• 제목/요약/키워드: stable transfection

검색결과 52건 처리시간 0.049초

말의 LH/CGR를 발현하는 CHO 세포와 PathHunter Parental 세포에서 유전자 재조합 eCGβ/α의 생화학적 특성 (Biochemical Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG), Using CHO Cells and PathHunter Parental Cells Expressing Equine Luteinizing Hormone/Chorionic Gonadotropin Receptors (eLH/CGR))

  • 이소연;?바락차 뭉흐자야;김정수;성훈기;강명화;민관식
    • 생명과학회지
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    • 제27권8호
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    • pp.864-872
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    • 2017
  • eCG는 다른 포유동물에서 FSH와 LH의 활성을 나타내기 때문에 성선자극 호르몬 family에서 아주 특이적이고 많은 당쇄가 수식되어진 알파와 베타의 비공유결합으로 구성되어 있다. 유전자 재조합 $eCG{\beta}/{\alpha}$의 생물학적 기능을 규명하기 위하여 말의 LH/CGR의 포유동물발현용 벡터를 구축하였다. 재조합 $eCG{\beta}/{\alpha}$의 활성분석은 말의 LH/CGR가 일시적으로 발현되는 CHO-K1 세포와 지속적으로 발현되는 PathHunter Parental 세포를 이용하여 분석하였다. 유전자 재조합 $eCG{\beta}/{\alpha}$는 CHO-K1 부유세포의 상층으로 효율적으로 분비되었으며, 분비량은 transfection 후 1일에서 7일까지 약 200 mIU/ml이었다. Western blot 분석결과는 재조합 $eCG{\beta}/{\alpha}$의 분자량은 약 40-45 kDa으로 검출되었다. eLH/CGR가 발현되는 CHO-K1 세포에서의 cAMP분비량으로 재조합 $eCG{\beta}/{\alpha}$의 활성을 분석하였다. 그 결과 cAMP농도는 재조합 $eCG{\beta}/{\alpha}$의 농도의존적으로 증가하였다. eLH/CGR가 일시적으로 발현하는 CHO-K1 세포에서 $EC_{50}$ 값은 $8.1{\pm}6.5ng$이었다. 또한 일시적 및 지속적으로 eLH/CGR가 발현하는 PathHunter Parental 세포에서도 재조합 $eCG{\beta}/{\alpha}$의 LH 활성 분석결과 높은 활성을 나타내는 것으로 확인되었으며, 이들의 $EC_{50}$ 값은 각각 $5.0{\pm}4.7ng/ml$, $4.5{\pm}5.2ng/ml$으로 나타났다. 따라서 이러한 결과에 의하면 재조합 $eCG{\beta}/{\alpha}$는 말의 LH/CGR가 발현하는 세포에서 생물학적 활성을 나타난다는 것을 확인하였으며, PathHunter Parental 세포에서 지속적으로 발현되는 세포의 확보는 당쇄제거에 의한 재조합 eCG의 돌연변이등에 관한 기능적인 메커니즘을 밝히는데 유용할 것으로 사료된다.

Effect of Bcl-2 on Apoptosis and Transcription Factor NF-κB Activation Induced by Adriamycin in Bladder Carcinoma BIU87 Cells

  • Zhang, Guo-Jun;Zhang, Zhe
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권4호
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    • pp.2387-2391
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    • 2013
  • Resistance to apoptosis is a major obstacle preventing effective therapy for malignancies. Bcl-2 plays a significant role in inhibiting apoptosis. We reconstructed a stable human Bcl-2 transfected cell line, BIU87-Bcl-2, that was derived from the transfection of human bladder carcinoma cell line BIU87 with a plasmid vector containing recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. A cell line transfected with the plasmid alone [pcDNA3.1(+)-neo] was also established as a control. BIU87 and BIU87-neo proved sensitive to adriamycin induced apoptosis, while BIU87-Bcl-2 was more resistant. In view of the growing evidence that NF-${\kappa}B$ may play an important role in regulating apoptosis, we determined whether Bcl-2 could modulate the activity of NF-${\kappa}B$ in bladder carcinoma cells. Stimulation of BIU87, BIU87-neo and BIU87-Bcl-2 with ADR resulted in an increase expression of NF-${\kappa}B$ (p<0.001). The expression of NF-${\kappa}B$ in BIU87-Bcl-2 was higher than in the other two cases, with a concomitant reduction in the $I{\kappa}B{\kappa}$ protein level. These results suggest that the overexpression of Bcl-2 renders human bladder carcinoma cells resistant to adriamycin-induced cytotoxicity and there is a link between Bcl-2 and the NF-${\kappa}B$ signaling pathway in the suppression of apoptosis.

Gelsolin Induces Promonocytic Leukemia Differentiation Accompanied by Upregulation of p21CIP1

  • Shirkoohi, Reza;Fujita, Hisakazu;Darmanin, Stephanie;Takimoto, Masato
    • Asian Pacific Journal of Cancer Prevention
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    • 제13권9호
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    • pp.4827-4834
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    • 2012
  • Tumor suppressor genes have received much attention for their roles in the development of human malignancies. Gelsolin has been found to be down-regulated in several types of human cancers, including leukemias. It is, however, expressed in macrophages, which are the final differentiation derivatives for the monocytic myeloid lineage, implicating this protein in the differentiation process of such cells. In order to investigate the role of gelsolin in leukaemic cell differentiation, stable clones over-expressing ectopic gelsolin, and a control clone were established from U937 leukaemia cells. Unlike the control cells, both gelsolin-overexpressing clones displayed retarded growth, improved monocytic morphology, increased NADPH and NSE activities, and enhanced surface expression of the ${\beta}$-integrin receptor, CD11b, when compared with the parental U937 cells. Interestingly, RT-PCR and western blot analysis also revealed that gelsolin enhanced p21CIP1 mRNA and protein expression in the overexpressing clones. Moreover, transient transfection with siRNA silencing P21CIP1, but not the control siRNA, resulted in a reduction in monocytic differentiation, accompanied by an increase in proliferation. In conclusion, our work demonstrates that gelsolin, by itself, is capable of inducing monocytic differentiation in U937 leukaemia cells, most probably through p21CIP1 activation.

Bag-1L is a Stress-withstand Molecule Prevents the Downregulation of Mcl-1 and c-Raf Under Control of Heat Shock Proteins in Cisplatin Treated HeLa Cervix Cancer Cells

  • Ozfiliz, Pelin;Arisan, Elif Damla;Coker-Gurkan, Ajda;Obakan, Pinar;Eralp, Tugce Nur;Dinler-Doganay, Gizem;Palavan-Unsal, Narcin
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권11호
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    • pp.4475-4482
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    • 2014
  • Background: Cisplatin, a DNA damaging agent, induces apoptosis through increasing DNA fragmentation. However, identification of intrinsic resistance molecules against Cisplatin is vital to estimate the success of therapy. Bag-1 (Bcl-2-associated anthanogene) is one anti-apoptotic protein involved in drug resistance impacting on therapeutic efficiency. Elevated levels of this protein are related with increase cell proliferation rates, motility and also cancer development. For this reason, we aimed to understand the role of Bag-1 expression in Cisplatin-induced apoptosis in HeLa cervix cancer cells. Cisplatin decreased cell viability in time- and dose-dependent manner in wt and Bag-1L+HeLa cells. Although, $10{\mu}M$ Cisplatin treatment induced cell death within 24h by activating caspases in wt cells, Bag-1L stable transfection protected cells against Cisplatin treatment. To assess the potential protective role of Bag-1, we first checked the expression profile of interacting anti-apoptotic partners of Bag-1. We found that forced Bag-1L expression prevented Cisplatin-induced apoptosis through acting on Mcl-1 expression, which was reduced after Cisplatin treatment in wt HeLa cells. This mechanism was also supported by the regulation of heat shock protein (Hsp) family members, Hsp90 and Hsp40, which were involved in the regulation Bag-1 interactome including several anti-apoptotic Bcl-2 family members and c-Raf.

Development of recombinant human chorionic gonadotropin (hCG) using high-density culture technique of suspension-adapted chinese hamster ovary (CHO) cells

  • Na, Kyu-Heum;Kim, Seung-Chul;Seo, Kwang-Seok;Lee, Sung-Hee;Kang, Soo-Hyung
    • 한국생물공학회:학술대회논문집
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    • 한국생물공학회 2005년도 생물공학의 동향(XVI)
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    • pp.37-37
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    • 2005
  • Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of non-covalently linked two subunits, the ${\alpha}$ and ${\beta}$ subunit. It has been used as a infertility drug for ovulation to mimic luteinizing hormone $(LH).^{1)}$ A stable cell line was established by transfection of Rc/CMV-i-dhfr-hCG, expression vector containing hCG ${\alpha}-$ and ${\beta}-genes$, into dihydrofolate reductase-deficient CHO cells and subesquent methotrexate-mediated gene amplification. Anchorage-dependent CHO cells were adapted into a serum-free and/or animal component-free suspension medium through gradual serum weaning for the hCG production. The established cell line showed typical morphological characteristics and growth profile of CHO cells, and could produce FSH with passage-to-passage consistency. The high density perfusion culture of the CHO cells was carried out in Celligen Plus bioreactor equipped with a spin-filter as a internal cell retention device. The cell density reached up to $>1x10^{7}$ cells/ml in less than 7 days and a perfusion-control strategy based on cellular consumption rates of glucose was $established.^{2)}$ Biologically active recombinant hCG was purified by a series of chromatographic steps including anion exchange chromatography and hydrophobic interaction chromatography to homogeneity. The highly purified recombinant hCG was characterized for physicochemical, immunological and biological properties.

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인간 신장암 Caki세포에서 Par-4에 의한 MMP-2 활성 저해를 통한 세포 이동 조절 (Par-4 Modulates Cell Migration through Inhibition of MMP-2 Activity in Human Renal Carcinoma Caki Cells)

  • 우선민;권택규
    • 생명과학회지
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    • 제26권5호
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    • pp.614-619
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    • 2016
  • Par-4는 다양한 세포사멸 자극에 세포 사멸을 조절하고, 종양 억제기능을 가지고 있다. 그러나, Par-4에 의한 암세포의 이동 및 침윤에 대한 연구는 수행되지 않았다. 본 연구에서 Par-4단백질의 과발현이 인간 신장암 Caki세포에서 MMP-2의 활성화를 억제하지만 MMP-9 활성에는 영향을 주지 않았다. Par-4에 의한 MMP-2의 활성 억제는 leucine zipper domain이 결실된 Par-4 에서는 확인되지 않았다. Par-4 siRNA를 이용한 knock-down 실험에서 PMA 처리 시 세포이동 및 침윤 증가함을 확인하였다. Par-4의 과발현과 knock-dwon에서 MMP-2 mRNA 발현의 변화를 확인 할 수 없었다. 이 점은 Par-4 매개의 MMP-2 활성 억제는 전사 후 조절을 통하여 야기됨을 추측 할 수 있다.

IDH1 Overexpression Induced Chemotherapy Resistance and IDH1 Mutation Enhanced Chemotherapy Sensitivity in Glioma Cells in Vitro and in Vivo

  • Wang, Ju-Bo;Dong, Dan-Feng;Wang, Mao-De;Gao, Ke
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권1호
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    • pp.427-432
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    • 2014
  • Isocitrate dehydrogenase (IDH) is of great importance in cell metabolism and energy conversion. IDH mutation in glioma cells is reported to be associated with an increased overall survival. However, effects biological behavior of therapy of gliomas are unclear. Here, we investigated the influence of wild-type and mutated IDH genes on glioma cell biological behavior and response to chemotherapy. Relevant mechanisms were further explored. We designed our study on the background of the IDHR132H mutation. Stable cell lines were constructed by transfection. The CCK-8 method was used to assess cell proliferation, flow cytometry for the cell cycle and cell apoptosis, and the transwell method for cell invasion. Nude mouse models were employed to determine tumorigenesis and sensitivity to chemotherapy. Western blotting was used to detect relevant protein expression levels. We found that overexpression of wild IDH1 gene did not cause changes in the cell cycle, apoptosis and invasion ability. However, it resulted in chemotherapy resistance to a high dose of temozolomide (TMZ) in vivo and in vitro. The IDH1 mutation caused cell cycle arrest in G1 stage and a reduction of proliferation and invasion ability, while raising sensitivity to chemotherapy. This may provide an explanation for the better prognosis of IDH1 mutated glioma patients and the relative worse prognosis of their wild-type IDH1 counterparts. We also expect IDH1 mutations may be optimized as new targets to improve the prognosis of glioma patients.

Overexpression of RUNX3 Inhibits Malignant Behaviour of Eca109 Cells in Vitro and Vivo

  • Chen, Hua-Xia;Wang, Shuai;Wang, Zhou;Zhang, Zhi-Ping;Shi, Shan-Shan
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권4호
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    • pp.1531-1537
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    • 2014
  • Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.

Expression and Function of GSTA1 in Lung Cancer Cells

  • Pan, Xue-Diao;Yang, Zhou-Ping;Tang, Qi-Ling;Peng, Tong;Zhang, Zheng-Bing;Zhou, Si-Gui;Wang, Gui-Xiang;He, Bing;Zang, Lin-Quan
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권20호
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    • pp.8631-8635
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    • 2014
  • Glutathione S-transferase A1 (GSTA1) appears to be primarily involved in detoxification processes, but possible roles in lung cancer remain unclear. The objective of this study was to investigate the expression and function of GSTA1 in lung cancer cells. Real-time PCR and Western blotting were performed to assess expression in cancer cell lines and the normal lung cells, then verify the A549 cells line with stable overexpression. Localization of GSTA1 proteins was assessed by cytoimmunofluorescence. Three double-strand DNA oligoRNAs (SiRNAs) were synthesized prior to being transfected into A549 cells with Lipofectamine 2000, and then the most efficient SiRNA was selected. Expression of the GSTA1 gene in the transfected cells was determined by real-time PCR and Western blotting. The viability of the transfected cells were assessed by MTT. Results showed that the mRNA and protein expression of A549 cancer cells was higher than in MRC-5 normal cells. Cytoimmunofluorescence demonstrated GSTA1 localization in the cell cytoplasm and/or membranes. Transfection into A549 cells demonstrated that down-regulated expression could inhibit cell viability. Our data indicated that GSTA1 expression may be a target molecule in early diagnosis and treatment of lung cancer.

도담탕(導痰湯)이 $C_{2}C_{12}$세포주로부터 myostatin발현에 의한 심근에 미치는 영향 (Effect of Differentiation for Mouse Myoblast $C_{2}C_{12}$ Cells against Myostatin expression from Dodamtang)

  • 이유승;신유정;박종혁;김승모;백경민;박치상
    • 대한한방내과학회지
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    • 제29권1호
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    • pp.243-257
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    • 2008
  • Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. To determine MyoD expression by Dodamtang treatment, we compared the expression pattern of $C_{2}C_{12}$ mouse myoblasts that constitutively express myostatin with control cells. In vitro, increasing concentrations of Dodamtang reversibly prevented the myogenic blockage of myoblasts by myostatin expression. ELISA assay, Western and confocal analysis indicated that treatment of Dodamtang to the low serum culture media increased the levels of MyoD leading to the inhibition of myogenic differentiation by myostatin. The stable transfection of $C_{2}C_{12}$ myoblasts with myostatin expressing constructs did rescue MyoD-induced myogenic differentiation. Consistent with this, the treatment of Dodamtang rescued the expression of a MyoD in $C_{2}C_{12}$ myoblasts treated with myostatin. Taken together, these results suggest that induction of MyoD by Dodamtang inhibits myostatin activity and expression via SMAD3 resulting in the rescue of the myoblasts to differentiate into myotubes. Thus we propose that myostatin action by Dodamtang plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that blockage of functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

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