• Title/Summary/Keyword: single cell analysis

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Induction of c-Jun Expression by Breast Cancer Anti-estrogen Resistance-3 (BCAR3) in Human Breast MCF-12A Cells (정상적인 인간유방상피세포인 MCF-12세포에서 유방암 항에스토젠 내성인자-3 (BCAR3)에 의한 c-Jun 발현 유도 연구)

  • Oh, Myung-Ju;Kim, Ji-Hyun;Jhun, Byung Hak
    • Journal of Life Science
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    • v.26 no.12
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    • pp.1383-1391
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    • 2016
  • Anti-estrogen drugs such as tamoxifen have been used for treating patients with ER-positive, early breast cancer. However, resistance to anti-estrogen treatment is inevitable in most patients. Breast cancer anti-estrogen resistance-3 (BCAR3) has been identified as the protein responsible for the induction of tamoxifen resistance in estrogen-dependent human breast cancer. We have previously reported that BCAR3 regulates the cell cycle progression and the signaling pathway of EGF and insulin leading to DNA synthesis. In this study, we investigated the functional role of BCAR3 in regulating c-Jun transcription in non-tumorigenic human breast epithelial MCF-12A cells. A transient transfection of BCAR3 increased both the mRNA and protein of c-Jun expression, and stable expression of BCAR3 increased c-Jun protein expression. The overexpression of BCAR3 directly activated the promoter of c-jun, AP-1, and SRE but not that of $NF-{\kappa}B$. Furthermore, single-cell microinjection of BCAR3 expression plasmid in the cell cycle-arrested MCF-12A cells induced c-Jun protein expression, and co-injection of dominant negative mutants of Ras, Rac, and Rho suppressed the transcriptional activity of c-Jun in the presence of BCAR3. Furthermore, stable expression of BCAR3 increased the proliferation of MCF-12A cells. The microinjection of inhibitory materials such as anti-BCAR3 antibody and siRNA BCAR3 inhibited EGF-induced c-Jun expression but did not affect IGF-1 induced upregulation of c-Jun. Taken together, we propose that BCAR3 plays a crucial role in c-Jun protein expression and cell proliferation and that small GTPases (e.g., Ras, Rac, and Rho) are required for the BCAR3-mediated activation of c-Jun expression.

Clinical Significance of Lymph Node Micrometastasis in Patients with Stage 1 Non-Small Cell Lung Cancer (제1기 비소세포폐암 환자에서 임파절 미세전이와 예후와의 상관관계)

  • 최필조;노미숙;이재익
    • Journal of Chest Surgery
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    • v.36 no.5
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    • pp.348-355
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    • 2003
  • Background: The prognostic significance of lymph node micrometastasis in non-small cell lung cancer remains controversial. We therefore investigated the clinicopathologic factors related to lymph node micrometastsis and evaluated the clinical relevance of micrometastasis with regard to recurrence. Material and Method: Five hundred six lymph nodes were obtained from 41 patients with stage 1 non-small ceil lung cancer who underwent curative resection between 1994 and 1998. Immunohistochemical staining using anti-cytokeratin Ab was used to detect micrometastasis in these lymph nodes. Result: Micrometastatic tumor cells were identified in pN0 lymph nodes in 14 (34.1%) of 41 patients. The presence of lymph node micrometastasis was not related to any clinicopathoiogic factor (p) 0.05). The recurrence rate was higher in patients with micrometastasis (57.1%) than in those without (37.0%), but the difference was not significant (p=0.22). Patients with micrometastasis had a lower 5-year recurrence-free survival rate (48.2%) than those without micrometastasis (64.1%), with a borderline significance (p=0.11), The S-year recurrence-free survival rate (25.0%) in the patients with 2 or more micrometastatic lymph nodes was significantly lower than that in the patients with no or single micrometastasis (p=0.02). In multivariate analysis, multiple lymph node micromestasis us was a significant independent predictor of recurrence (p=0.028, Risk ratio=3.568). Conclusion: Immunehistochemical anti-cytokeratin staining was a rapid, sensitive, and easy way of detecting lymph node micrometastasis. The presence of lymph node micrometastasis was not significantly associated with the recurrence, but had a tendency toward a poor prognosis in stage 1 non-small cell lung cancer. Especially, the presence of multiple micrometastatic lymph nodes was a significant and independent predictor of recurrence.

Noninvasive Prenatal Diagnosis using Cell-Free Fetal DNA in Maternal Plasma: Clinical Applications

  • Yang, Young-Ho;Han, Sung-Hee;Lee, Kyoung-Ryul
    • Journal of Genetic Medicine
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    • v.8 no.1
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    • pp.1-16
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    • 2011
  • Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

Enhanced Growth Inhibition by Combined Gene Transfer of p53 and $p16^{INK4a}$ in Adenoviral Vectors to Lung Cancer Cell Lines (폐암세포주에 대한 p53 및 $p16^{INK4a}$의 복합종양억제유전자요법의 효과)

  • Choi, Seung -Ho;Park, Kyung-Ho;Seol, Ja-Young;Yoo, Chul-Gyu;Lee, Choon-Taek;Kim, Young-Whan;Han, Sung-Koo;Shim, Young-Soo
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.1
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    • pp.67-75
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    • 2001
  • Background : Two tumor suppressor genes, p53 and p16, which have different roles in controlling the cell cycle and inducing apoptosis, are frequently inactivated during carcinogenesis including lung cancer. Single tumor suppressor gene therapies using either with p53 or p16 have been studied extensively. However, there is a paucity of reports regarding a combined gene therapy using these two genes. Methods : The combined effect of p53 and p16 gene transfer by the adenoviral vector on the growth of lung cancer cell lines and its interactive mechanism was investigated. Results : An isobologram showed that the co-transduction of p53 and p16 exhibited a synergistic growth in hibitory effect on NCI H358 and an additive effect on NCI H23. Cell cycle analysis demonstrated the induction of a synergistic G1/S arrest by a combined p53 and p16 transfer. This synergistic interaction was again confirmed in a soft agar confirmed in a soft agar clonogenic assay. Conclusion : These observations suggest the potential of a p53 and p16 combination gene therapy as another potent strategy in cancer gene therapy.

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Glass Dissolution Rates From MCC-1 and Flow-Through Tests

  • Jeong, Seung-Young
    • Proceedings of the Korean Radioactive Waste Society Conference
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    • 2004.06a
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    • pp.257-258
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    • 2004
  • The dose from radionuclides released from high-level radioactive waste (HLW) glasses as they corrode must be taken into account when assessing the performance of a disposal system. In the performance assessment (PA) calculations conducted for the proposed Yucca Mountain, Nevada, disposal system, the release of radionuclides is conservatively assumed to occur at the same rate the glass matrix dissolves. A simple model was developed to calculate the glass dissolution rate of HLW glasses in these PA calculations [1]. For the PA calculations that were conducted for Site Recommendation, it was necessary to identify ranges of parameter values that bounded the dissolution rates of the wide range of HLW glass compositions that will be disposed. The values and ranges of the model parameters for the pH and temperature dependencies were extracted from the results of SPFT, static leach tests, and Soxhlet tests available in the literature. Static leach tests were conducted with a range of glass compositions to measure values for the glass composition parameter. The glass dissolution rate depends on temperature, pH, and the compositions of the glass and solution, The dissolution rate is calculated using Eq. 1: $rate{\;}={\;}k_{o}10^{(ph){\eta})}{\cdot}e^{(-Ea/RT)}{\cdot}(1-Q/K){\;}+{\;}k_{long}$ where $k_{0},\;{\eta}$ and Eaare the parameters for glass composition, pH, $\eta$ and temperature dependence, respectively, and R is the gas constant. The term (1-Q/K) is the affinity term, where Q is the ion activity product of the solution and K is the pseudo-equilibrium constant for the glass. Values of the parameters $k_{0},\;{\eta}\;and\;E_{a}$ are the parameters for glass composition, pH, and temperature dependence, respectively, and R is the gas constant. The term (1-Q/C) is the affinity term, where Q is the ion activity product of the solution and K is the pseudo-equilibrium constant for the glass. Values of the parameters $k_0$, and Ea are determined under test conditions where the value of Q is maintained near zero, so that the value of the affinity term remains near 1. The dissolution rate under conditions in which the value of the affinity term is near 1 is referred to as the forward rate. This is the highest dissolution rate that can occur at a particular pH and temperature. The value of the parameter K is determined from experiments in which the value of the ion activity product approaches the value of K. This results in a decrease in the value of the affinity term and the dissolution rate. The highly dilute solutions required to measure the forward rate and extract values for $k_0$, $\eta$, and Ea can be maintained by conducting dynamic tests in which the test solution is removed from the reaction cell and replaced with fresh solution. In the single-pass flow-through (PFT) test method, this is done by continuously pumping the test solution through the reaction cell. Alternatively, static tests can be conducted with sufficient solution volume that the solution concentrations of dissolved glass components do not increase significantly during the test. Both the SPFT and static tests can ve conducted for a wide range of pH values and temperatures. Both static and SPFt tests have short-comings. the SPFT test requires analysis of several solutions (typically 6-10) at each of several flow rates to determine the glass dissolution rate at each pH and temperature. As will be shown, the rate measured in an SPFt test depends on the solution flow rate. The solutions in static tests will eventually become concentrated enough to affect the dissolution rate. In both the SPFt and static test methods. a compromise is required between the need to minimize the effects of dissolved components on the dissolution rate and the need to attain solution concentrations that are high enough to analyze. In the paper, we compare the results of static leach tests and SPFT tests conducted with simple 5-component glass to confirm the equivalence of SPFT tests and static tests conducted with pH buffer solutions. Tests were conducted over the range pH values that are most relevant for waste glass disssolution in a disposal system. The glass and temperature used in the tests were selected to allow direct comparison with SPFT tests conducted previously. The ability to measure parameter values with more than one test method and an understanding of how the rate measured in each test is affected by various test parameters provides added confidence to the measured values. The dissolution rate of a simple 5-component glass was measured at pH values of 6.2, 8.3, and 9.6 and $70^{\circ}C$ using static tests and single-pass flow-through (SPFT) tests. Similar rates were measured with the two methods. However, the measured rates are about 10X higher than the rates measured previously for a glass having the same composition using an SPFT test method. Differences are attributed to effects of the solution flow rate on the glass dissolution reate and how the specific surface area of crushed glass is estimated. This comparison indicates the need to standardize the SPFT test procedure.

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Apoptotic Effect of Co-Treatment with Valproic Acid and 17AAG on Human Osteosarcoma Cells (Valproic acid와 17AAG의 병용처리가 사람골육종세포에 미치는 세포자멸사 효과에 대한 연구)

  • Park, Jun-Young;Park, Se-Jin;Kim, In-Ryoung;Park, Bong-Soo;Jeong, Sung-Hee;Ko, Myung-Yun;Ahn, Yong-Woo
    • Journal of Oral Medicine and Pain
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    • v.36 no.1
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    • pp.11-20
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    • 2011
  • Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent. And it is known that antitmor activity of VPA is associated with its targeted at histone deacetylases. 17AAG, Inhibition of HSP90 leads to the proteasome degradation of the HSP90 client proteins, such as Akt, Raf/Ras, Erk, VEGF, cyclin D and p53, and causes potent antitumor activity. It is reported that 17AAG-induced HSP90 inhibition results in prevention of cell proliferation and induction of apoptosis in several types of cancer. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and the HSP90 inhibitor, 17AAG on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and 17AAG showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-3, caspase-7 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or 0.5 ${\mu}M$ 17AAG for 48 h did not induce apoptosis, the co-treatment with them induced prominently apoptosis. Therefore our data in this study provide the possibility that combination therapy with VPA and 17AAG could be considered as a novel therapeutic strategy for human osteosarcoma.

The Porcine FoxO1, FoxO3a and FoxO4 Genes: Cloning, Mapping, Expression and Association Analysis with Meat Production Traits

  • Yu, Jing;Zhou, Quan-Yong;Zhu, Meng-Jin;Li, Chang-Chun;Liu, Bang;Fan, Bin;Zhao, Shu-Hong
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.5
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    • pp.627-632
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    • 2007
  • FoxO1, FoxO3a and FoxO4 belong to the FoxO gene family, which play important roles in the PI3K/PKB pathway. In this study, we cloned the porcine FoxO1, FoxO3a and FoxO4 sequences and assigned them to SSC11p11-15, SSC1p13 and SSC xq13 using somatic cell hybrid panel (SCHP) and radiation hybrid panel (IMpRH). RT-PCR results showed that these three genes are expressed in multiple tissues. Sequencing of PCR products from different breeds identified a synonymous T/C polymorphism in exon 2 of FoxO3a. This FoxO3a single nucleotide polymorphism (SNP) can be detected by AvaII restriction enzyme. The allele frequencies of this SNP were investigated in Dahuabai, Meishan, Tongcheng, Yushan, Large White, and Duroc pigs. Association of the genotypes with growth and carcass traits showed that different genotypes of FoxO3a were associated with carcass length and backfat thickness between 6th and 7th ribs (BTR) and drip loss (p<0.05).

Electrochemical Performances of Acid-Treated and Pyrolyzed Cokes According to Acid Treatment Time (산처리 시간별 산화 코크스와 열분해 코크스의 전기화학적 거동)

  • Kim, Ick-Jun;Yang, Sunhye;Jeon, Min-Je;Moon, Seong-In;Kim, Hyun-Soo
    • Applied Chemistry for Engineering
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    • v.19 no.4
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    • pp.407-412
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    • 2008
  • As an activation procedure, in this study, the oxidation treatment of needle cokes with a dilute nitric acid and sodium chlorate $(NaClO_3)$, combined with heat treatment, was attempted. The structures of acid-treated and pyrolyzed coke were examined with XRD, FESEM, elemental analyzer, BET, and Raman spectroscopy. The behavior of double layer capacitance was investigated with the analysis of charge and discharge. The structure of needle coke treated with acid was revealed to a single phase of (001) diffraction peak after 24 h. On the other hand, thecoke oxidized by heat treatment was reduced to a graphite structure of (002) at $300^{\circ}C$. The distorted graphene layer structure, derived from the process of oxidation and reduction of the inter-layer, makes the pores by the electric field activation at the first charge, and generates the double layer capacitance from the second charge. The cell using pyrolyzed coke with 24 h acid treatment and $300^{\circ}C$ heat treatment exhibited the maximum capacitance per weight and volume of 33 F/g and 30 F/mL at the two-electrode system in the potential range of 0~2.5 V.

X-ray Powder Diffraction Structural Phase-transition Study of $(Na_{0.7}Sr_{0.3})(Ti_{0.3}Nb_{0.7})O_3$using the Rietveld Method of Analysis (분말 X-선 회절의 리트벨트 해석법을 이용한 $(Na_{0.7}Sr_{0.3})(Ti_{0.3}Nb_{0.7})O_3$계에서의 구조 상전이 특성연구)

  • Jeong, Hun-Taek;Kim, Ho-Gi
    • Korean Journal of Materials Research
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    • v.5 no.6
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    • pp.748-753
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    • 1995
  • Solid solution of NaNb $O_3$70 mol% and SrTi $O_3$30 mol% was single phase. A broad dielectric peak was found at about l00K. Crystal structure was analysed at room temperature and 12K using Rietveld analysis. The unit cell was assigned to have a a doubled lattice parameter of simple perovskite sturcture at room temperatue, the structure was orthorombic with space group Pmmn. At 12K, the structure was also orthorombic with space group Pnma. This structure change with temperature was due to the distortion of oxygen octahedron. This distortion of oxygen octahedron was made by the decrease of (Ti, Nb)-O bounds length with no variation of (Ti, Nb)-O-(Ti, Nb) bound angle. Therefore the broad dielectirc peak about l00K was attributed to the structural change casued by oxygen octahedron distortion.

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Comparison of Inflammatory Markers Changes in Patients Who Used Postoperative Prophylactic Antibiotics within 24 Hours after Spine Surgery and 5 Days after Spine Surgery

  • Youn, Gun;Choi, Man Kyu;Kim, Sung Bum
    • Journal of Korean Neurosurgical Society
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    • v.65 no.6
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    • pp.834-840
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    • 2022
  • Objective : C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and white blood cell (WBC) count are inflammatory markers used to evaluate postoperative infections. Although these markers are non-specific, understanding their normal kinetics after surgery may be helpful in the early detection of postoperative infections. To compliment the recent trend of reducing the duration of antibiotic use, this retrospective study investigated the inflammatory markers of patients who had received antibiotics within 24 hours after surgery according to the Health Insurance Review & Assessment Service guidelines and compared them with those of patients who had received antibiotics for 5 days, which was proven to be non-infectious. Methods : We enrolled 74 patients, divided into two groups. Patients underwent posterior lumbar interbody fusion (PLIF) at a single institution between 2019 and 2020. Group A included 37 patients who received antibiotics within 24 hours after the PLIF procedure, and group B comprised 37 patients who had used antibiotics for 5 days. A 1 : 1 nearest-neighbor propensity-matched analysis was used. The clinical variables included age, sex, medical history, body mass index, estimated blood loss, and operation time. Laboratory data included CRP, ESR, and WBC, which were measured preoperatively and on postoperative days (POD) 1, 3, 5, and 7. Results : CRP dynamics tended to decrease after peaking on POD 3, with a similar trend in both groups. The average CRP level in group B was slightly higher than that in group A; however, the difference was not statistically significant. Multiple linear regression analysis revealed operation time, number of fused levels, and estimated blood loss as significant predictors of a greater CRP peak value (r2=0.473, p<0.001) in patients. No trend (a tendency to decrease from the peak value) could be determined for ESR and WBC count on POD 7. Conclusion : Although slight differences were observed in numerical values and kinetics, sequential changes in inflammatory markers according to the duration of antibiotic administration showed similar patterns. Knowledge of CRP kinetics allows the assessment of the degree of difference between the clinical and expected values.