• Tuberculosis and Respiratory Diseases
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• 제56권5호
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• pp.450-464
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• 2004

연구배경 : FHIT 유전자의 homozygous deletion과 이와 관련된 mRNA 발현 이상, 단백질의 발현 결손은 폐암에서 매우 높은 빈도로 관찰되고 있다. 일부 연구에 의하면 FHIT 유전자를 폐암 세포 내에 이입시켰을 때 apoptosis가 유발되었고, 세포 주기의 이상 소견이 관찰되었으며, 종양형성 능력이 억제됨이 관찰되었다. 하지만 아직까지 FHIT 단백질의 기능에 대한 지식은 미진한 상태이다. 본 연구에서는 FHIT 유전자를 폐암 세포에 이입시켰을 때 유발되는 apoptosis의 기전을 규명하고자 하였다. 방 법 : FHIT 유전자가 결손된 NCI-H358 세포주에 FHIT 유전자를 stable transfection 시킨 후, cisplatin 혹은 paclitaxel을 가하고 apoptosis가 항진되어 나타나는가를 DAPI staining과 flow cytometry로 관찰해 보았다. 또한 이 과정에서 나타나는 caspase system의 변화와 Bcl-2 family의 변화를 Western blotting으로 조사해 보았다. 결 과 : FHIT를 발현시킨 세포에서는 cisplatin 혹은 paclitaxel을 투여하였을 때 유의하게 생존율이 감소하였으며, 이는 apoptosis 증가에 의한 것으로 확인 되었다. 이 과정에서 FHIT가 발현된 세포는 caspase-3, caspase-7의 활성화가 유의하게 증가되었으며, Bcl-2와 Bcl-xL 발현은 유의하게 감소하고 Bax와 Bad 발현은 유의하게 증가하였다. 결 론 : FHIT가 발현된 폐암 세포에 항암제를 투여하였을 때 유의하게 증가한 apoptosis는 caspase system과 Bcl-2 family의 활성화와 관련되어 있다.

• Journal of Chest Surgery
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• 제32권8호
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• pp.726-731
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• 1999

목적 및 배경: 세포사를 조절하는 암유전자 산물인 bcl-2 단백과 암억제유전자 산물인 p53 단백은 암의 발생 에 관여하는 것으로 알려져 있다. 최근 종양 조직내에서 bcl-2 단백 및 p53 단백의 발현과 종양의 악성도 및 예후와의 관련성에 대한 연구가 활발하게 진행되고 있으나 흉선종에서의 연구는 미흡한 실정이다. 대상 및 방법: 1984년부터 1997년까지 고신대학교 복음병원 흉부외과에서 수술 치험한 흉선상피종 중 병리 조직의 보관이 비교적 양호하고 병록 기록지가 충실한 30례를 대상으로 Rosai씨 분류법 및 Masaoka 병기와 중증근 무력증의 동반 여부에 따라 분류하고 종양조직을 이용하여 면역조직화학 검사를 시행하여 각 분류에 따른 bcl-2 단백과 p53 단백의 발현율, 그리고 bcl-2 단백과 p53 단백의 발현간에 상관관계를 조사하였다. 결과: bcl-2 단백은 비침습성 흉선종에는 발현된 경우가 없었고 침습성 흉선종 10례 중 3례(30%), 흉선암 11례 중 8 례(61.5%)에서 발현되어 악성도가 높을수록 발현율이 높게 나타났다(p=0.021). p53 단백은 비침습성 흉선종에 서 1례(14.3%), 침습성 흉선종 5례(50%), 흉선암 8례(61.5%)에서 발현되어 악성도가 높을수록 발현율이 높아 지는 경향을 보였으나 통계학적 유의성은 없었다(p=0.126). 17례의 흉선종의 Masaoka 병기(1기 5례, 2기 7례, 3기 2례, 4a기 3례)에 따른 bcl-2 단백은 1기와 4a기에서는 발현 예가 없었고, 2기 1례(14.3%), 3기 2례(100%) 에서 발현되어 병기가 진행할 수록 높은 발현율을 보였고(p=0.011), p53 단백은 1기 1례(20%), 2기 2례 (28.6%), 3기 2례(100%), 4a기 1례(33.3%) 발현되어 p53 단백 역시 병기가 진행할 수록 발현율이 높아지는 경 향을 보였으나 통계학적 유의성은 없었다(p=0.229). 중증근무력증의 동반 여부와 bcl-2단백, p53 단백의 발현 과는 무관하였다. bcl-2 단백과 p53 단백의 발현간의 상관관계는 bcl-2 단백과 p53 단백의 발현이 일치하는 경우가 23례(76.7%), 불일치하는 경우가 7례(23.3%)로 상관관계가 있는 것으로 나타났다(kappa치=0.525). 결 론: 흉선상피종 조직내 bcl-2 단백의 발현은 종양의 악성도를 반영하는 것으로 나타났고 p53 단백은 종양의 악성도와 관련이 없는 것으로 나타났다. 그럼에도 두 단백의 발현간에 상관관계가 있는 것으로 나타나 이의 규명을 위해서는 향후 p53 단백의 유전자 변이와 두 단백의 발현과 환자의 생존율과의 상관관계에 대한 연 구가 요할 것으로 사료된다.

• Molecules and Cells
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• 제21권1호
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• pp.1-6
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• 2006

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a mitochondrial pro-apoptotic protein that has a single Bcl-2 homology 3 (BH3) domain and a COOH-terminal transmembrane (TM) domain. Although it belongs to the Bcl-2 family and can heterodimerize with Bcl-2, its pro-apoptotic activity is distinct from those of other members of the Bcl-2 family. For example, cell death mediated by BNIP3 is independent of caspases and shows several characteristics of necrosis. Furthermore, the TM domain, but not the BH3 domain, is required for dimerization, mitochondrial targeting and pro-apoptotic activity. BNIP3 plays an important role in hypoxia-induced death of normal and malignant cells. Its expression is markedly increased in the hypoxic regions of some solid tumors and appears to be regulated by hypoxia-inducible factor (HIF), which binds to a site on the BNIP3 promoter. Silencing, followed by methylation, of the BNIP3 gene occurs in a significant proportion of cancer cases, especially in pancreatic cancers. BNIP3 also has a role in the death of cardiac myocytes in ischemia. Further studies of BNIP3 should provide insight into hypoxic cell death and may contribute to improved treatment of cancers and cardiovascular diseases.

• 한국진공학회지
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• 제14권3호
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• pp.138-142
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• 2005

액상 x-선 소각산란법을 이용하여 단백질의 구조를 분석하였다. 사용한 단백질은 구조가 이미 알려진 Lysozyme과 $Bcl-XL(\vartriangle TM/\vartriangle loop)$ 그리고 $Bcl-XL(\vartriangle TM/\vartriangle loop))$에 자유롭게 움직이는 고리를 가진 $Bcl-XL(\vartriangleTM))$이다. Lysozyme와 $Bcl-XL(\vartriangle TM/\vartriangle loop)$에 대한 소각산란결과는 단백질 결정학으로부터 알려진 분자구조에서 얻은 이론적인 결과와 농도에 의한 차이정도를 제외하고는 잘 일치하였다. $Bcl-XL(\vartriangleTM))$의 경우는 단백질 결정산란 신호에서 볼 때 $Bcl-XL(\vartriangle TM/\vartriangle loop)$와 차이가 없는 것으로 알려져 있으나, 소각산란에서는 뚜렷한 차이를 나타내는 결과를 얻어 loop와 같이 쉽게 움직이는 부분을 가진 단백질을 연구하는 경우 소각산란의 장점을 확인할 수 있었다. 위 실험을 통하여 포항 가속기 연구소 4C1 빔라인의 성능은 적어도 해상도 $\sim2.2\;nm$까지 용액상의 단백질 구조를 분석할 수 있다는 것을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.926-936
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• 2003

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.

• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7065-7068
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• 2014

To investigate the anti-proliferative mechanism of mangiferin in a human nasopharyngeal carcinoma cell line, CNE2 cells were incubated with different concentrations of mangiferin (12.5, 25, 50, 100, 150 and $200{\mu}M$) or with PBS as a control for 72 hours. Analyses were made of the cell cycle and apoptosis with measurement of mRNA and protein levels of two apoptosis-related genes, Bcl-2 and Bax. Flow cytometry assays showed mangiferin could inhibit CNE2 cell proliferation via G2/M arrest and induction of early apoptosis. Real time PCR and Western blotting showed the mRNA and protein level of Bcl-2 to be down-regulated, while those of Bax were upregulated, when CNE2 cells were treated with mangiferin. This investigation indicated anti-proliferation effects of mangiferin through induction of cell apoptosis regulated by Bcl-2 and Bax expression.

• 대한약학회:학술대회논문집
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• 대한약학회 2000년도 NEW STRATEGY FOR DRUG DEVELOPMENT IN POST-GENOMIC ERA(대한약학회)
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• pp.86-87
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• 2000

• 한국동물학회:학술대회논문집
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• 한국동물학회 1998년도 한국생물과학협회 학술발표대회
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• pp.360-360
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• 1998

C11, a chloride-containing VK3 analog, acts as a mediator of programmed cell death in SK-Hep-1 cell lines, but its molecular mechanisms linked to cell death are not understood. In this study, we investigated the expression of p21 gene and its relationship to apoptosis induced by C11. In SK -hep-1 cells, the addition of C11 resulted in time-dependent growth suppression and DNA fragmentation characteristics of apoptosis. p21 protein was induced during this process, while the protein level of p53 was not changed at the same condition. This apoptotic cell death with p21 induction was also observed in the Hep3B cells lacking functional p53 after treatment of C11. These results suggest that C11-induced apoptosis is associated with up-regulation of p21 protein in p53-independent pathway. Next, in order to confirm whether the p53-independent p21 induction is required for C11-induced apoptosis, we introduced the p21 gene into Hep3B. Overexpression of p21 did not affect the expression of the bcl-2 gene, but DNA fragmentation and PARa cleavage were significantly increased. These data indicate that p21 is involved in C11-induced apoptosis. Although Bcl-2 has been implicated to interfere with an essential signaling molecule involved in the apoptosis pathway, its molecular mechanism and target molecule are poorly understood. To determine the effects of bcl-2 overexpression on apoptosis and to investigate whether BcI-2 interfers with the p53-independent p21 pathway, we transfected the bcl-2 expression vector into SK - Hep-1 cels. Overexpression of Bcl-2 prevented C11-induced apoptosis. Taken together, C11-induced apoptosis is regulated by p52-independent p21 pathway and bcl-2 may inhibit functional activity of p21, therebe may inhibit the C11-induced apoptosis.ptosis.

• Molecules and Cells
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• 제24권3호
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• pp.378-387
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• 2007

The Bcl-2 family of proteins interacts at the mitochondria to regulate apoptosis. However, the anti-apoptotic Bcl-2 and $Bcl-X_L$ are not completely localized to the mitochondria. In an attempt to generate Bcl-2 and $Bcl-X_L$ chimeras that are constitutively localized to the mitochondria, we substituted their C-terminal transmembrane tail or both the C-terminal transmembrane tail and the adjacent loop with the equivalent regions from Bak or Bax mutant (BaxS184V) as these regions determine the mitochondrial localization of Bak and Bax. The effects of these substitutions on subcellular localization and their activities were assessed following expression in HeLa and CHO K1 cells. The substitution of the C-terminal tail or the C-terminal tail and the adjacent loop of Bcl-2 with the equivalent regions from Bak or the Bax mutant resulted in its association with the mitochondria. This change in subcellular localization of Bcl-2 chimeras triggered cells to undergo apoptotic-like cell death. The localization of this Bcl-2 chimera to the mitochondria may be associated with the disruption of mitochondrial membrane potential. Unlike Bcl-2, the loop structure adjacent to the C-terminal tail in $Bcl-X_L$ is crucial for its localization. To localize the $Bcl-X_L$ chimeras to the mitochondria, the loop structure next to the C-terminal tail in $Bcl-X_L$ protein must remain intact and cannot be substituted by the loop from Bax or Bak. The chimeric $Bcl-X_L$ with both its C-terminal tail and the loop structure replaced by the equivalent regions of Bak or Bax mutant localized throughout the entire cytosol. The $Bcl-X_L$ chimeras that are targeted to the mitochondria and the wild type $Bcl-X_L$ provided same protection against cell death under several death inducing conditions.

• The Korean journal of internal medicine
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• 제34권1호
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• pp.146-155
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• 2019

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of $NF-{\kappa}B$ was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt in HK-2 cells. $NF-{\kappa}B$ promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, $NF-{\kappa}B$, and Akt signaling pathway in HK-2 cells.