The Identification of Somatic Mutations in Interferon-g Signal Molecules in Human Uterine Leiomyosarcoma

Human uterine leiomyosarcoma (LMS) is neoplastic malignancy that typically arises in tissues of mesenchymal origin. The identification of novel molecular mechanism leading to human uterine LMS formation and the establishment of new therapies has been hampered by several critical points. We earlier reported that mice with a homozygous deficiency for proteasome beta subunit 9 (PSMB9)/b1i, an interferon (IFN)-g inducible factor, spontaneously develop uterine LMS. The use of research findings of the experiment with mouse model has been successful in increasing our knowledge and understanding of how alterations, in relevant oncogenic, tumour suppressive, and signaling pathways directly impact sarcomagenesis. The IFN-g pathway is important for control of tumour growth and invasion and, has been implicated in several malignant tumours. In this study, experiments with human tissues revealed a defective PSMB9/b1i expression in human uterine LMS that was traced to the IFN-g pathway and the specific effect of somatic mutations of Janus kinase (JAK1) molecule or promoter region on the transcriptional activation of PSMB9/b1i gene. Understanding the molecular mechanisms of human uterine LMS may lead to identification of new diagnostic candidates or therapeutic targets in human uterine LMS. DOI : 10.14302/issn2572-3030.jcgb-16-1276 Freely Available Online www.openaccesspub.org | JCGB CC-license DOI : 10.14302/issn2572-3030.jcgb-16-1276 Vol-1 Issue 1 Pg. no.30 Introduction Uterine mesenchymal tumours have been traditionally divided into benign tumour leiomyomas (LMA) and malignant tumour leiomyosarcomas (LMS) based on cytological atypia, mitotic activity and other criteria. Uterine LMS, which are some of the most common neoplasms of the female genital tract, are relatively rare uterine mesenchymal tumour, having an estimated annual incidence of 0.64 per 100,000 women (1). They account for approximately one-third of uterine sarcomas, of only 53% for tumours confined to the uterus (2,3). Generally, patients with uterine LMS typically present with vaginal bleeding, pain, and a pelvic mass. Gynecological cancer, for instance breast cancer and endometrial carcinomas, are strongly promoted by female hormones, but the rate of hormone receptor expression is reported to be significantly less in human uterine LMS compared with normal myometrium. These low receptor expressions were found to not correlate with the promotion of initial disease development or with the overall survival of patients with uterine LMS. As uterine LMS is resistant to chemotherapy and radiotherapy, and thus surgical intervention is virtually the only means of treatment for this disease (4,5,6), however, molecular targeting therapies against tumours have recently shown remarkable achievements (7,8). It is noteworthy that, when adjusting for stage and mitotic count, uterine LMS has a significantly worse prognosis than carcinosarcoma (9); developing an efficient adjuvant therapy is expected to improve the prognosis of the disease. A trend towards prolonged disease-free survival is seen in patients with matrix metalloproteinase (MMP)-2-negative tumours (10). Although typical presentations with hypercalcemia or eosinophilia have been reported, this clinical abnormality is not an initial risk factor for uterine LMS. To the best of our knowledge, little is known regarding the biology of uterine LMS; therefore, the risk factors that promote the initial development of uterine LMS and regulate their growth in vivo remain poorly understood. The mice with a targeted disruption of proteasome beta subunit 9 (PSMB9)/b1i, which is interferon (IFN)-g -inducible proteasome subunit, exhibited a defect in tissueand substratedependent proteasome function, and female PSMB9-deficient mice shown to develop uterine LMS, with a disease prevalence of 37% by 14 months of age (11,12). Defective PSMB9/b1i expression is likely to be one of the risk factors for the development of human uterine LMS, as it is in PSMB9/b1i-deficient mice (12). Recent report shows that stable PSMB9/b1i expression contributes to cell proliferation, which directly correlates to the progressive deterioration with increasing stage and grade of the tumour. As the importance and involvement of the IFN-g signal pathway in the transcriptional regulation of the transporter associated with antigen processing (TAP1) and PSMB9/b1i promoter have been established, it is demonstrated that the defective PSMB9/b1i expression was attributable to G871E somatic mutation in the ATPbinding region of JAK1 molecule in SKN cell line, which is established from patient with uterine LMS. It is (Continued on page 31) Corresponding Author: Takuma Hayashi, Dept. of Obstetrics and Gynecology, Shinshu University School of Medicine, Japan, 3-1-1, Asahi, Matsumoto, Nagano 390-8621, Japan. Tel: 81-263-37-2719, e-mail: takumah@shinshu-u.ac.jp


Introduction
Uterine mesenchymal tumours have been traditionally divided into benign tumour leiomyomas (LMA) and malignant tumour leiomyosarcomas (LMS) based on cytological atypia, mitotic activity and other criteria.Uterine LMS, which are some of the most common neoplasms of the female genital tract, are relatively rare uterine mesenchymal tumour, having an estimated annual incidence of 0.64 per 100,000 women (1).They account for approximately one-third of uterine sarcomas, of only 53% for tumours confined to the uterus (2,3).Generally, patients with uterine LMS typically present with vaginal bleeding, pain, and a pelvic mass.Gynecological cancer, for instance breast cancer and endometrial carcinomas, are strongly promoted by female hormones, but the rate of hormone receptor expression is reported to be significantly less in human uterine LMS compared with normal myometrium.These low receptor expressions were found to not correlate with the promotion of initial disease development or with the overall survival of patients with uterine LMS.
As uterine LMS is resistant to chemotherapy and radiotherapy, and thus surgical intervention is virtually the only means of treatment for this disease (4,5,6), however, molecular targeting therapies against tumours have recently shown remarkable achievements (7,8).It is noteworthy that, when adjusting for stage and mitotic count, uterine LMS has a significantly worse prognosis than carcinosarcoma (9); developing an efficient adjuvant therapy is expected to improve the prognosis of the disease.A trend towards prolonged disease-free survival is seen in patients with matrix metalloproteinase (MMP)-2-negative tumours (10).Although typical presentations with hypercalcemia or eosinophilia have been reported, this clinical abnormality is not an initial risk factor for uterine LMS.To the best of our knowledge, little is known regarding the biology of uterine LMS; therefore, the risk factors that promote the initial development of uterine LMS and regulate their growth in vivo remain poorly understood.
The mice with a targeted disruption of proteasome beta subunit 9 (PSMB9)/b1i, which is interferon (IFN)-g -inducible proteasome subunit, exhibited a defect in tissue-and substrate-dependent proteasome function, and female PSMB9-deficient mice shown to develop uterine LMS, with a disease prevalence of 37% by 14 months of age (11,12).Defective PSMB9/b1i expression is likely to be one of the risk factors for the development of human uterine LMS, as it is in PSMB9/b1i-deficient mice (12).Recent report shows that stable PSMB9/b1i expression contributes to cell proliferation, which directly correlates to the progressive deterioration with increasing stage and grade of the

Uterine LMS
The effects of IFN-γ on expression of PSMB9/b1i was examined using five cell lines (13).The expression of PSMB9/b1i were not markedly induced by IFN-γ treatment in human uterine LMS cell lines, although cervical epithelial adenocarcinoma cell lines and normal human uterus smooth muscle cells underwent strong induction of PSMB9/b1i following IFN-γ treatment (13).(Supplementary Figure 1).Tyrosine phosphorylated STAT1 forms homodimers that translocate to the nucleus and bind GAS (IFN-γ-activated site) elements in the promoters of IFN-γ-regulated genes (14,15) (Supplementary Figure 1).The defect was localized to JAK1 activation, which acts upstream in the IFN-γ signal pathway since IFN-γ treatment could not strongly induce JAK1 kinase activity in human uterine LMS cell lines.Sequence analysis demonstrated that the loss of IFN-γ responsiveness in the human uterine LMS cell line was attributable to the inadequate kinase activity of JAK1 due to a G781E somatic mutation in the ATP-binding region (13).
Genetic alterations in tyrosine kinases have previously been firmly implicated in tumourigenesis, but only a few serine/threonine kinases are known to be mutated in human cancers (18,19,20,21,22)

Gene Analyses of Human Uterine Lyomyosarcoma
Uterine LMS are relatively rare mesenchymal tumours, having an estimated annual incidence of 0.64 per 100 000 women.They account for approximately    transmembrane tyrosine kinase receptor, c-kit, may be associated with the pathogenesis of human uterine LMS (28,29,30).However, in terms of the tumourigenesis of human uterine LMS, merely comparing the expression of potential pro-oncogenic factors between normal and malignant tissues is not sufficient because the results obtained may be the consequence of malignant transformation and, therefore, not necessarily the cause.
In addition, dysregulation of apoptotic mechanisms has also been implicated in many human malignancies.
Although the significant differential expression of The down regulation of major histocompatibility complex (MHC) expression, including the TAP1 and PSMB9/b1i genes, is one of the biological mechanisms tumour cells use to evade host immunosurveillance (37,38,39).Recently, the incidence of IFN-g unresponsiveness in human tumours was examined in several malignant tumours, and revealed that approximately 33% of each group exhibited a reduction in IFN-g sensitivity (40).Nevertheless, the expression of PSMB9/ b1i, rather than providing an escape from immune surveillance, seems to play an important role in the negative regulation of human uterine LMS cell growth.
Defective expression of PSMB9/b1i is likely to be one of the risk factors for the development of human uterine neoplasm, as it is in the PSMB9/b1i-deficient mouse.
Thus, gene therapy with PSMB9/b1i expression vectors may be a new treatment for human uterine LMS that exhibits a defect in the expression of PSMB9/b1i.
Because there is no effective therapy for unresectable human uterine LMS, our results may bring us to specific molecular therapies to treat this disease.

Tissue Collection
A total of 51 patients aged between 32 and 83 years who were diagnosed with smooth muscle tumours in the uterus were selected from pathological files.Serial Madison, WI) was added and incubated as previously reported (45,46).

Sequencing of the
tumour.As the importance and involvement of the IFN-g signal pathway in the transcriptional regulation of the transporter associated with antigen processing (TAP1) and PSMB9/b1i promoter have been established, it is demonstrated that the defective PSMB9/b1i expression was attributable to G871E somatic mutation in the ATPbinding region of JAK1 molecule in SKN cell line, which is established from patient with uterine LMS.It is furthermore likely that the expressions of PSMB9/b1i are down-regulated in human uterine LMS tissues such like human uterine LMS cell line.We demonstrate that there are serious mutational defects in the factors on the IFNγ signal pathway, which is the key cell-signaling pathway for PSMB9/b1i expression and promoter region of PSMB9/b1i gene, in human uterine LMS tissues.The somatic mutational defects in the IFN-γ signal molecules may induce the initial development of human uterine LMS.Recent advances in our understanding of the biological characters of uterine LMS have concentrated on the impaired IFN-γ signal pathway.It is clear that somatic mutations in key regulatory factors (tumour suppressors and proto-oncogenes) alter the behavior of cells and can potentially lead to the unregulated growth seen in malignant tumour.Therefore, continued improvement of our knowledge of the molecular biology of uterine LMS may ultimately lead to novel therapies and improved outcome.
serious loss in the ability to induce expression of PSMB9/b1i in human uterine LMS tissues in comparison with normal myometrium tissues located in same tissue sections and other 4 mesenchymal tumour types (Figure1 in human uterine LMS (Figure1C).The both research experiments strongly supported the research findings obtained from IHC experiments.Somatic Mutations in IFN-γ Signal Pathway inHuman Uterine LMS Tissues IFN-γ treatment markedly increased the expression of PSMB9/b1i, a subunit of the immunoproteasome, which alters the proteolytic specificity of proteasomes.After binding of IFN-γ to the type II IFN receptor, which is constructed by two components, IFNγ receptor subunit 1 (IFNGR1) and IFN-γ receptor subunit 2 (IFNGR2), Janus-activated kinase 1 (JAK1) and JAK2 are activated and phosphorylate the signal transducer and activator of transcription 1(STAT1) on the tyrosine residue at position 701 (Tyr701) and the serine residue at position 727 (Ser727)(14,15) analysis also revealed that somatic mutations, which were identified in the catalytic domains of these molecules, resulted in impaired activations of tyrosine kinases or transcriptional factor(24).In a recent report, a comparative genomic hybridization (CGH)-based analysis of human LMS using a high resolution genome-wide array gave gene-level information about the amplified and deleted regions that may play a role in the development and progression of human uterine LMS.Other reports showed that among the most intriguing changes in genes were losses of JAK1 (1p31-p32) and PSMB9/b1i (6p21.3)(25,26).It has also been demonstrated that a correlation exists between the development of malignant tumours and ethnic background, so we conducted CGH experiments with tissue samples obtained from Japanese patients in order to obtain gene-level information.Our results showed that human uterine LMS having a clear functional loss at JAK1 (1p31-p32) and PSMB9/b1i (6p21.3)also harbored one nonsense mutation and one deletion, suggesting a possible homozygous loss of function.The discovery of these mutational defects in a key cell-signaling pathway may be important in understanding the pathogenesis of human uterine LMS.

Table 1 :
Identification of somatic (tumour-specific) mutations in the cataclytic domains of JAK1, JAK2 kinases, STAT1 or activation region of the promoter region of PSMB9/b1i gene in human uterine leiomyosarcoma (total 23 cases).The gemonic DNA was extracted from the human uterine leiomyosarcoma tissues and normal uterine smooth muscle tissues using the protocol indicated in the materials and methods section of this manuscript.The restricted DNA fragments for the direct sequence analysis were amplified by PCR procedure with the appropriate primers for the ATP-binding region and kinase activation domain of the JAK1 molecule, the promoter region of PSMB9/b1i gene, the Tyr701 and Ser727 aminoacid of STAT1 molecule, and the ATP-binding region and kinase activation domain of the JAK2 molecule.Information of human uterine leiomyosarcomas and the primer sets was indicated in Supplementary

Figure 1 :
Figure 1: Differential expression of PSMB9/b1i expression in human normal myometrium and several mesenchymal tumour types.(A) Immunohistchemistry of PSMB9/b1i in normal myometrium, usual leiomyoma, Bizarre leiomyoma, smooth muscle tumour of uncertain malignant potential (STUMP) and uterine leiomyosarcoma tissues located in same tissue.For all samples, 5-mm sections of tissues specimens were stained with anti-PSMB9/b1i antibody revealed by peroxidase-cojugated anti-rabbit IgG antibody.(B) IHC experiments individually performed at several medical facilities revealed a marked loss in the ability to induce PSMB9/b1i expression in human uterine LMS tissues compared to that in normal human myometrium located in the same tissue section, as well as to that in LMA tissues.Normal total: 58 cases, LMA total: 50 cases, Bizarre Leiomyoma total: 3 cases, LMS total: 56 cases.The experiments were performed three times with similar results.(C) Examinations of mRNA expression for PSMB9/b1i and b-ACTIN in normal human myometrium (Myo.), uterine usual leiomyoma (LMA), uterine leiomyosarcoma (LMS) and IFN-g-treated HeLa cells by reverse transcription-polymerase chain reaction (RT-PCR).RT-PCR was performed with the appropriate primers indicated in the materials and methods section of this manuscript.The DNA products amplified by RT-PCR were loaded in agarose gel.The expression levels of PSMB9/b1i and b-ACTIN were examined by western blotting with appropriate antibodies.The cytosolic extracts were prepared from normal human myometrium, uterine usual leiomyoma (LMA) and uterine leiomyosarcoma (LMS), and IFN-g-treated HeLa cells, and 50 mg of cytosolic extracts were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).PSMB9/b1i levels were significantly decreased in human uterine LMS compared with normal human myometrium and human usual LMA.Student's t-test.*P < 0.005.(n = 8 per group).

2 (
apoptotic and cell cycle regulatory factors in human uterine LMS, such as B-cell Lymphoma-2 (BCL-2), BCL-2-Associated X protein (BAX), P16 Inhibts CDK4 (P16/ INK4a), P21 Cyclin-Dependent Kinase Inhibitor 1 (P21/ CIP1), P27 Kinase Inhibitor Protein 1 (P27/KIP1), Cellular v-KIT Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), Mitogen-Inducible Gene-MIG-2), MDM2, Tumour Protein 53 (TP53), have all been reported and compared to normal myometrium, there exists no scientific evidence to show that abnormal expression of these factors directly correlates to the initiation and promotion of human uterine LMS.PSMB9/ b1i-deficient mice were reported to be prone to the development of uterine LMS, but not in their parental mice, C57BL/6 mice (12).The percentage of mice with overt tumours increased with age after six months, with a cumulative prevalence of disease in female mice of 37% by 14 months of age and no apparent plateau at this late observation time.Histopathological examinations of PSMB9/b1i-deficient uterine neoplasms revealed common characteristic abnormalities of uterine LMS.In addition, recent research reports show the loss in the IFN-g-inducible ability of PSMB9/b1i expressions in SKN cell line and other primary human uterine LMS cells established from patients.The histopathological experiments demonstrated a serious loss in the ability to induce the expression of PSMB9/b1i in human uterine LMS tissues in comparison with normal myometrium tissues located in same tissue sections (Fig. 1).IFN-g treatment markedly increased the expression of PSMB9/b1i, a subunit of the proteasome, which alters the proteolytic specificity of proteasomes.Sequence analysis demonstrated that the loss of IFN-g responsiveness in the human uterine LMS cell line was attributable to the inadequate kinase activity of JAK1 due to a G781E somatic mutation in the ATP-binding region(13).The defect was localized to JAK1 activation, which acts upstream in the IFN-g signal pathway since IFN-g treatment could not strongly induce JAK1 kinase activity in human uterine LMS cell lines.Genetic alterations in tyrosine kinases have previously been firmly implicated in tumourigenesis, but only a few serine/threonine kinases are known to be mutated in human cancers(31,32,33,34).For instance, mice carring homozygous deletion of Phosphatase and Tensin Homolog Deleted from Chromosome 10 (Pten) alleles developed widespread smooth muscle cell hyperplasia and abdominal LMS(35), and JUN oncogene amplification and over-expression block adipocytic differentiation in highly aggressive sarcomas(22).Most frequently, LMS have appeared in the uterus, retroperitoneum or extremities, and although histologically indistinguishable, they have different clinical courses and chemotherapeutic responses.The molecular basis for these differences remains unclear, therefore, the examination of human uterine LMS tissues (23 LMS tissue sections and normal tissue sections located in the same tissue) was performed to detect somatic mutations in the IFN-g signal molecules.In a recent report, high-resolution genomewide array comparative genomic hybrodization (CGH) analysis of LMS cases gave gene-level information about the amplified and deleted regions that may play a role in the development and progression of human uterine LMS.Among the most intriguing genes, whose copy number sequence was revealed by CGH, were loss of JAK1 (1p31-p32) and PSMB9/b1i (6p21.3)(25,26).The discovery of these mutational defects in a key cellsignaling pathway may be an important development in the pathogenesis of human uterine LMS.The growth of JAK1-deficient cell lines reportedly is unaffected; similarly, the cell cycle distribution pattern of freshly explanted tumour cells derived from JAK1-deficient tumours shows no response to IFN-g signaling(36).The growth of the original SKN cells, which had defective JAK1 activity, was unaffected by IFN-g treatment.In contrast, the growth of JAK1transfected SKN cells, which had strong exogenous JAK1 activity, was prevented by IFN-g treatment.Interesting-ly, when PSMB9/b1i-transfected SKN cells, which have marked the expression of PSMB9/b1i, were analyzed, expression of exogenous PSMB9/b1i resulted in cell growth inhibition.Conversely, the growth of PSMB9/b1itransfected SKN cells was unaffected by IFN-g signal pathway.Taken together, IFN-g response to cell growth inhibition may be attributable to the physiological significance of PSMB9/b1i.
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 Freely Available Online www.openaccesspub.org| JCGB CC-license DOI : 10.14302/issn2572-3030.jcgb-16-1276 Vol-1 Issue 1 Pg.no.-38 obtaining written consent from each patient.The pathological diagnosis of human uterine mesenchymal tumours was performed using established criteria with some modifications (41,42).Briefly, usual leiomyoma (usual LMA) was defined as a tumour 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 tumour with significantly increased cellularity (>2000 myoma cells/HPF) and a MI<5, but without cytologic atypia.Bizarre leiomyoma (BL) was defined as a tumour either with diffuse nuclear atypia and a MI<2 or with focal nuclear atypia and a MI<5 without coagulative tumour cell necrosis.A tumour of uncertain malignant potential (UMP) was defined as a tumour with no mild atypia and a MI<10, but with coagulative tumour cell necrosis.Leiomyosarcoma (LMS) was diagnosed in the presence of a MI>10 with either diffuse cytologic atypia, coagulative tumour cell necrosis, or both.Of the 113 uterine mesenchymal tumours, 52 cases were diagnosed as uterine LMA, 3 cases were Bizarre LMA, and 58 cases were uterine LMS.Protein expression studies with cervical epithelium and carcinoma tissues were performed using tissue arrays (Uterus cancer tissues, AccuMax Array, Seoul, Korea).Details regarding tissue sections are indicated in the manufacturer's literature (AccuMax Array).Immunohitochemistry (IHC) Immunohistochemical staining for PSMB9/b1i, Estrogen Receptor (ER), Progesterone Receptor (PR), TP53, and Ki-67/MIB1 was performed on the serial human uterine LMS sections.Antibodies for ER(ER1D5), PR(PR10A), TP53(DO-1), and Ki-67(MIB-1) were purchased from Immunotech (Marseille, France).Antihuman PSMB9 antibody was produced by SIGMA-Aldrich collaboration Laboratory (SIGMA-Aldrich, Japan Science and Technology Agency (JST) and Shinshu University).IHC was performed using the avidin-biotin complex method previously described.Briefly, one representative 5-mm tissue section was cut from a paraffin-embedded sample of the radical hysterectomy specimen from patients with uterine LMS.Sections were deparaffinized and rehydrated in graded alcohols and then incubated with normal mouse serum for 20 min.Sections were incubated at room temperature for 1 h with primary antibody.Afterwards, sections were incubated with a biotinylated secondary antibody (Dako, Carpinteria, CA, USA) and then exposed to a streptavidin complex (Dako).Complete reaction was revealed by 3, 3¢diaminobenzidine, and the slide was counterstained with hematoxylin.Normal USM portions in the specimens were used as positive controls.Negative controls consisted of tissue sections also incubated with normal rabbit IgG instead of the primary antibody.These studies are registered, at Shinshu University in accordance with local guidelines (approval no.M192) The expressions PSMB9/b1i and b-ACTIN transcripts were examined using RT-PCR.Total RNA was prepared from human uetine LMS tissues and normal myometrim tissues using TRIzol reagent according to the manufacturer's protocol (Invitrogen Co., CA).The RNA was reverse-transcribed with Superscript II enzyme (Invitrogen), the single strand cDNA was used to amplify.PSMB9/b1i and b-ACTIN transcripts using PCR analysis with the appropriate primer sets following a program of 35 cycles of 94°C for 30s, 60°C for 30s and 72°C for 1.5min with additional 5min for extention of transcripts (13,43,44).The primers used for PCR are as follows: PSMB9/b1i; 5'-GGGATAGA ACTGGAGGAACC-3' and 5'-AGATGACACCCCCGCTTGAG-3', b2-MICROGLOBULIN; 5'-T GAAGCTGACAGCATTCG-3' and 5'-TGCGGCATCTTCAAACCTCC-3', b-ACTIN; 5'-TCCGG AGACGGGGTCA-3' and 5'-CCTGCTTGCTGATCCA-3'.These studies are registered, at Shinshu University in accordance with local guidelines (approval no.4737, no.Freely Available Online www.openaccesspub.org| JCGB CC-license DOI : 10.14302/issn2572-3030.jcgb-16-1276 Vol-1 Issue 1 Pg.no.-39 150 and no.M192) onto a poly-vinylidene 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 the primary antibodies were mouse monoclonal or rabbit polyclonal, obtained from several industries, and were used at different final dilutions (1:1000~1:500) in the blocking buffer.These antibodies were raised using the following proteins as immunogens: PSMB9/b1i (23.4 kDa protein), PSMB8/b5i (30.3 kDa protein), b-ACTIN (41.7 kDa protein).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 alkaliphosphatase conjugated goat-anti-mouse IgG antibody or anti-rabbit IgG antibody (Promega, Madison, WI) diluted in 5% non -fat milk in wash buffer.The PVDF membranes were washed with wash buffer three times for 30 min, and Western Blue Stabilized Substrate (Promega Co. Catalytic Domains of JAK1, STAT1, JAK2, and PSMB9/b1i Promoter Region To demonstrate whether the somatic mutations in the ATP-binding region and kinase activation domain of the JAK1 molecule, promoter region of PSMB9/b1i gene, the Tyr701 and Ser727 aminoacid of STAT1 molecule, and the ATP-binding region and kinase activation domain of the JAK2 molecule playing in human uterine LMS were identified, the isolating of gemonic DNA and direct sequencing was carried out.The gemonic DNA was extracted from the human uterine LMS tissues and normal USM tissues using the standard protocol.The gemonic DNA was subjected to PCR procedure.The restricted DNA fragments for the direct sequence analysis were amplified by PCR according to the published sequencing oligonuclrotide primers.Polymerase chain reaction products were directly sequenced using DYEnamic Terminator Cycle sequencing Kit (Amersham-Biosciences, Piscataway, NJ, USA) by ABI Prism 3100 Genetic Analyzer (Applied Biosystem, Foster City, CA, USA).The sequences of mutant JAK1, STAT1, and promoter region of PSMB9/b1i gene derived from the individual human uterine LMS tissue sections were registered at DDBJ (Accession: AB219242, DJ055380, DJ055379, DJ055378, DJ055377, DJ055376).Details of direct sequencing are indicated in Supplementary material.

Table 2 :
Somatic mutations in IFN-g signaling pathway in human uterine leiomyosarcoma.The data of somatic mutations in table 1 was shown separately with respect to each gene, JAK1, JAK2, STAT1 and activation region of the promoter of PSMB9/b1i gene.