1
Supplementary Information
Potential role of LMP2 as tumor-suppressor defines new targets for uterine
leiomyosarcoma therapy
Takuma Hayashi, Akiko Horiuchi, Kenji Sano, Nobuyoshi Hiraoka, Mari Kasai, Tomoyuki Ichimura,
Tamotsu Sudo, Youichi Tagawa, Ryuichiro Nishimura, Osamu Ishiko, Yae Kanai, Nobuo Yaegashi,
Hiroyuki Aburatani, Tanri Shiozawa, Ikuo Konishi
Supplementary Methods
Supplementary Figures
Supplementary Tables
1
2
Supplementary Materials and Methods
Tissue Collection. A total of 101 patients between 32 and 83 years of age and diagnosed as having
smooth muscle tumors of the uterus were selected from pathological files. Serial sections were cut
from at least 2 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 after
obtaining written consent from each patient. The pathological diagnosis of uterine smooth muscle
tumors was performed using established criteria (Hendrickson and Kempson, 1995) with some
modification. Briefly, usual leiomyoma (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 (MFs) in 10 high-power fields (HPFs)] of <5 MFs per 10 HPFs. Cellular leiomyoma (cellular
LMA) was defined as a tumor with significantly increased cellularity (>2000 myoma cells / HPF) and
a MI<5, but without cytologic atypia. Bizarre leiomyoma (BL) 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. A tumor of uncertain malignant potential (UMP) was defined as tumor with no mild
atypia and a MI<10 but with coagulative tumor cell necrosis. Leiomyosarcoma (LMS) was diagnosed
in the presence of a MI>10 with either diffuse cytologic atypia, coagulative tumor cell necrosis, or
both. Of the 105 smooth muscle tumors, 48 were diagnosed as LMA, 3 were BL, and 54 were LMS.
Of the 54 LMS, 43 were histologically of the spindle-cell type and 5 were of the epithelioid type. The
clinical stage of the LMS patients was stage I in 13 cases, stage II or III in 25 cases, and stage IV in 16
cases. Protein expression studies with cervix epithelium and carcinoma tissues were performed using
tissue array (Uterus cancer tissues, AccuMax Array, Seoul, Korea). Details about tissue sections are
indicated in manufacture’s information (AccuMax Array).
Construction of Wild Type and Mutant JAK1 Expression Vectors. Point mutations in the JAK1
coding sequence (G871E, G876R, C881F, E986P, Y987S, and R995S) were constructed using the
Site-Directed Mutagenesis System according to the manufacturer’s protocol (Promega Co., WI, USA).
2
3
The DNA sequence of mutagenic oligonucleotides for the JAK-mutant expression vectors were as
follows:
G871E
5’-gaggattcgtgacttggaagagggtcactttggg-3’
(g2612a),
G876R
5’-ggagagggc
cactttaggaaggttgagctc-3’ (g2626a), C881F 5’-gggaaggttgagctcttcaggtatgacccc-3’ (g2642t), E986P
5’-tatttgggttctcggccatacgttcaccgg-3’ (a2967c), Y987S 5’-gggttctcggcaatccgttcaccgggactt-3’ (a2970c),
and R995S 5’-gacttggcagcaagcaatgtccttgttgag-3’ (a2985t). SKN cells and JAK1 null cells were then
transfected with a pRK5 control expression vector (2 g), JAK1wt expression vector (2 g), or one of
the JAK1-mutant expression vectors (2 g) (a kind gift from Dr. J. Ihle, St. Jude Children’s Research
Hospital, Memphis, TN, USA). Interferon- (100U/ml) (PeproTech, NJ, USA) was added 24h after
transfection and the cells were incubated for an additional 24h prior to harvesting. Co-transfections
with pCMV-Gal were performed to normalize transfection efficiency.
Immunoblotting and Electrophoresis Mobility Shift Assay (EMSA). A total of 2 g of the
wild-type or mutant JAK1 expression vectors were transfected into JAK1-deficient MEFs, which were
donated by Dr. Robert D. Schreiber (St. Jude Children’s Research Hospital, Memphis, TN, USA) with
FuGENE6 Transfection Reagent (Roche, IN, USA) according to the manufacturer’s recommendations.
Cytosolic and nuclear extracts were prepared from 5x106 cells and treated with or without 250 U/ml of
human IFN- for the periods indicated in each figure 24 h after transfection, essentially as described
previously(1). The cells were collected by centrifuging for 10 min at 1200 r.p.m., washed in 5ml of
ice-cold PBS, and centrifuged again for 5 min at 12000 r.p.m. at 4°C. The cells were pelleted and
washed once in 0.4ml of buffer A [10mM Hepes, pH 7.8; 10mM KCl; 2mM MgCl 2; 1mM DTT;
0.1mM EDTA; complete protease inhibitor cocktail (Kirkegaard & Perr Lab., MD, USA)] and
incubated for 2 h at 4°C. Then, 25 l of a 10% Nonidet P-40 solution was added, and the cells were
vigorously mixed for 1 h at 4°C and centrifuged for 5 min at 12000 r.p.m.. The supernatant was
collected as cytosolic extracts and stored at -80°C. Pelleted nuclei were resuspended in 40 ml of buffer
C (50mM Hepes, pH 7.8; 50mM KCl; 300mM NaCl; 0.1mM EDTA; 1mM DTT; 10% (v/v) glycerol),
mixed for 2 h at 4°C, and centrifuged for 5 min at 12000 r.p.m. at 4°C. The supernatant containing the
3
4
nuclear proteins was harvested and stored at -80°C.
To detect the expression of STAT1, phospho-STAT1(Tyr701), JAK1, phospho-JAK1 (Tyr1022),
NF-Bp50, NF-Bp65(RelA), and LMP2, whole cell lysates, nuclear extracts or cytosolic extracts
were resolved by 10% SDS-PAGE and immunoblotting was performed using anti-STAT1 antibody or
anti-phospho-STAT1(Tyr701) antibody (Santa Cruz, CA, USA), anti-JAK1 antibody (Chemicon Int’l,
CA, USA), anti-phospho-JAK1 (Tyr1022) antibody or anti-NF-Bp50 antibody (Santa Cruz
Biotechnology, CA, USA), anti-NF-Bp65(RelA) antibody (Applied Biological Materials, Inc. BC,
Canada), or anti-LMP2 monoclonal antibody (SIGMA-Aldrich Israel Ltd., Rehovot, Israel).
Nuclear extracts were prepared from JAK1-transfected cells treated with IFN- as already
described(1). EMSA was performed as reported previously(1) using DNA probes containing the
Stat1-binding sequence, IRF-E, SP1 binding sequence, or NF-B binding sequence. The DNA
sequence of the oligonucleotides for the Stat1-binding site was 5’-aagcattcctgtaaggact-3’, that for
IRF-E was 5’-ggaagcgaaaatgaaattgact-3’, that for the SP1-binding site was 5’-attcgatcggggcggggcg
agc-3’, and that for the NF-B-binding site was 5’-gatctagggactttccgctggggactttccag -3’.
1. Hayashi, T., Kobayashi, Y., Kohsaka, S. & Sano, K. The mutation in the ATP-binding region of JAK1, identified in
human uterine leiomyosarcomas, results in defective interferon-gamma inducibility of TAP1 and LMP2.
Oncogene 25, 4016-4026 (2006).
Comparative Genomic Hybridization (CGH) and Digital Image Analysis. DNA was extracted
from paraffin-embedded tissue sections from tumor samples following the standard procedure(2). CGH
was performed using direct fluorochrome-conjugated DNA for all samples as described elsewhere(3,4).
Briefly, DNA isolated from tumor and normal tissue sections from the same patient was labeled with
fluorescein-iso-thiocyanate (FITC)-conjugated dCTP and dUTP (Dupont, Boston, MA, USA) and
Texas red-conjugated dCTP and dUTP (Dupont) by nick translation, respectively, to obtain fragments
ranging from 600 to 2000 bp, as previously published(4,5). The hybridization mixture consisted of 400
ng tumor DNA, 400 ng normal DNA, and 10 g unlabeled human Cot-1 DNA (Gibco/BRL, Life
Technologies, Gaithersburg, MD, USA) dissolved in 10 l of hybridization buffer (50% formamide,
4
5
10% dextran sulfate, 2xSSC). The hybridization mixture was denatured at 75°C for 5 min and then
hybridized to a slide with normal metaphase spreads denatured in 70% formamide/2xSSC (pH7) at
68°C for 2 min. Hybridization was carried out at 37°C for 48 h. The slides were then washed three
times in 50% formamide/2xSSC (pH7.0), twice in 2xSSC, and once in 0.1xSSC at 45°C, followed by
washing in 2xSSC, 0.1M NaH2PO4, 0.1M Na2HPO4, 0.1% Nonidet P-40 (pH 8), and distilled water
at room temperature for 10 min each. After airdrying, the slides were counterstained with DAPI
(Sigma, St Louis, MO, USA) and mounted using an antifading medium (Vector Laboratories,
Burlingame, CA, USA).
Hybridization was analyzed using an Olympus fluorescence microscope and the ISIS digital image
analysis system (MetaSystems GmbH, Altlussheim, Germany) based on an integrated high-sensitivity
monochrome charge-coupled device (CCD) camera and automated CGH analysis software.
Three-color images (green for tumor DNA, red for normal DNA, and blue for counterstaining) were
acquired from 12 metaphases per sample. The chromosomal regions were interpreted as
over-represented when the green-to-red ratio exceeded 1.17 (gains) or 1.5 (high-level amplifications),
and as under-represented (losses) when the ratio was less than 0.85. In each CGH experiment, a
negative (normal DNA) and positive (tumor DNA with known copy number changes) control were
included and run simultaneously with the tumor samples. Telomeric and heterochromatinic regions
were excluded from the analysis when they appeared as the sole aberration present in the sample, as
these regions cannot be evaluated reliably by CGH as already described(5,6). All results were
confirmed using a 99% confidence interval with a 1% error probability. The intraexperimental s.d. for
all positions in the CGH ratio profiles were calculated from the variation of the ratio values of all
homologous chromosomes within the experiment. Confidence intervals for the ratio profiles were then
computed by combining them with an empirical interexperimental s.d. and estimating the error
probability based on the t-distribution.
2. Isola J, DeVries S, Chu L, et al. Analysis of changes in DNA sequence copy number by comparative genomic
hybridization in archival paraffin-embedded tumor samples. Am J Pathol 145, 1301–1308 (1994).
5
6
3. Larramendy ML, Tarkkanen M, Valle J, et al. Gains, losses, and amplifications of DNA sequences evaluated
by comparative genomic hybridization in chondrosarcomas. Am J Pathol 150, 685–691 (1997).
4. El-Rifai W, Larramendy ML, Bjo¨rkqvist AM, et al. Optimization of comparative genomic hybridization
using fluorochrome conjugated to dCTP and dUTP nucleotides. Lab Invest 77, 699–700 (1997).
5. Larramendy ML, El-Rifai W, Knuutila S. Comparison of fluorescein isothiocyanate- and Texas red-conjugated
nucleotides for direct labeling in comparative genomic hybridization. Cytometry 31, 174–179 (1998).
6. Kallioniemi OP, Kallioniemi A, Piper J, et al. Optimizing comparative genomic hybridization for analysis of
DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 10, 231–243 (1994).
Sequencing of the Catalytic Domains of the JAK1, STAT1, and JAK2 genes and the LMP2
promoter region. To determine whether somatic mutations exist in the ATP-binding region or kinase
activation domain of JAK 1 and JAK2, in the LMP2 promoter region at Tyr701 or Ser727 of STAT1,
or in the ATP-binding region and kinase activation domain of JAK2 in human uterine LMS, genomic
DNA was isolated and direct sequencing was carried out. Genomic DNA was extracted from
consecutive paraffin-embedded human uterine LMS tissue and normal myometrium tissue sections
using the microwave-based DNA extraction method for PCR amplification(7). To avoid contamination
of normal myometrium or inflammatory cells, the tumor areas were confirmed using a hematoxylin
and eosin-stained glass slide as a template. The tumor tissues were scraped by razor-micro dissection
from paraffin-embedded consecutive tissue sections. The genomic DNA was subjected to PCR, and
restricted DNA fragments for direct sequencing analysis were amplified using published
oligonucleotide primers. PCR products were directly sequenced using a DYEnamic Terminator Cycle
Sequencing Kit (Amersham-Biosciences, Piscataway, NJ, USA) with an ABI Prism 3100 Genetic
Analyzer (Applied Biosystem, Foster City, CA, USA). The sequences of mutant JAK1, STAT1, and
the LMP2 promoter region derived from individual uterine LMS tissue sections are registered in the
DDBJ (Accession: AB219242, DJ055380, DJ055379, DJ055378, DJ055377, DJ055376).
7. Banerjee SK, Makdisi WF, Weston AP, et al. Microwave-based DNA extraction from paraffin-embedded tissue for
PCR amplification. BioTechniques 1995; 18: 768-774.
Primer Sets for Direct Sequence Analysis.
JAK1: (F, 5’-caccaaatctttaaaccggaccccagcctt-3’, R, 5’-tacgatggggcttccctgataacagcacat-3’), (F, 5’-atg
6
7
gctttctgtgctaaaatgaggagctcc-3’, R, 5’-tccatcctgctcggtcttggggtctcgaat-3’), (F, 5’-attcgagaccccaagacc
gagcaggatgga-3’, R, 5’-tccactggattccaagattcccagtcacca-3’), (F, 5’-tggtgactgggaatcttggaatccagtgg a-3’,
R, 5’-ggcggctcatgaggtctcccaagctgggga-3’), (F, 5’-tccccagcttgggagacctcatgagccacc-3’, R, 5’-cc
gtaatggggatgccggggtcactgagct-3’), and (F, 5’-agctcagtgaccccggcatccccattacgg-3’, R, 5’-cagatcagctat
gtggttacctccactctc-3’)
JAK2: (F, 5’-cagattatgggtaatgattaaaggctccca-3’, R, 5’-cacagcatttctccaacatctgacaaccaaacc-3’), (F,
5’-gacagtctgctaattccagctactagaa-3’, R, 5’-gcctctccctctgggcattggcataagtcc-3’), and (F, 5’-atgaagcaac
cgtgttgaagtagacattag-3’, R, 5’-cccacgtggactataaccatgactataagacc-3’), Primer sets for the nested-PCR:
(F, 5’-gaaactatttgagtttccctgtatcatttag-3’, R, 5’-ctacaagcactccttaaaatgtt gtagaaag-3’), (F, 5’-gtaatttgcct
tgaaaacttggtatttcc-3’, R, 5’-gcataagtccagatcgttaagacattgtac-3’), and (F, 5’-gaagtagaca ttaggaaatcatct
agacg-3’, R, 5’-cactgttactgtaaatatagaaatggcaaac-3’)
STAT1: (Ser727 F, 5’-cacttattgagagctacacacaggccagcc-3’, R, 5’-ggctggggacatgagaatcccatgagctgt-3’)
and (Tyr701 F, 5’-tgctgataggcagtaacacggggatctcaa-3’, R, 5’-aggaggctaagctgtctagaaacacagtag-3’)
Primer sets for the nested-PCR: ( Ser727 F, 5’-ttgagagctacacacaggccagccgtggta-3’, R, 5’-gggacatga
gaatcccatgagctgtacttt-3’) and (Tyr701 F, 5’-tgctgataggcagtaacacggggatctcaa-3’, R, 5’-gtctagaaacaca
gtagaactttaatcccc-3’)
LMP2 promoter region: (F, 5’-cgagaagctcagccatttaggggaaagcga-3’, R, 5’-cgcccgcagcatccctgcaaggc
accgctc-3’). Primer sets for the nested-PCR: (F, 5’-aagcgaaatcgaaagcggccgcctgctcac-3’, R, 5’-ctctcc
tcgccgcctggggcactggtttcc-3’)
DNA Transfection and Isolation of Flat Revertants.
Transfection
of
pCEM9-LMP2wt,
pCEM9-LMPK33A,
pCEM9-NF-Bp50,
pCEM9-NF-B
p65(RelA) (AIDS program/NIH, MD, USA), pshRNA-Calponin h1, pshRNA-control (Santa Cruz
7
8
Biotechnology Inc.) or its empty vector pCEM9 was carried out with FuGENE6 Transfection Reagent
(Roche, IN, USA) according to the manufacturer’s recommendations with 5 g of plasmid DNA and 5
x 105 SKN cells (from Dr. I. Ishiwata, Ishiwata Clinic, Ibaraki, Japan) and 5 x 105 HeLa cells (from Dr.
Y. Adachi, Shinshu University, Nagano, Japan) plated into 6-well tissue culture dishes (Corning NY,
USA) on the previous day. Forty-eight hours after transfection, cells were treated with trypsin and
replated onto 100-mm dishes with 15 ml of growth medium containing 1 mg of G418 per ml
(SIGMA-Aldrich, MO, USA). The cells were incubated at 37°C for an additional 6-8 days, with
medium changes on day 1 and day 3 or 4. The number of G418-resistant colonies at this stage was
counted.
Transfected G418-resistant colonies from 2-10 dishes were pooled after trypsin treatment, and
aliquots of 1 x 105 cells were subjected to one of the following enrichment procedures:
(i) Adhesion enrichment: This procedure selects for cells with increased attachment to an Ultra Low
attachment dish (Coster, NY, USA). Cells were plated onto 100-mm tissue culture dishes (Nunc, NY,
USA) with 10 ml of G418 medium and incubated for 4 days at 37°C. Cells that were weakly adherent
to the plastic were mechanically removed by being sharply blown off the medium with a pipette onto a
cell sheet. The cultures were washed extensively with phosphate-buffered saline without Ca2+ or
Mg2+.
(ii) Soft agar enrichment: This procedure selects for cells unable to grow in soft agar medium.
Suspended cells were mixed with 4 ml of melted agar medium, which consisted of growth medium
containing 0.33% Agarose (SIGMA-Aldrich, MO, USA) and G418 described above. The cells in the
agar medium were poured onto a base layer (4 ml per 60-mm bacterial dish) consisting of growth
medium containing 0.8% agar and G418. After 48 hr of incubation at 37°C, the top agar portion was
transferred to a 15-ml test tube and suspended in 5 ml of serum-free medium after passing them
several times through a G21 needle. The cells together with small agar blocks were then recovered by
centrifugation at 200 x g for 15 min, rinsed twice with 10 ml of serum-free medium, resuspended and
plated with 5 ml of growth medium in 60 mm tissue culture dishes, and incubated overnight at 37°C.
8
9
After each of the above treatments, cultures were refed with G418 medium and incubated for an
additional 5-7 days. One or more cycles of adhesion enrichment were applied to further facilitate the
detection of small flat colonies among the vast majority of large transformed colonies. Flat colonies
found by microscopic scanning of the dishes were picked up with stainless cloning cylinders and
further purified by repeated recloning.
Xenograft Studies.
Nude mice (BALB/cSlc-nu/nu, Female, 7-8 weeks old, Japan SLC, Shizuoka, Japan) were injected
intracutaneous with 1×107 cells of the SKN-CEM9 (T type) clones, SKN-LMP2wt (F type) clones,
SKN-LMP2K33A (F type) clones, SKN-p50p65 (T type) clones, SKN-LMP2wt/shRNA control (F
type) clones, or SKN-LMP2wt/shRNA Calponin h1 (T type) clones with BD Matrigel Matrix (BD
Biosciences, MA, USA) in 5 mg/ml of culture medium containing 15% FCS plus SmGM-2
SingleQuots (CAMBREX, MD, USA) at a volume of 100 μl. Nude mice (BALB/cSlc-nu/nu, Female,
7-8 weeks old, Japan SLC) were also injected intracutaneous with 1×107 cells of the HeLa-CEM9 (T
type) clones, HeLa-LMP2wt (T type) clones, HeLa-LMP2K33A (T type) clones with BD Matrigel
Matrix (BD Biosciences, MA, USA) in 5 mg/ml of culture medium containing 15% FCS at a volume
of 100 μl. Tumor formation was assessed every day. At 7 weeks after injection, the tumors were
dissected for RT-PCR experiments. Tumor volumes were calculated as (L×W×W)/2, where W
represents width and L represents length. Statistical analysis was performed on mean tumor volumes
at the end of the study using Dunnett’s test.
Flow Cytometry.
Cells were incubated in culture dishes in F-12 medium (GIBCO, NY, USA) without FCS to allow
analysis of cell cycle. After an overnight culture to allow cellular attachment to the plate, the medium
was removed and fresh medium with 15% FCS was added. Adherent cells were collected and washed
with PBS 3 times at 16 hr, 24 hr, and 36 hr later. The cells were fixed in 70% ethanol and stored at
9
10
4°C for 20 min, and then resuspended in a DNA-stain solution containing propidium iodide (20
mg/ml; Calbiochem, CA, USA) and RNase (20 mg/ml; Novagen, Darmstadt, Germany). The cells
were analyzed with a FACScan (Fluorescence Activated Cell Sorter) flow cytometer in combination
with BD lysis II software (488 nm; Becton Dickinson Immunocytometry System, Mountain View, CA,
USA).
Sub-purification of 20S Proteasomes and Proteasome Assay.
A rapid method was performed for the isolation of biologically active 20S proteasomes from SKN
transfectant clones (2 x 107 cells) using affinity matrix beads comprised of a GST-fusion protein
containing an ubiquitin-like domain (UbL) bound to GST-agarose (Proteasome Isolation Kit, Merck
Ltd., Darmstadt, Germany). The proteasome subunit proteins were identified by loading the beads
directly onto an SDS-PAGE gel and immunoblotting with subunit-specific antibodies (Enzo Life
Science, International, Inc. PA, USA). Alternatively, proteasome-bound UbL beads were used in
proteolytic assays using proteasome substrates. The 20S proteasomes were also partially purified from
SKN transfectant clones (5 x 107 cells) by differential centrifugation as published previously(8) and
used in proteolytic assays using proteasome substrates. Protein degradation activities of the
sub-purified 20S proteasomes from SKN transfectant clones were examined using a 20S proteasome
assay kit following the manufacturer’s recommendations (Enzo Life Sciences International, Inc.).
8. Tanaka, K., Li, K., Ichihara, A., Waxman, L., and Goldberg, A.L. A high molecular weight protease in the cytosol
of rat liver. I. Purification, enzymatic properties, and tissue distribution. J Biol Chem 261, 15197-15203 (1986).
IFN--Deficient Mice and TNF--Deficient mice.
IFN- gene-deficient chimeric mice were generated by injecting IFN- gene-disrupted A3-1 ES cells
from a 129/SvJ mouse (9). Genotypes of the IFN- locus were also checked by PCR. After establishing
the IFN- deficient mouse lines, these mice were crossed to C57BL/6J mice, and the F2 generations
were used for the preparation of the homozygous IFN--deficient mice that were used in the
experiments. TNF- gene-deficient chimeric mice were also generated by injecting TNF-
10
11
gene-disrupted TT2 ES cells into 8-cell stage embryos from C57BL/6J mice(9). Then, the chimeric
mice were crossed with C67BL/6J mice, and the F2 generations were used for the preparation of
homozygous the TNF--deficient mice that were used in the experiments.
All wild-type and homozygous mice examined in our experiments were littermates of
IFN--deficient F10 mice or TNF--deficient F10 mice generated by backcrossing with C57BL/6J
mice, which were purchased from CLEA Japan, Inc. (Tokyo, Japan). The mice used in the present
study were female mice of 2 months of age. Mice were kept under specific pathogen-free conditions in
an environmentally controlled clean room at the Laboratory Animal Research Center, Shinshu
University School of Medicine. All equipment and supplies, including cages, water bottles, wooden
chips, and food pellets, were sterilized. The experiments were conducted according to institutional
ethical guidelines for animal experiments and safety guidelines (approval no.03-28-008).
9. Tagawa, Y., Sekikawa K., & Iwakura, Y. Suppression of Concanavalin A-induced hepatitis in IFN--/- mice,
but not in TNF--/- mice. J. Immunol. 159, 1418–1428 (1997).
11
12
Supplementary Figure S1 Development of uterine neoplasms in LMP2-deficient mice. Histological
analysis shows the abnormal cells of a uterine leiomyosarcoma (LMS) from LMP2-deficient mice (8
months of age) and the uterine smooth muscle cells from the age-matched normal myometrium of
parental C57BL/6 mice. Immunostaining of uterine tissue sections from LMP2-deficient and C57BL/6
mice with an antibody to Ki-67 (x200). Presentation of these histological data in this manuscript was
approved by Prof. Susumu Tonegawa (Picore Inst. and Dept. of Biology, M.I.T., MA).
12
13
Supplementary Figure S2 Specificity of anti-human LMP2 mouse monoclonal antibodies to human
LMP2. Anti-human LMP2 mouse monoclonal antibody (clone#LMP2-12.3 Lot#120709) for
immunoblotting and immunohistochemistry (IHC) analyses were produced under the Shinshu
University and SIGMA-Aldrich Collaboration Laboratory Project managed by Japan Science and
Technology Agency (Project#S2006-0097). A, Immunoblotting analysis with T1 cells, T2 cells, and
genetically modified T2 cells demonstrate that the anti-human LMP2 mouse monoclonal antibody
specifically recognizes human LMP2 as double bands, which indicate pre-LMP2 (upper band) and
mature LMP2 (lower band). RT-PCR experiments with T1 cells, T2 cells, and genetically modified T2
cells were also performed. T1, T2, T2/LMP7(T2/L7), and T2/LMP2LMP7(T2/L2/7) cells were gifts
from Dr. Peter M. Kloetzel (Medical Faculty, Humboldt University, Berlin, Germany). B, IHC
experiments with 2 samples derived from individual patients (patients #4 and #9, details in Table S1
and Table S2) with uterine LMS clearly demonstrate that the monoclonal antibodies to human LMP2
specifically bind to the antigen. The experiments were performed four times with similar results.
13
14
Supplementary Figure S3 LMP2 expression was also examined in skeletal and rectum metastases
from individual uterine leiomyosarcoma (LMS) patient samples (patients #3 and #18, details in Table
S1 and Table S2). The antibody for ER (ER1D5) was purchased from Immunotech (Marseille,
France). The antibody for -Smooth muscle actin (SMA) was purchased from Covance Research
Products, Inc. (Princeton, NJ, USA). The anti-LMP2 antibody was produced by SIGMA-Aldrich
Israel Ltd. (Rehovot, Israel; dilution 1/100). IHC was performed using the avidin-biotin complex
method as described previously. Briefly, representative 5-m tissue sections were cut from
paraffin-embedded samples of radical hysterectomy specimens of skeletal muscle and rectum
metastases from individual uterine LMS patients. (magnification x20, x40) The experiments were
performed three times with similar results.
14
15
Supplementary Figure S4 Key role of the IFN- signaling pathway on LMP2 expression in human
myometrium. After the binding of interferon- (IFN-) 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--activated site
(GAS) elements that are present in the promoters of IFN-γ-regulated genes. The IFN--activated JAKs
also regulate, through yet unknown intermediates, activation of the catalytic subunit (p110) of
phosphatidylinositol 3-kinase (PI3K). PI3K activation ultimately results in downstream activation of
protein kinase C-δ (PKC-δ), which in turn regulates phosphorylation of STAT1 on the serine residue
at position 727 (Ser727). 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- receptor subunit 1; IFNGR2, IFN- receptor subunit 2.
15
16
Supplementary Figure S5 Basal LMP2 expression in several tissues derived from 2-month-old
experimental IFN--deficient, TNF--deficient, and wild-type female mice. Immunohistochemical
experiments on tissue sections of heart, esophagus, ventriculus, and liver were carried out, revealing
similar basal expression of LMP2 in tissues (heart, ventriculus, esophagus, liver) obtained from IFN-and TNF--deficient mice as that of age-matched wild type mice. The experiments were performed
three times with similar results.
16
17
Supplementary Figure S6 Loss of IRF-1 association with the LMP2 enhancer/promoter region in
human uterine LMS. ChIP assays show that human genomic DNA of the LMP2 enhancer/promoter
region is markedly amplified using immunoprecipitated LMA tissue (patients UL2, UL3, and UL4) as
well as normal myometrium tissue (patients #13, #15, and #16, details in Table S1 and Table S2) with
anti-IRF-1 antibody. No DNA amplification was detected in the immunoprecipitated LMS samples
(patients #13, #15, and #16, details in Table S1 and Table S2) with anti-IRF-1 antibody. DNA
amplifications were detected in all immunoprecipitated samples with anti-SP1 antibody as an internal
positive control. The experiments were performed three times with similar results.
17
18
Supplementary Figure S7 Identified somatic mutations in JAK1 catalytic sites by direct sequence
experiments.
18
19
19
20
Supplementary Figure S8 Summary of gains and losses in DNA sequence copy number in 4 primary
uterine leiomyosarcoma (LMS) samples (patients #14, #15, #16, and #18, details in Table S1 and
Table S2) analyzed by comparative genomic hybridization (CGH). Losses are shown on the left side
of each chromosome (red) and gains on the right (green). Each vertical line represents a genetic
alteration seen in one sample. High-level amplifications of small chromosomal regions are shown as
thick horizontal bands.
20
21
Supplementary Figure S9 Normal expression of IFN-R1 in uterine leiomyosarcoma (LMS) tissues.
Western blot (W.B.) analysis shows that the expression of IFN-R1 is detected in normal myometrium
tissue (patient #17, details in Table S1 and Table S2), all uterine LMS tissues (patients #14, #15, #16,
#17, #18, details in Table S1 and Table S2), and the uterine LMS culture cell line (SKN cells) at a
similar basal expression level. The experiments were performed three times with similar results.
21
22
Supplementary Figure S10 Immunohistochemical (IHC) experiments with antibodies to JAK1 and
STAT1 show strong positive staining in uterine leiomyosarcoma (LMS) tissue sections, as well as in
normal myometrium tissue sections (patients #15 and #17, details in Table S1 and Table S2).
Conversely, although p-STAT1 is weakly positive in normal myometrium tissue sections, it is
essentially negative in uterine LMS tissue sections derived from the same patients (patients #15 and
#17, details in Table S1 and Table S2). Thus, the loss of IFN- responsiveness in uterine LMS is likely
attributable to the inadequate kinase activity of JAK1 due to mutations in its catalytic domains. The
result from western blotting (W.B.) strongly supports the IHC findings. The experiments were
performed three times with similar results.
22
23
Supplementary Figure S11 Incorporation of LMP2 and LMP7 into 20S proteasomes. A. Cell lysates
that were prepared from SKN-transfectant clones were mixed with GST-UbL beads (Merck Ltd.) for
12 hours at 4°C. After washing, 20S proteasomes were sub-purified as GST-UbL bead complexes. A
total of 1/50 of the input of the reaction mixture (I) and supernatant (S) and 1/5 of the last wash (W)
and pellet (P) were subjected to SDS-PAGE and analyzed by Western blot using specific antibodies. B.
20S proteasomes were partially purified by differential centrifugation of cell lysates. The sub-purified
20S proteasome complexes were subjected to SDS-PAGE and analyzed by Western blot using specific
antibodies. The SKN-transfectants used in sub-purification of 20S proteasomes were SKN-CEM9#1,
SKN-LMP2wt#121, and SKN-LMP2K33A#1. Details of SKN- transfectants are shown in Table 1.
The experiments were performed four times with similar results.
23
24
Supplementary Figure S12 Morphology of SKN transfectants. Phase-contrast micrographs of the
colonies obtained after transfection of the pCEM9 control plasmid or pCEM9-LMP2 plasmid into
SKN cells. The transformed (T type) colony, partially flat (P/F type) colony, and flatrevertant (F type)
colony are shown. Photographs were taken 20 days after G418 selection. a Details are shown in Table
1.
24
25
Supplementary Figure S13 Morphology of the parental SKN cell line and its transfectants.
Phase-contrast micrographs of SKN cells. The primary transformed and flatrevertant clones are
obtained after transfection of the pCEM9 control plasmid, pCEM9-LMP2wt plasmid, or
pCEM9-LMP2K33A plasmid into SKN cells. Transformed clones and flatrevertant clones are shown.
Photographs of clones were taken 20 days after selection with G418. a Details are shown in Table 1.
25
26
Supplementary Figure S14 Cell cycle analysis of the SKN-CEM9 (T type) clone and SKN-LMP2 (F
type) clone. The y axis denotes cell count and the x axis represents DNA content. The percentages of
cells in the G0/G1, S, and G2/M phases of the cell cycle were calculated using Multicycle software.
The experiments were repeated at three times with similar results. a Details are shown in Table 1.
26
27
Supplementary Figure S15 Changes in the human uterine LMS cell line, SKN transfectants, the
SKN-CEM9#2 (T type) clone, and the SKN-LMP2K33A#1 (F type) clone xenograft volumes in mice
(n=4). Representative photographs of xenografts in mice (Left panel). Tumor growth of the
SKN-LMP2K33A (F type) clone is markedly reduced in comparison with that of the transfectant
SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9
control (T type) clone and SKN-LMP2 (F type) clone (Right panel). RT-PCR experiments reveal
hLMP2 mRNA expression in tumors (Lower right panel). The experiments were performed four times
with similar results. Details of the SKN-transfectant clones are indicated in Table 1.
27
28
Supplementary Figure S16. No toxic effect from either wild type LMP2 or mutant LMP2K33A
overexpression on tumorigenesis. Immunohistochemical experiments with anti-hLMP2 antibody were
performed to examine LMP2 expression in gynecologic tumor tissues. LMP2 was clearly detected in
both normal cervical epithelium and carcinoma tissues as well as in myometrium, but not in
leiomyosarcoma tissues (Upper left panel). Changes in HeLa transfectant, HeLa-CEM9#1 (T type)
clone, HeLa-LMP2wt#1 (T type) clone, and HeLa-LMP2K33A#1 (T type) clone xenograft volumes in
mice (n=4). Representative photographs of xenografts in mice (Lower left panel). Tumor growth of
the HeLa-LMP2wt clone and HeLa-LMP2K33A clone are similar to that of the transfectant
SKN-CEM9 clone. Tumor growth kinetics after subcutaneous injection of the HeLa-CEM9 control
clone, HeLa-LMP2wt clone, and HeLa-LMP2K33A clone (lower right panel). RT-PCR experiments
reveal hLMP2 mRNA expression in tumors (Upper right panel). The experiments were performed four
28
29
times with similar results. Details of the HeLa transfectant clones are indicated in Supplementary
Table 6 and Supplementary Table 7.
29
30
Expression of ER, PR, p53, Ki-67, p53 and LMP2 in human uterine leiomyosarcoma
Patient
No.
Age
in
yrs
TNM
stage
Immunohistochemical staining
MF
CCN
ER
PR
Ki-67
p53
Mutation
LMP2
p53
JAK1
JAK2
STAT1
1
37
T4N1M0
97
+
3000 +++
M
ND
ND
2
58
T3N0M0
24
+
3500
+
+/M
ND
ND
3
45
T2N1M0
32
+
+/+/2150 +++
M
M
ND
4
65
T1N0M0
30
+
+/+/1700 +++
M
M
ND
5
52
T1N1M0 107
+
+
2600
++
+
ND
M
ND
6
49
T1N0M0
46
+
4300
+
ND
ND
ND
7
55
T1N1M0
75
+
4000 +++
ND
ND
ND
8
43
T3N0M0
57
+
+
2000
+/ND
ND
ND
9
67
T1N1M0
13
+
+/1430
ND
M
ND
10
67
T1N0M0
37
+
2100
ND
ND
ND
11
51
T1N1M0
93
+
4500
ND
ND
ND
12
48
T1N0M0
14
+
900
+++
+
ND
ND
ND
13
51
T1N1M0
22
+
+/+
450
+
ND
M
ND
14
67
T1N0M0
64
+
+
1450
++
ND
ND
ND
15
52
T1N1M0
65
+
1780
++
ND
M
ND
16
42
T3N0M0
73
+
2130
++
ND
ND
ND
17
80
T1N1M0
98
+
1980 +++
ND
M
ND
18
56
T1N0M0
78
+
1860
++
ND
ND
ND
19
58
T1N0M0
40
+
1750
++
ND
ND
ND
ER, estrogen receptor; PR, progesterone receptor; Ki-67, positive cell number/10 high power fields;
detected; D, died of disease; A, alive; MF, mitotic figure/10 high power fields.
LMP2-pro
ND
ND
ND
M
M
M
ND
ND
ND
ND
ND
ND
M
M
ND
ND
ND
ND
M
M
M
ND
ND
ND
ND
M
ND
ND
M
ND
ND
M
ND
ND
ND
ND
ND
ND
M, mutation; ND,
Follop-up
(months)
D(1)
D(23)
D(24)
D(20)
D(13)
D(24)
D(18)
D(10)
A(34)
A(15)
A(94)
A(58)
A(34)
A(15)
A(23)
A(21)
A(19)
A(11)
A(10)
not
Supplementary Table S1 Expression of ER, PR, p53, Ki-67, p53 mutation, and LMP2 in human
uterine leiomyosarcoma (LMS). Immunohistochemistry of ER, PR, p53, and LMP2 in normal
myometrium and uterine LMS tissues located in the same tissue section was performed. The isolation
of genomic DNA from normal myometrium and patient-matched uterine LMS tissues and direct
sequencing for p53, JAK1, JAK2, STAT1, and LMP2 promoter were carried out to demonstrate
somatic mutations. This table shows our current results combined with previous research findings
[Zhai YL, et al., (1999) Intl J Gynecol Pathol 18, 20-28], which was published by the Dept. of
Obstetrics and Gynecology, Shinshu University School of Medicine, by a clinical facility member of
our research project.
30
31
Mutations in JAK1 kinase, LMP2 promoter region, and STAT1 in human uterine leiomyosarcoma
Ptient #
JAK1 kinase
LMP2 promoter region
STAT1(701Y,727S)5
JAK2 kinase
LMP27
#1
wt
wt
wt
wt
Neg.
#2
wt
A210G, C214T(IRF-E)3
wt
wt
P.Posi.
#3
G986P(active)R995S(active)1
C214T, G219A(IRF-E)
(S710A)6
wt
Neg.
#4
G876R(ATP)2
wt
wt
wt
Neg.
#5
C881F(ATP)
wt
wt
wt
P.Posi.
#6
wt
wt
wt
wt
Neg.
#7
wt
A216G(IRF-E)
(L693R)6
wt
Neg.
#8
wt
wt
wt
wt
F.Posi.
#9
Y987S(active)
wt
wt
wt
Neg.
#10
wt
A217G(IRF-E)
(R716S)6
wt
Neg.
#11
wt
wt
(I702L)6
wt
Neg.
#12
wt
wt
wt
wt
F.Posi.
#13
Y987S(active)
A216G(IRF-E)
wt
wt
Neg.
#14
Wt
wt
wt
wt
Neg.
#15
G871E(ATP)
wt
(I702L)6
wt
Neg.
#16
wt
G239A(HSF)4
wt
wt
Neg.
#17
C881F(ATP)
wt
wt
wt
Neg.
#18
wt
wt
wt
wt
Neg.
#19
wt
wt
wt
wt
Neg.
1 Kinase activation site of JAK1, 2 ATP binding region of JAK1, 3 Interferon-g regulatory factor-enhancer site, 4 Heat
Schock Factor binding site, 5 Tyr701 or Ser727 phosphorylation of STAT1, 6 Mutation is not located in major functional
regions of STAT1, 7 The results of LMP2 expression are immunohistochemical staining, Neg., Negative; P.Posi., Partial
Positive; F.Posi., Focally Positive.
Supplementary Table S2 Identification of somatic mutations in the catalytic domains of JAK1 and
JAK2 kinases, STAT1, and the activation region of the LMP2 promoter in human uterine
leiomyosarcoma (LMS). Genomic DNA was extracted from human LMS and normal myometrium
tissues as described in the Materials and Methods section. The restricted DNA fragments for direct
sequencing analysis were amplified by PCR with the appropriate primers for the ATP-binding region
and kinase activation domain of JAK1, the LMP2 promoter region, Tyr701 and Ser727 of STAT1, and
the ATP-binding region and kinase activation domain of JAK2. Information on the primer sets is
indicated in the Supplemental Materials and Methods section. The experiments were performed three
times with similar results.
31
32
Mutations in the IFN- pathway in human uterine leiomyosarcoma
Gene
Name
JAK1
Locus
GenBank
Accession
HUMPTKJAK1
M64174.1
*147795
JAK2
AF005216
AF005216.1
NM_007315
X62741
STAT1
LMP21
Evoluationary4
conservation
Nucleotide
Amino
Acid
Domain
ULMS
G2612A
G2626A
G2642A
A2957C
A2960C
A2985T
G871E
G876R
C881F
Q986P
Y987S
R995S
ATP binding
ATP binding
ATP binding
active site
active site
active site
p,c,m,r,g,d
+147796
ULMS
ND2
ND
ND
p,c,b,m,r,g,d
NM_007315
+600555
ULMS
A2104C
T2128G
T2078G
A2148C
I702L
S710A
L693R
R716S
NA3
NA
NA
NA
c,b,m,r,g,d
X62741.1
*177045
ULMS
MIM ID
Tumor
A210G
IRF-E site
p,c,b,m,r,d
C214T
IRF-E site
A216G
IRF-E site
A217G
IRF-E-site
G219A
IRF-E site
G239A
HSF site
1LMP2 promoter region, NCBI Reference Sequence NT_007592.15 Homo sapiens Chromosome 6. 2 not detected. 3
non-kinase activation region. 4 Evolutionary conservation refers to the species in which an identical residue was observed
in the homolog (p, pan troglodytes; c, canis lupus familiaris; b, bos Taurus; m, mus musculus; r, rattus norvegicus; g,
gallus gallus; d, danio rerio)
Supplementary Table S3 Somatic mutations in the IFN- pathway in human uterine leiomyosarcoma.
The data on somatic mutations in Table S2 are shown individually with respect to JAK1, JAK2,
STAT1, and LMP2.
32
33
Biological activity of human LMP2 and NF-B in uterine LMS
Number of Coloniesa
Morphology
G418R(total)
pCEM9
pLMP2wt
59.2
54.3
pLMP2K33A
56.4
p50 and p65
51.3
flatb
0
26.3(48.4%) 28.7(50.9%)
Transformedc
0
11.0(20.3%) 13.3(23.3%) 50.0(99.4%)
Flatrevertantc
0
16.0(29.5%) 14.0(24.8%)
Partially
0
0
Five micrograms of pCEM9, pLMP2wt, pLMP2K33A, pNF-Bp50, or pFN-Bp65 DNA
was transfected into 5 x 105 SKN cells and selected in a growth medium containing
1.0 mg/ml of G418, respectively. Total five microgram of p50 (2.5 g) and p65(RelA)
(2.5 g) DNA were cotransfected into 5 x 105 of SKN cells and selected in a growth
medium containing 1.0 mg/ml of G418 respectively.
a
Total number of colonies observed in the experiment is shown. Number in parentheses
indicates percent of total G418 resistant (G418) colonies.
b Observed after 1 week of selection with G418.
c Observed after 3 weeks of selection with G418.
d No colonies of flatrevertant and partially flat. morphology was observed in the cultures
transfected with NF-Bp50, NF-Bp65, the combination of NF-Bp50 with p65. The
percentage of found colonies consisting of SKN-p50p65 transformed (T type) cells is
indicated in Table S3. The experiments were repeated at three times with similar results.
Supplementary Table S4 Biological activity of LMP2 and NF-B in human uterine leiomyosarcoma
(LMS).
33
34
Biological Properties of the Transfectants
Cell line
aMorphology
PDTb
SoftAgarColony
Efficiency(%)cSized
Protein Expressionf
Tumorigenecitye
LMP2
LMP7
Calpo.
SKN-CEM9#1 hTransformed
SKN-CEM9#2
Transf Transformed
SKN-CEM9#3
Transformed
SKN-CEM9#4
Transformed
15.2
15.2
15.3
15.4
67
63
53
65
10-26
11-28
11-29
9-25
+++
++
+++
+++
W
W
W
W
L
L
L
L
-
SKNp50p65#1
Transf Transformed
SKNp50p65#2
Transformed
SKNp50p65#3
Transformed
SKNp50p65#4 Transformed
15.6
15.0
15.4
15.2
47
49
50
52
9-24
10-23
8-19
11-20
++
++
++
++
W
W
W
W
M
M
M
M
-
jCase
Fibro
.
P50/
p65
Inco
LMP2f
iTNF-
Cell death
L
L
L
L
gL
L
L
L
-
Sen
Sen
Sen
Sen
L
L
L
L
L
L
L
L
-
Sen
Sen.
Sen
Sen
Protein Expressionk
no.
LMP2
LMP7 Calpo.
p50/
p65
#14 Myo
H
M
M
H
#14 LMS
L
W
#15 Myo
H
M
M
H
#15 LMS
L
W
#16 Myo
H
M
M
H
#16 LMS
L
W
#17 Myo
H
M
M
H
#17 LMS
L
W
a Morphology. After 2 to 3 weeks, when most of the colonies outgrew and detached from the substrate, some colonies consisting of
very flat cells were found at frequencies of a few percent of the total number of G418-resistant colonies initially observed in Table S4.
Microscopic characteristics, such as flat cell morphology and transformed cell morphology, were analyzed. The number of flat cells or
transformed cells in 10 consecutive high-power-fields was counted. Transformed, no evidence of flat cells. b PDT Population Doubling
Time. c Rotio(%) of soft agar colonies/number of cells plated that have an ability to form colonies on plastic substrate. d Diameter of
colonies as expressed by the number of cells lined up across the colonies; determined on day 21. The experiments were repeated
three times with similar results. e Cells (1x107) were inoculated subcutaneously into 7-8-weeks old nude mice and periodically
examined for evidence of tumors: -, no evidence of tumor; +/-, tumor of <0.5 cm diameter; +, tumor of 0.5-1.0 cm diameter; ++, tumor
of 1.0-1.5 cm diameter, +++, tumor of 1.5-2.0 cm diameter; in two inoculated mice. Experiments were terminated at 5 weeks after
inoculation. f Estimated by immunoblot analysis; W, weak expression; L, low expression; M, medium expression, H, high expression;
-, no evidence of expression. g L, p65 expression was clearly detected, but p50 was weakly detected by immunoblot analysis. h
Incorporation of LMP2 into proteasome complexes. Proteasomes were isolated by GST-UbL beads in accordance with manufacturer’
s recomendations (Merck Ltd. Darmstadt, Germany). Immunoblot analysis was performed. -, no evidence of expression; +,
Detectable expression of LMP2 by immunoblot analysis. i TNF- induced cell death Sen., Sensitive to TNF--induced cell death;
Resi., Resistant to TNF--induced cell death. j Case number. Details in Table S1 and Table S2. k Estimated by
immunohistochemistry with appropriate antibodies. W, weak expression; L, low expression; M, medium expression; H, high
expression; -, no evidence of expression.
Supplementary Table S5 Biological Properties of the SKN-Transfectants with NF-B.
34
35
Biological activity of human LMP2 in cervical cancer
Number of Coloniesa
Morphology
G418R(total)
pCEM9
pLMP2wt
pLMP2K33A
49
51
46
flatb
0
0(0.0%)
0(0.0%)
Transformedc
49
50(98.0%)
44(95.6%)
Flatrevertantc
0
0(0.0%)
0(0.0%)
Partially
Five micrograms of pCEM9, pLMP2wt, pLMP2K33A DNA was transfected into 5 x 10 5
HeLa cells and selected in a growth medium containing 1.0 mg/ml of G418,
respectively.
aTotal number of colonies observed in the experiment is shown. Number in parentheses
indicates percent of total G418 resistant (G418) colonies.
b Observed after 1 week of selection with G418.
c Observed after 3 weeks of selection with G418.
No colonies of flatrevertant and partially flat. morphology was observed in the cultures
transfected with LMP2wt, LMP2K33A. The percentage of found colonies consisting of
HeLa-LMP2wt, HeLa-LMP2K33A transformed (T type) cells is indicated in Table S6.
The experiments were repeated at three times with similar results.
Supplementary Table S6 Biological activity of LMP2 in human cervical cancer.
35
36
Biological Properties of the Transfectants
SoftAgarColony
Efficiency(%)bSizec
iTNF-
Inco
f
LMP2 Cell death
LMP2
LMP7
+++
+++
++
+++
+++
Calponin
14-32
15-30
15-28
14-29
15-30
M
M
M
M
M
M
M
M
M
M
W
W
W
W
W
L
L
L
L
L
+
+
+
+
+
Resi.
Resi.
Resi.
Resi
Resi.
67
70
68
69
15-31
14-28
14-30
15-29
+++
+++
+++
++
H
H
H
H
M
H
M
M
W
W
W
W
L
L
L
L
+
+
+
+
71
68
70
69
14-33
15-29
14-30
15-31
+++
++
+++
+++
L
L
L
L
+
+
+
+
Resi.
Resi.
Resi.
Resi
i
Resi.
Resi.
Resi.
Resi
PDTa
HeLa
hTransformed
HeLa-CEM9#1
HeLa-CEM9#2
HeLa-CEM9#3
HeLa-CEM9#4
Transformed
Transformed
Transformed
Transformed
14.8
15.0
15.3
14.7
14.8
70
68
67
69
71
Tran
LMP9wt#1
LMP2wt#2
LMP2wt#3
LMP2wt#4
Transformed
Transformed
Transformed
Transformed
15.0
15.4
14.9
15.1
LMP2K33A#1
LMP2K33A#2
LMP2K33A#3
LMP2K33A#4
Transformed
Transformed
Transformed
Transformed
15.4
14.8
15.1
14.9
jCase
Protein Expressione
Tumorigenecityd
Molphology
Cell line
no.
Cancer tissue#1
Cancer tissue#2
Cancer tissue#3
Cancer tissue#4
H
M
W
H
M
W
H
M
W
H
H
W
Protein Expressionk
LMP2
LMP7
Calponin
M
M
H
M
M
M
H
M
W
W
W
W
Fibronectin
a PDT: Population Doubling Time. b Ratio(%) of soft agar colonies/number of cells plated that have an ability to form colonies on
plastic substrate. c Diameter of colonies as expressed by the number of cells lined up across the colonies; determined on day 21. The
experiments were repeated three times with similar results. d Cells (1x107) were inoculated subcutaneously into 7-8-weeks old nude
mice, and the mice were periodically examined for evidence of tumors, -, no evidence of tumors; +/-, tumors of <0.5 cm diameter; +,
tumors of 0.5-1.0 cm diameter; ++, tumors of 1.0-1.5 cm diameter, +++, tumors of 1.5-2.0 cm diameter, in two inoculated mice.
Experiments were terminated at 5 weeks after inoculation. e Estimated by immunoblot analysis. W, weak expression; L, low
expression; M, medium expression; H, high expression; -, no evidence of expression. f Incorporation of LMP2 into proteasome
complexes. Proteasome was isolated by GST-UbL beads in accordance with manufacturer’s recomendation (Merck Ltd. Darmstadt,
Germany). Immunoblot analysis was performed. -, no evidence of expression; +, Detectable expression of LMP2 by immunoblot
analysis. g Cryo UtSMC: normal human uterine liomyosarcoma cell line (Cambrex BioScience Walkersville, Inc. MD, USA). h
Morphology. After 2 to 3 weeks, when most of the colonies outgrew and detached from the substrate, some colonies consisting of very
flat cells were found at frequencies of around a few percent of the total number of G418-resistant colonies initially observed in Table
S4. Microscopic characteristics such as flat cell morphology and transformed cell morphology were analyzed. The number of flat cells
or transformed cells in 10 consecutive high-power-fields was counted. Transformed, no evidence about appearance of flat cells;
Flatrevertant, transformed cell number is less than 10% of total cell number; P.Flat., Partially Flatrevertant, transformed cell
number is less than 30% of total cell number; P.Tras., Partially Transformed, transformed cell number is higher than 30% of total
cell number. i TNF- induced cell death. Sen., Sensitive to TNF--induced cell death; Resi., Resistant to TNF--induced cell death.
j Case number, protein expression studies were performed using tissue array (Uterus cancer tissues, AccuMax Array, Seoul, Korea).
Details in manufacture’s information (AccuMax Array). k Estimated by immunohistochemistry with appropriate antibodies. W,weak
expression; L, low expression; M, medium expression, H, high expression; -, no evidence of expression.
Supplementary Table S7 Biological Properties of the HeLa-Transfectants with LMP2.
36