 kDa), STAT1 (87.2 kDa), LMP2 (23.4 kDa), LMP7 (30.3 kDa), LMP3 (29.1 kDa), LMP10 (29.1 kDa), β-actin (41.7 kDa), CDK2 (39.0 kDa), CDK4 (33.8 kDa), CDK6 (37.0 kDa), cyclin D (33.4 kDa), RB (105.4 kDa), E2F1 (46.3 kDa), and p21^CIP1 (17.8 kDa). The blots were washed three times for 30 min each with wash buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween-20, pH 7.5) and then incubated with an alkaline phosphatase-conjugated goat-anti-mouse IgG antibody or anti-rabbit IgG antibody (Promega, Madison, WI, USA) diluted in 5% non-fat milk in wash buffer. PVDF membranes were washed with wash buffer three times for 30 min, Western Blue Stabilized Substrate (Promega) was then added, and membranes were then incubated at room temperature until proteins were appeared as blue color bands.

Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared from cells treated and untreated with IFN-γ, as described above. EMSA was performed as previously described (15), using DNA probes containing the STAT1-binding sequence or IRFE. The DNA sequences of these synthetic oligonucleotides for the STAT1-binding site or IRFE were as follows. STAT1: 5’-AAGCATTCCTGTA AGGACT-3’, IRF-E: 5’-GGAAGCGAAAATGAAATTGACT-3’.

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Figure 3.

Signal transducer and activator of transcription 1 (Stat1) was essential for IFN1-induced IRF1 expression. A: The key role of the IFN-γ signaling pathway on LMP2 expression in the human myometrium. After the binding of IFN-γ to the type-II IFN receptor, Janus activated kinase 1 (JAK1) and JAK2 are activated and phosphorylate STAT1 on the tyrosine residue at position 701 (Tyr701). The tyrosine-phosphorylated form of STAT1 forms homodimers that translocate to the nucleus and bind to IFN-γ-activated site (GAS) elements that are present in the promoters of IFN-γ-regulated genes. The phosphorylation of Ser727 is not essential for the translocation of STAT1 to the nucleus or the binding of STAT1 to DNA, but is required for full transcriptional activation. IFNγR1, IFN-γ receptor subunit 1; IFNγR2, IFN-γ receptor subunit 2. B: Defect in IFN-γ-induced IRF1 expression in mouse embryonic fibroblasts (MEFs), which were isolated from homozygous or heterozygous mice deficient in Stat1, and parental mice (wild-type). Cytosolic extracts were prepared from MEFs treated with IFN-γ (250 U/ml) for the individual times indicated, and 50 μg of cytosolic extracts was resolved using 10% SDS-PAGE. The expression levels of p-STAT1, STAT1, LMP2, LMP7, LMP3, LMP10, IRF1, IRF2, and β-actin were examined by western blotting analysis with appropriate antibodies. The DNA-binding activities of IRF1 or IRF2 were examined using an electrophoretic mobility shift assay (EMSA).

DNA transfection and isolation of Irf1 stable transformants. Primary murine Ut-LMS cells were co-transfected with the pcDNA1 control (2 μg) or Irf1 expression vector (pcDNA1-IRF1) (2 μg), which were a generous donation from Dr. Tadatsugu Taniguchi (The University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan), by FuGENE 6 Transfection Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's recommendation in serum-free PromoCell Smooth Muscle Cell Growth Medium 2 at 2.0 μg DNA per 1×10⁶ cells, which were plated on 6-well tissue culture dishes the previous day. Transfected cells were grown in PromoCell Smooth Muscle Cell Growth Medium 2 with FCS plus (final concentration 5%) for 48 h before the addition of the selection antibiotic, G418 sulfate (Gibco). Selected murine Ut-LMS were maintained in DMEM with 10% FCS with G418 sulfate at 0.4 mg/ml for the pcDNA1 plasmid. The transfected G418-resistant colonies were isolated as previously described (16). IFN-γ was added when the plates were 60% confluent, for 48 h prior to harvesting. After the IFN-γ treatment, cells were scraped from the plates. Viable cells were counted by trypan blue exclusion (17).

Xenograft studies. Nude mice (BALB/cSlc-nu/nu, female, 7-8 weeks old; Japan SLC, Meguro, Tokyo, Japan) were injected intracutaneously with 1×10⁷ cells of the mUt-LMS-pcDNA1 clone or mUt-LMS-pIRF-1 clone with BD Matrigel Matrix (BD Biosciences, MA, USA) in 5 mg/ml of culture medium (PromoCell Smooth Muscle Cell Growth Medium 2) containing 5% FCS and PromoCell supplement solution plus SmGM-2 SingleQuots (CAMBREX) at a final volume of 100 μl. Tumor formation was assessed every day. Seven weeks after the injection, the tumors were dissected for reverse transcription-polymerase chain reaction analysis (RT-PCR) and western blotting experiments. Tumor volumes were calculated as (L×W×W)/2, where W represents the width and L the length. These mice were kept in a specific pathogen-free environment at Shinshu University's animal facilities, in accordance with institutional guidelines (approval no. 03–28–008). Statistical analysis was performed on mean tumor volumes at the end of the study using Dunnett's test.

RT-PCR. The expression of Irf1 and the β-actin transcripts was examined using RT-PCR. All tested cell types were both untreated and treated with 250 units/ml human or mouse IFN-γ (Pepro Tech, Inc., NJ, USA) for 48 h before the RNA harvest. Total RNA was prepared from 5×10⁶ cells using TRIzol reagent (Invitrogen Co.) according to the manufacturer's protocol. The RNA was reverse transcribed with the Superscript II enzyme (Invitrogen Co.), single-strand cDNA was used for amplification of Irf1 or β-actin DNA fragments by PCR analysis, following a program of 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min with an additional 5 min for the extraction of the transcripts. The PCR-amplified products were run on 2.0% agarose gels as described previously (18). Irf1: forward primer 5’-CAGAGGAAAGAGAGAAAGTCC-3’, reverse primer 5’-CACACGGTGACAGTGCTGG-3’; β-actin: forward primer 5’-TCCGGAGA CGGGGTCA-3’, reverse primer 5’-CCTGCTTGCTGATCCA-3’. These experiments were conducted at Shinshu University in accordance with local guidelines (approval no. 4737, no. 150, and no. M192).

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Figure 4.

Defective IRF1 expression in the IFN-γ signal cascade in mouse uterine smooth muscle cells derived from Lmp2-deficient mice. A: The key role of the IFN-γ signaling pathway on LMP2 expression in the human myometrium. After the binding of Ifn-γ to the type-II Ifn receptor, Janus activated kinase 1 (JAK1) and JAK2 are activated and phosphorylate signal transducer and activator of transcription 1 (Stat1) on the tyrosine residue at position 701 (Tyr701). The tyrosine-phosphorylated form of Stat1 forms homodimers that translocate to the nucleus and bind to Ifn-γ-activated site (GAS) elements that are present in the promoters of Ifn-γ-regulated genes. The phosphorylation of Ser727 is not essential for the translocation of Stat1 to the nucleus or for the binding of Stat1 to DNA, but is required for full transcriptional activation. IfngR1, Ifn-γ receptor subunit 1; IfngR2, Ifn-γ receptor subunit 2. (B) Defect in Ifn-γ-induced Irf-1 expression in mouse embryonic fibroblasts (MEFs), which were isolated from homozygous or heterozygous mice deficient in Lmp2, and parental mice (wild type). Cytosolic extracts were prepared from MEFs treated with Ifn-γ (250 U/ml) for the individual times indicated in the Figure, and 50 mg of cytosolic extracts was resolved using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The expression levels of Irf-1, Irf-2, and β-actin were examined by western blotting analysis with appropriate antibodies. (C) Defect in Ifn-γ-induced Irf-1 expression in mouse uterine smooth muscle cells (mUt-SMCs), which were isolated from the uterii of homozygous or heterozygous mice deficient in Lmp2, and parental mice (wild type). Cytosolic extracts were prepared from mUt-SMCs treated with Ifn-γ (250 U/ml) for the individual times indicated in the figure, and 50 μg of cytosolic extracts was resolved using 10% SDS-PAGE. The expression levels of p-Stat1, Stat1, Lmp2, Lmp7, Lmp3, Lmp10, Irf-1, Irf2, and β-actin were examined by western blotting analysis with appropriate antibodies. The DNA binding activities of Irf-1 or Irf-2 were examined using an electrophoretic mobility shift assay (EMSA). The details of MEFs and mUt-SMCs clones are indicated in Table I.

Tissue collection. A total of 51 patients aged between 32 and 83 years who were diagnosed with smooth muscle tumors in the uterus were selected from pathological files. Serial sections were cut from at least two tissue blocks from each patient for hematoxylin and eosin staining and immunostaining. All tissues were used with the approval of the Ethical Committee of Shinshu University (approval number: no. 150) after obtaining written consent from each patient. The pathological diagnosis of uterine smooth muscle tumor was performed using established criteria with some modifications (14). Briefly, usual LMA was defined as a tumor showing typical histological features with a mitotic index (MI) [obtained by counting the total number of mitotic figures in 10 high-power fields (HPFs)] of <5 mitotic figures per 10 HPFs. Cellular LMA was defined as a tumor with significantly increased cellularity (>2000 myoma cells/HPF) and a MI<5, but without cytologic atypia. Bizarre LMA was defined as a tumor either with diffuse nuclear atypia and a MI<2 or with focal nuclear atypia and a MI<5 without coagulative tumor cell necrosis. Tumor of uncertain malignant potential (UMP) was defined as a tumor with no mild atypia and a MI<10, but with coagulative tumor cell necrosis. LMS was diagnosed in the presence of a MI>10 with either diffuse cytologic atypia, coagulative tumor cell necrosis, or both. Of the 51 smooth muscle tumors, 23 were diagnosed as LMA, two were bizarre LMA, and 32 were LMS.

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Figure 5.

Defective expression of IRF1 in the IFN-γ-inducible cascade in proteasome-inhibitor-treated mouse embryonic fibroblasts (MEFs). A: Proteasomes are very large protein complexes inside all eukaryotes and archaea as well as some bacteria. In eukaryotes, they are located in the nucleus and cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry-out such reactions are called proteases. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a poly-ubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein. B: MEFs derived from wild-type C57BL/6 mice were treated with (+) or without (−) 50 μM mM MG115 or MG132, which are proteasome inhibitors. After the treatment with the proteasome inhibitors, the stimulation of MEFs with TNF-α (20 ng/ml) or IFN-γ (250 U/ml) was also performed for the indicated times at 37°C. Whole-cell lysates were analyzed by SDS-PAGE, and were then subjected to western blotting analysis with antibodies to IκBα or β-actin. The positions of unphosphorylated (lower arrowheads) and phosphorylated (upper arrowheads) IκBα are indicated. C: Defect in IFN-γ-induced IRF1 expression in MEFs which were isolated from wild-type C57BL/6 mice. Cytosolic and nuclear extracts were prepared from MEFs treated with IFN-γ (250 U/ml) for the individual times indicated, and 50 μg of cytosolic extracts were resolved by 10% SDS-PAGE. The expression levels of p-STAT1, STAT1, LMP2, LMP7, LMP3, LMP10, IRF1, IRF2, and β-actin were examined by western blotting analysis with appropriate antibodies. The DNA binding activities of IRF1 and IRF2 were examined using an electrophoretic mobility shift assay (EMSA). The details of MEFs are indicated in Table I.

Immunohistochemistry (IHC). IHC staining for LMP2, IRF1, ER, PR, TP53, and Ki-67 was performed on serial human Ut-LMS sections. Antibodies for ER (ER1D5), PR (PR10A), TP53 (DO-1), and Ki-67(MIB-1) were purchased from Immunotech (Marseille, France). The antibody for α-SMA was purchased from Covance Research Products, Inc. (Princeton, NJ, USA). Anti-LMP2 and anti-IRF1 were produced by SIGMA-Aldrich Israel Ltd. (Rehovot, Israel). IHC was performed using the avidin-biotin complex method as described previously. (14). Briefly, one representative 5-μm tissue section was cut from a paraffin-embedded sample of a radical hysterectomy specimen from each patients with Ut-LMS patient. Sections were deparaffinized and rehydrated in graded concentrations of alcohol, incubated with normal mouse serum for 20 min, and then incubated at room temperature for 1 h with a primary antibody. Sections were then incubated with a biotinylated secondary antibody (DAKO Japan, Minoto-ku, Tokyo, Japan) (Dako, CA, USA) and exposed to a streptavidin complex (Dako). The completed reaction was revealed by 3,3’-diaminobenzidine, and the slide was counterstained with hematoxylin. Normal myometrium portions in each specimen were used as positive controls. To examine the expression levels of target molecules, we performed LMP2 and IRF1 immunofluorescence experiments on paraffin-embedded human myometrium or Ut-LMS. These sections were then incubated with the appropriate antibodies at 4°C overnight. Regarding primary antibodies, we used a rabbit polyclonal antibody to LMP2 (1:200;SIGMA-Aldrich Israel Ltd.) or a mouse monoclonal antibody to IRF1 (1:200; SIGMA-Aldrich Israel Ltd.). After incubation with secondary antibody of Alexa Fluor 488- or 546-conjugated anti-goat, anti-rabbit, or anti-mouse IgG (1:200; Santa Cruz Biotechnology), respectively, the sections were coverslipped with mounting medium and DAPI (VECTASHILD, Vector Laboratory, Burlingame, CA, USA)(VECTASHILD; Vector Laboratory, CA) and visualized using a confocal microscope (Carl Zeiss, Thornwood, NY, USA). Negative controls consisted of tissue sections incubated with normal rabbit IgG instead of the primary antibody. These experiments were conducted at Shinshu University in accordance with institutional guidelines (approval no. M192).