BubR1 in the Coordination of Kinetochore-Microtubule Attachment and Spindle Assembly Checkpoint Signaling

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 2. 이현숙.BubR1 is a key protein constituting the mitotic checkpoint complex (MCC). During mitosis, the spindle assembly checkpoint (SAC) acts to delay anaphase onset until all chromosomes are attached to mitotic spindles at kinetochores. The SAC works through generation of MCC, which inhibits the anaphase-promoting complex/cyclosome E3 ligase (APC/C) in the cytoplasm. This study addresses on first, how BubR1 coordinated kinetochore-microtubule (KT-MT) attachments and SAC signaling. Secondly, it also addresses on how a regulator in mitosis, tumor suppressor BRCA2 serves as a signaling platform during SAC. In order to understand the physiological role of BubR1 acetylation, acetylation deficient knock-in mice (K243R/+) were generated. After 60 weeks, 38% of mice spontaneously developed tumors at various tissues. K243R/+ Mouse Embryonic Fibroblasts (MEFs) were highly aneuploid and had weakened SAC. At kinetochores, unstable KT-MT attachments were observed due to reduced recruitment of PP2AB56a. Insufficient amount of PP2AB56a could not counterbalance the excessive Aurora B activity at kinetochores. All together, unstable KT-MT attachment and failure in maintaining MCC formed in mitosis led to accumulation of chromosome instability (CIN) in K243R/+. These CIN manifested in various forms in the acetylation deficient mice generated a mutation-prone cellular environment favoring tumorigenesis. Previous works have shown that in prometaphase, BubR1 acetylation occurs at kinetochores, only if BRCA2 is present to support the BubR1-P300/CBP-associated factor (PCAF) interaction (Choi et al., 2012). My research showed that BubR1 was deacetylated when SAC was silenced. Deacetylation of BubR1 was a cue to SAC silencing, as cells expressing acetylation mimetic form of BubR1 could not exit mitosis after the metaphase delay. Also, acetylation of BubR1 diminished when SAC silencing was mimicked in mitotic cells. Deacetylation of BubR1 was mediated by class I HDACs, and BRCA2 was needed for HDACs to interact with BubR1 at prometaphase. Hence, one can conclude that BRCA2 not only regulates BubR1 acetylation, but it presents the acetylated BubR1 to HDACs at prometaphase for deacetylation. Analysis on the BRCA2 complex in mitosis suggested that BRCA2 acts as a signaling platform within the SAC signaling network by specifying the interaction and localization of essential proteins at kinetochores. In essence, my study provides further insight into the following key questions in mitosis. First, the question of how KT-MT attachment is sensed and relayed to SAC was further explained by elucidating the role of BubR1 in coordinating KT-MT attachment and SAC signaling. Second, the question of how complex SAC signaling is punctually regulated by multiple proteins during metaphase to anaphase transition was addressed through elucidating the role of BRCA2 as a scaffold for BubR1 acetylation/deacetylation at kinetochores.I INTRODUCTION 9 I-1. Mitosis and chromosome instability (CIN) 9 I-2. Mouse models for CIN 14 I-3. Spindle assembly checkpoint (SAC) activity and regulation 18 I-4. Sensors in SAC 25 I-5. Roles of BubR1 in mitosis 29 I-6. A Novel function of BRCA2 in mitosis 37 I-7. SAC silencing 39 II. MATERIALS AND METHODS 42 II-1. Genotyping 42 II-2. Cell culture, drugs and transfection 42 II-3. Constructs, antibodies and siRNA 43 II-4. Immunoprecipitation (IP) and western blot (WB) 45 II-5. Immunofluorescence assay (IFA) 45 II-6. Chromosome spreads 46 II-7. Histopathology 46 II-8. Microscope image acquisition and processing 47 II-9. Cold stable microtubule assay and scoring of the immunofluorescence intensity 47 II-10. Cell cycle analysis 48 III. RESULTS 49 BUBR1 ACETYLATION IS REQUIRED FOR KINETOCHORE-MICROTUBULE ATTACHMENT AND STABLE MITOTIC CHECKPOINT COMPLEX FORMATION 49 III-1. Loss of BubR1 acetylation leads to spontaneous tumorigenesis in mice 49 III-2. CIN in BubR1 acetylation deficient mice 58 III-3. SAC is weakened in BubR1 acetylation deficient mice 62 III-4. Intact initialization of SAC in BubR1 acetylation deficient mice 65 III-5. BubR1 acetylation deficiency leads to premature mitotic checkpoint complex (MCC) disassembly 68 III-6. BubR1 acetylation deficiency leads to abrupt kinetochore-microtubule attachment 72 III-7. Cell cycle profile of cells with BubR1 acetylation deficiency 86 EXTENDED STUDY ON THE ROLE OF BRCA2 IN MITOSIS: SPINDLE ASSEMBLY CHECKPOINT SIGNALING REGULATING BUBR1 91 III-8. BRCA2 complex in mitosis 91 III-9. BRCA2 mediated BubR1 acetylation is required for the BubR1-HDAC interaction 94 III-10. BubR1 deacetylation is a cue to SAC silencing 99 IV. DISCUSSION 113 IV-1. Dual roles of BubR1 acetylation and tumorigenesis 113 IV-1-1. BubR1 acetylation deficiency results in premature disassembly of MCC 116 IV-1-2. BubR1 acetylation deficiency disrupts stable kinetochore-microtubule attachment 117 IV-2. BubR1 acetyl/deacetylation in SAC signaling network 120 IV-3. BRCA2 is a hub for SAC silencing: a hub for BubR1 modulation 122 V. REFERENCE 127 국문 초록 135Docto

A Multi-institutional Study of Interlaboratory Variance in the Estrogen and Progesterone Receptor Assays

Purpose The expression of hormone receptors is the most reliable factor for predicting the responsiveness to hormonal therapy At present, immunohistochemistry (IHC) is considered as a practically reliable method. This study was designed to examine the interlaboratory variance in immunohistochemical assays for estrogen receptor (ER) and progesterone receptor (PR) in Korea Methods The Korean Study Group for Breast Pathology (KSGBP) made a questionnaire to know the current situation in HR assay in Korea. The questionnaire was sent to the members of KSGBP by e-mail, which were included eight questions relating to tissue handling, ER/PR IHC procedure and interpretation method. Forty laboratories replied with the completed questionnaire Results All 40 laboratories were using formalin as a fixative. Pretreatment was performed using six different methods including autoclave (25%), microwave (30%) and full autostainer (15%). Primary antibodies for ER were SP1 in 40%, 6F11 in 27.5% and 1D5 in 32.5%. Primary antibodies for PR were more variable (seven clones) than those for ER Interpretation method used was Allred system in 20%, modified Allred system in 15%, report the % of positive tumor cells in 45%, positive/negative in 15% and others in 5% The expression rate of ER was ranged from 45.6% to 93% (mean 635%) and the expression rate of PR was ranged from 27% to 90% (mean 59 1%) The differences according to the numbers of breast cancer in each institute, primary antibodies, detection systems and interpretation methods did not influence to the expression rate of ER/PR, statistically (p>0 05) Conclusion In Korea, the interlaboratory variance in ER/PR IHC procedure was too huge to make a standardized method We suggest the proper quality control program such as ER/PR staining with positive internal and external controls and negative control might be better to aim at getting similar results among the different laboratones rather than trying to standardize the procedure.Yun YH, 2007, BREAST CANCER RES TR, V106, P245, DOI 10.1007/s10549-006-9490-7UMEMURA S, 2006, BREAST CANC, V13, P232JOO HJ, 2006, MANUAL QUALITY CONTR, P139Goldhirsch A, 2005, ANN ONCOL, V16, P1569, DOI 10.1093/annonc/mdi326Mann GB, 2005, J CLIN ONCOL, V23, P5148, DOI 10.1200/JCO.2005.02.076Rudiger T, 2002, AM J SURG PATHOL, V26, P873Rhodes A, 2001, AM J CLIN PATHOL, V115, P44Fitzgibbons PL, 2000, ARCH PATHOL LAB MED, V124, P966Rhodes A, 2000, J CLIN PATHOL, V53, P292Rhodes A, 2000, J CLIN PATHOL, V53, P125Harvey JM, 1999, J CLIN ONCOL, V17, P1474Taylor CR, 1999, AM J CLIN PATHOL, V111, P443Barnes DM, 1998, EUR J CANCER, V34, P1677Barnes DM, 1996, BRIT J CANCER, V74, P1445Alberts SR, 1996, CANCER, V78, P764GREENE GL, 1982, J STEROID BIOCHEM, V16, P353GREENE GL, 1980, P NATL ACAD SCI-BIOL, V77, P5115

Histologic Alterations in the Ipsilateral and Contralateral Testes and Epididymides of Rats following Unilateral Torsion and Detorsion of the Testes

PURPOSE: This investigation was undertaken to determine the damage to the testes and epididymides following torsion and detorsion of the testes. MATERIALS AND METHODS: The right testes of 8-week-old male rats(n=30) were subjected to torsion for 10 min. At 0, 1, 4, 8, and 24 hours, and 1 week after the repair of a torsion, the ipsilateral and contralateral testes and epididymides were harvested. The mean number of spermatids per tubule, the mean seminiferous tubular diameter(MSTD), and the germinal epithelial cell thickness(GECT) were used to evaluate changes to the testes. The histologic changes to the epididymal ductal epithelium were also evaluated. RESULTS: The mean number of spermatids per tubule, GECT, and MSTD were significantly decreased in the 24-hour ipsilateral detorsion group, but minimal changes to ipsilateral testes were observed in the 1-week detorsion group. There was no evidence of histologic changes to the testes in any of the contralateral detorsion groups. The interstitial fibroblast proliferation and hemorrhage of the ipsilateral epididymis were found in the 4-hour detorsion group and increased in the 8-hour detorsion group. Interstitial fibroblast proliferation was prominent in the ipsilateral epididymis of the 24-hour detorsion group, but was only occasionally observed in the contralateral epididymides. Shortening of the tubular epithelial cell height and tubule dilatation were observed in the ipsilateral and contralateral epididymis 1 week after detorsion. CONCLUSIONS: Torsion/detorsion damage was found earlier and at a higher intensity in the epididymides than in the testes. This finding may be due to the protection afforded by the blood-testis barrier

Factors Affecting the Ipsilateral Breast Tumor Recurrence after Breast Conserving Therapy in Patients with T1 and T2 Tumors

본 논문은 2008년 대한외과학회 추계학술대회에서 구연 발표되었음.Purpose: Nearly half of all breast cancers are treated with breast conserving therapy (BCT). The purpose of this study was to identify the risk factors for ipsilateral breast tumor recurrence (IBTR) after BCT in T1 and T2 breast cancer patients. Methods: The medical records of 294 T1 or T2 breast cancer patients who underwent BCT at Seoul National University Hospital between January 1998 and December 2002 were retrospectively reviewed. Kaplan-Meier curves and Cox proportional regression analysis were used to identify the significant clinicopathologic factors that influence IBTR. Results: Among the 294 patients, 12 patients (4.8%) developed IBTR after a median follow-up of 82 months. Univariate analysis demonstrated that younger age (<= 35 year) had significant associations with IBTR (p=0.006). Tumor size, lymph node status, histologic grade, extensive intraductal component, lymphovascular invasion, and close resection margins were not significant factor associated with IBTR. The triple negative breast cancer subtype also did not have significant association with IBTR. Multivariate analysis showed that the younger age at diagnosis was a significant predictor of IBTR with a FIR of 3.86 (p=0.036; 95% CI, 1.09-13.60). Conclusion: Younger age at diagnosis (<= 35) may be associated with an increased risk of IBTR in patients who underwent BCT.Han W, 2010, BREAST CANCER RES TR, V119, P193, DOI 10.1007/s10549-009-0388-zBenson JR, 2009, LANCET, V373, P1463Luini A, 2009, BREAST CANCER RES TR, V113, P397, DOI 10.1007/s10549-008-9929-0Nguyen PL, 2008, J CLIN ONCOL, V26, P2373, DOI 10.1200/JCO.2007.14.4287Lee JW, 2007, J BREAST CANCER, V10, P206Dent R, 2007, CLIN CANCER RES, V13, P4429, DOI 10.1158/1078-0432.CCR-06-3045KANG SH, 2007, J KOREAN SURG SOC, V73, P385Haffty BG, 2006, J CLIN ONCOL, V24, P5652, DOI 10.1200/JCO.2006.06.5664Ahn SH, 2006, BREAST CANCER RES TR, V99, P209, DOI 10.1007/s10549-006-9188-xWapnir IL, 2006, J CLIN ONCOL, V24, P2028, DOI 10.1200/JCO.2005.04.3273Komoike Y, 2006, CANCER, V106, P35, DOI 10.1002/cncr.21551Abe O, 2005, LANCET, V366, P2087Noh WC, 2005, WORLD J SURG, V29, P1001, DOI 10.1007/s00268-005-7928-4Kim KJ, 2005, JPN J CLIN ONCOL, V35, P126, DOI 10.1093/jjcolyhi039Han WS, 2004, BMC CANCER, V4, DOI 10.1186/1471-2407-4-82MORROW M, 2004, DIS BREAST, P719Arriagada R, 2003, ANN ONCOL, V14, P1617, DOI 10.1093/annonc/mdg452Singletary SE, 2002, AM J SURG, V184, P383Veronesi U, 2002, NEW ENGL J MED, V347, P1227Fisher B, 2002, NEW ENGL J MED, V347, P1233Freedman GM, 2002, J CLIN ONCOL, V20, P4015, DOI 10.1200/JCO.2002.03.155Haffty BG, 2002, LANCET, V359, P1471Jobsen JJ, 2001, EUR J CANCER, V37, P1820Sasson AR, 2001, CANCER, V91, P1862Voogd AC, 2001, J CLIN ONCOL, V19, P1688Park CC, 2000, J CLIN ONCOL, V18, P1668Freedman G, 1999, INT J RADIAT ONCOL, V44, P1005Peterson ME, 1999, INT J RADIAT ONCOL, V43, P1029SUH CO, 1997, J KOREAN SOC THER RA, V15, P331BORGER J, 1994, J CLIN ONCOL, V12, P653WAZER DE, 1992, J CLIN ONCOL, V10, P356SOLIN LJ, 1991, INT J RADIAT ONCOL, V21, P279JACQUEMIER J, 1990, BRIT J CANCER, V61, P873VERONESI U, 1990, EUR J CANCER, V26, P671FOURQUET A, 1989, INT J RADIAT ONCOL, V17, P719LOCKER AP, 1989, BRIT J SURG, V76, P890

Development of in vitro 3D assays for functional evaluation of immunogenic cell death

MasterIn vitro evaluation of cancer drug efficacy is an important process to screen cancer drug candidates. Conventional cancer drug screening methods based on the measurement of cancer cell cytotoxicity using 2-dimensional (2D) culture can provide limited information as cancer drug can not only trigger cancer cell death but also initiate anti-cancer immune responses by inducing immunogenic cell death (ICD) of cancer cells. New drug evaluation methods that can assess the effects of cancer drugs on ICD as well as cancer cell death need to be developed. As a first step to address this issue, we developed a 3-dimensional (3D) platform that allow monitoring dynamic cancer cell-dendritic cell (DC) interactions with cancer drug treatment. DCs are immune cells that engulfs dying/dead cancer cells to initiate anti-cancer immune responses, thus interactions between cancer cells and DCs critical to evaluating ICD. To develop this platform, we first tested various cytotoxicity assays and identified 2D and 3D IC50s of various cancer drugs, of which ~ 50% of cancer cells die, for cancer cells and DCs. Then, cancer cells in 3D collagen gels were treated with various concentrations of cancer drugs, and DCs were added to sides of collagen gel blocks. DCs were recruited into the collage gels, presumably by ATP released from dying cancer cells, and interacted with cancer cells to engulf them. DC migration and interaction with cancer cells were monitored by performing live cell imaging. This 3D platform will be useful in evaluating ICD of cancer drugs and studying basic mechanisms of ICD

젊은 여성의 유방에 발생한 원발성 활막 육종 1예

활막 육종은 주로 관절 주위 조직에 호발하는 악성 종양이다. 종종 관절 이외의 다른 부위에 발생하는 경우도 보고되어있는데, 원발성 유방 활막 육종은 세계적으로도 보고된 예가 드물다. 본 증례는 원발성 유방 활막 육종으로 진단된 15세 여자 환자의 1예이다. 진단 후 유방 종괴에 대한 광범위 절제술을 시행하였으나 9개월 후 국소 재발하였고, 이에 재발 부위에 대한 광범위 절제술을 다시 시행하였다. 그러나, 27개월 후 폐의 우상엽에 고립성 폐 전이가 발견되었다. 이에 폐 병변에 대한 절제술 및 고식적 화학요법으로 doxorubicin 및 ifosfamide의 병합 화학요법을 시행하였다. 현재 항암화학요법 종료 후 28개월째 재발의 증거 없이 경과관찰 중이다. 이에 저자들은 본 증례를 문헌고찰과 함께 보고하는 바이다

Multi-functional Microparticles for the Evaluation of Multiple NK Cell Functions

2