• BMB Reports
• /
• 제30권2호
• /
• pp.119-124
• /
• 1997

Transforming growth $factor-{\beta}\;(TGF-{\beta})$ is a multifunctional polypeptide that exerts biological roles including cell proliferation, differentiation, extracellular matrix deposition and apoptosis in many different cell types. $TGF-{\beta}$, although known as a negative growth regulator, has not been tested in human embryo lung (HEll cells. This study attempts to understand the role of $TGF-{\beta}$ on growth control of HEL cells in relationship to tyrosine phosphorylation pattern of cellular proteins. In density-arrested HEL cells treated with $TGF-{\beta}$, analysis of Western immunoblot showed induction of tyrosine phosphorylation of two major cellular proteins (15 kDa and 45 kDa). In normal proliferating HEL cells with different concentrations of serum, further analysis indicated that the increase in tyrosine phosphorylation of a 45 kDa protein was regulated in serum concentration-dependent manner. However, in proliferating HEL cells treated with $TGF-{\beta}$, tyrosine phosphorylation of 45 kDa was down-regulated. Calcium involvement in the regulation of tyrosine phosphorylation of 45 kDa and 15 kDa proteins was also examined. Tyrosine phosphorylation of 15 kDa protein but not of 45 kDa protein was regulated by exogenous calcium. The level of tyrosine phosphorylation of 15 kDa protein was low at reduced caclium concentration and high at elevated caclium concentration. $TGF-{\beta}$ reversed the pattern of tyrosine phosphorylation of 15 kDa protein. These results suggest that tyrosine phosphorylation of 45 and 15 kDa proteins in HEL cells may be controlled depending on the physiological status of the cells, i.e., low in arrested cells and high in proliferating cells. And the tyrosine phosphorylation of the two proteins appears to be down- or up-regulated by $TGF-{\beta}$.

• 대한바이러스학회지
• /
• 제26권1호
• /
• pp.1-8
• /
• 1996

Human cytomegalovirus (HCMV) replication and $Ca^{2+}$ response in human cell lines of neuronal origin were investigated. SK-N-SH (neuroblastoma cells) and A172 cells (glioblastoma cells) were used. SK-N-SH cells were permissive for HCMV multiplication with a delay of one day compared to virus multiplication in human embryo lung (HEL) cells. The delay of HCMV multiplication in SK-N-SH cells appeared to be correlated with a delay in the $Ca^{2+}$ response. The cytoplasmic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) began to increase at 12 h p.i. in HCMV-infected SK-N-SH cells, while $[Ca^{2+}]_i$ increase in HCMV-infected HEL cells was observed as early as 3 h p.i. On the whole, the level of the increase in $[Ca^{2+}]_i$ in SK-N-SH cells was about 30% of that in HEL cells. On the other hand, in A172 cells infected with HCMV, neither production of infectious virus nor detectable increase in $[Ca^{2+}]_i$ was observed. Treatment with TPA of HCMV-infected SK-N-SH cells resulted in $[Ca^{2+}]_i$ increase at 6 h p.i. The stimulatory effect of TPA on HCMV- induced $[Ca^{2+}]_i$ increase continued until 12 h p.i., but TPA failed to stimulate the $Ca^{2+}$ response in SK-N-SH cells at 24 h p.i., suggesting that the effect of TPA had disappeared in SK-N-SH cells at that time point. In conclusion, SK-N-SH cells are permissive for HCMV replication and the delay in $Ca^{2+}$ response may be a consequence of the lower responsiveness of SK-N-SH cells than HEL cells to HCMV infection.

• 미생물학회지
• /
• 제31권1호
• /
• pp.72-78
• /
• 1993

Human cytomegalovirus (HCMV) 의 immediate early(IE) 유전자의 promoter/enhancer 부위에 cAMP response element가 있다는 것으부터 caMP 가 HCMV 의 증식에 관여할 것이라고 생각할 수 있다. 이러한 가능성을 알아보기 위해 8-bromoadenosine 3', 5'-cyclic monophosphate (BrA) 와 papaverine 같은 세포내 cAMP 농도를 변화시키는 약제를 사용하여 HCMV 의 증식, DNA 합성 및 IE 유전자 발현에 대한 영향을 알아보았다. HCMV 증식과 DNA 합성은 papaverine 에 의해 억제된 반면, BrA 는 HCMV 의 증식에는 큰 영향을 주지 않았고 DNA 합성은 오히려 촉진시키는 것을 알 수 있었다. HCMV IE promoter 에 의해 작동되는 CAT 유전자를 함유한 plasmid pCMVIE/CAT 을 세포내로 transfection 시켰을 때, papaverine 을 처리한 세포에서는 CAT 효소 활성도가 감소한 반면 BrA 를 처리한 세포에서는 증가하였다. HCMV 에 감수성이 없는 HeLa 세포에서는 CAT 활성도가 감소성 세포인 HEL 세포에서보다 높게 나타난 반면, Vero 세포에서는 낮게 나타났다. 이들 비감수성 세포인 HEL 세포에서 보다 높게 나타난 반면 Vero 세포에서는 낮게 나타났다. 이 들 비감수성 세포에서의 CAT 활성도는 BrA 를 처리하여 주었을 때 모두 증가하였다. 이상과 같은 결과로부터 cAMP 는 HCMV 증식과 IE 유전자 발현에 어느 정도 관여한다는 것을 알 수 있다.

• KSBB Journal
• /
• 제22권2호
• /
• pp.67-72
• /
• 2007

형광을 이용한 암 진단을 위해 배양된 정상 폐세포 및 폐암 세포주에 광민감제인 5-ALA를 투여하고 세포 내 외에서 생성된 protoporphyrin IX (PpIX)의 형광을 측정하여 5-ALA 투여의 최적 농도를 조사하였다. 정상 폐세포주 (Hel299) 및 폐암 세포주 (A549, NCI-H460)에 5-ALA를 $0\sim800{\mu}g/mL$ 농도별로 투여하여 24시간 동안 배양한 다음 MTT assay로 세포증식 저해율 및 이때 생성되는 PpIX의 양을 형광의 강도로 측정하였다. 그 결과 Hel299 및 A549에서는 5-ALA의 처리농도가 증가할수록 세포 증식의 저해율이 증가하였으나 NCI-H460에서는 세포 증식이 저해되지 않았다. 그리고 폐암세포인 A549와 NCI-H460에 대한 5-ALA의 최적농도는 $100{\mu}g/mL$이며, 이때의 형광 (emission) 스펙트럼은 여기 파장이 410 nm일 때 세포 외에서는 615.8 nm와 660.8 nm, 616.7 nm와 660.2 nm, 세포 내에서는 603.2 nm와 661.4 nm, 603.5 nm와 661.4 nm에서 각각 형광 봉우리가 관찰되었다. 또한 PpIX를 형광 강도로 측정하면, PpIX는 정상세포에서는 낮은 농도로 축적이 되는 반면에 암세포에서 높은 농도로 축적되었으며, 세포 외보다는 세포 내에서 더 높은 농도로 축적됨을 알 수 있었다.

• IMMUNE NETWORK
• /
• 제20권4호
• /
• pp.35.1-35.13
• /
• 2020

Antigen delivery systems play critical roles in determining the quality and quantity of Ab responses in vivo. Induction of protective antibodies by B cells is essential in the development of vaccines against infectious pathogens, whereas production of IgE antibodies is prerequisite for investigation of allergic responses, or type 1 hypersensitivity reactions. Virus-like particles (VLPs) are efficient platforms for expression of proteins of interest in highly repetitive manners, which grants strong Ab responses to target antigens. Here, we report that delivery of hen egg lysozyme (HEL), a model allergen, through VLP could provoke strong HEL specific IgE Ab responses in mice. Moreover, acute allergic responses were robustly induced in the mice sensitized with VLPs that express HEL, when challenged with recombinant HEL protein. Our data show that antigen delivery in the context of VLPs could function as a platform for sensitization of mice and for subsequent examination of allergic reactions to molecules of interest.

• Natural Product Sciences
• /
• 제3권1호
• /
• pp.19-28
• /
• 1997

Constitutive cyclooxygenase (COX-I) is present in cells under physiological conditions, whereas inducible cyclooxygenase (COX-II) is induced by some cytokines, mitogens, and endotoxin presumably in pathological conditions such as inflammation. We have evaluated the inhibitory effects of solvent fractionated extracts of natural products on the activities of COX-I and COX-II. Oxygen uptake COX assay was performed, as a primary screening from the tissue extracts of bovine seminal vesicles (BSV), by monitoring the initial rate of oxygen uptake using an oxygen electrode. Additionally, we evaluated plant extracts for the inhibitory effects of COX-I (in HEL cells) and COX-II (in lipopolysaccharide activated J774A.1 macrophages) using thin layer chromatography of prostanoids produced from $^{14}C-labelled$ arachidonic acid (AA). The use of such models of COX-I and COX-II assay will lead to the identification of specific inhibitors of cyclooxygenases with presumably less side effects than present therapies. Inhibitory effects of 50 kinds of plant extracts on the COX-I and COX-II activities were determined and the active fractions were found in the ethyl acetate fractions of Dryopteris crassirhizoma (roots), Amomum cardamomum (roots), Triticum aestivum (seeds), Perilla sikokiana (leaves), Anemarrhena asphodeloides (roots). Especially, the ethyl acetate fraction of Dryopteris crassirhizoma (roots), which exhibited the strong inhibition against BSV COX $(IC_{50},\;65.4\;{\mu}g/ml)$, COX-I $(IC_{50},\;8.5\;{\mu}g/ml)$, and COX-II $(IC_{50},\;17.2\;{\mu}g/ml)$, is under investigation to isolate active principles using activity-guided fractionation method.

• BMB Reports
• /
• 제48권12호
• /
• pp.691-695
• /
• 2015

We report that phytosphingosine, a sphingolipid found in many organisms and implicated in cellular signaling, promotes megakaryocytic differentiation of myeloid leukemia cells. Specifically, phytosphingosine induced several hallmark changes associated with megakaryopoiesis from K562 and HEL cells including cell cycle arrest, cell size increase and polyploidization. We also confirmed that cell type specific markers of megakaryocytes, CD41a and CD42b are induced by phytosphingosine. Phospholipids with highly similar structures were unable to induce similar changes, indicating that the activity of phytosphingosine is highly specific. Although phytosphingosine is known to activate p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis, the signaling mechanisms involved in megakaryopoiesis appear to be distinct. In sum, we present another model for dissecting molecular details of megakaryocytic differentiation which in large part remains obscure.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
• /
• pp.81-81
• /
• 2003

The present study was taken to examine the inhibitory effect of extracts of Scytosiphon lomentaria, a marine alga growing in Jeju Island, on the growth of cancer cells and to develop an anti-cancer agent using components of S. lomemtaria. The effect was observed by the measurement of metabolic activity using colorimetric 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. In results, crude extract of this alga markedly inhibited the growth of leukemia cell lines such as HL-60 and KG-1, but could scarcely inhibit the growth of normal cells (HEL299) and adenocarcinoma cells (SNU-16 and HCT-I5). When HL-60 cells were treated with the extract, DNA fragmentation and the increase of proportion of sub-G1 hypodiploid cells were observed. Therefore, the inhibitory effect of S. lomemtaria on the growth of HL-60 cells seems to arise from the induction of apoptosis. In order to understand the mechanism of apoptosis inducton by S. lomemtaria, we examined the changes of Bcl-2 and Bax expression. The extract reduced Bcl-2, an anti-apoptotic protein, but increased Bax, a pro-apoptotic protein in a dose-dependent manner. When we examined the activation of caspase-3, an effector of apoptosis, the expression of active form(19 kDa) of caspase-3 was increased and the increase of their activities was demonstrated by the cleavage of poly(ADP-ribose)polymerase, a substrate of caspase-3, to 85 kDa. The results indicate that extract of S. lomentaria induces the apoptosis of HL-60 cells via the down-regulation of Bc1-2 and the activation of caspases.

• 한국약용작물학회지
• /
• 제12권1호
• /
• pp.36-42
• /
• 2004

사철쑥 추출물은 정상폐세포인 HEL299 세포주에 $0.5\;g/{\ell}$ 이하의 농도로 투여시는 정상세포 생존율을 80% 이상으로 유지시켜 정상세포에 대한 안정성이 유지되었으며, 암세포 생육억제활성은 증류수 및 에탄올 추출물 모두 $0.5\;mg/m{\ell}$ 이상의 농도에서 유방암세포주인 MCF7에 대하여 88%이상, 폐암세포주인 A549에 대하여서는 모든 추출물에서 $0.5\;mg/m{\ell}$의 농도 이상에서 65%이상의 저해를 나타내었다. 또한 간암세포주인 Hep3B에 대하여는, 에탄올 추출물이 87% 이상, 위암세포주인 AGS에 대하여 증류수 추출물이 약 85% 이상의 높은 암세포 생육억제활성을 나타내었다. 또한 정상세포독성에 대한 암세포의 억제율을 나타내는 selectivity에 있어서 각 추출물이 $0.1\;mg/m{\ell}$에서 $1.0\;mg/m{\ell}$의 농도에서 1.5 이상의 수치를 나타내었다. B와 T 세포주의 생육촉진 실험 결과 배양 기간이 증가함에 따라 세포의 생육이 촉진되었으며, 배양 5일째 $0.5\;mg/m{\ell}$의 농도에서 B세포의 생장을 최고 1.2배 이상 증가, T-cell을 $1.2{\sim}1.5$배 이상 생장을 증가시켰으며, 배양 6일째 에탄올 추출물이 IL-6를 $67\;pg/m{\ell},\;TNF-{\alpha}$를 물 추출물이 $68\;pg/m{\ell}$의 분비 하였다.

• 약학회지
• /
• 제52권1호
• /
• pp.1-6
• /
• 2008

The present study investigated the antiproliferative effects of Crnum asiaticum var. japonicum against HL-60 human leukemia cells. The 80% MeOH extract or several solvent fractions from the C. asiaticum inhibited the growth of HL-60 cells, whereas the growth of HEL-299 cells, human embryonic lung fibroblast, was scarcely inhibited. When the HL-60 cells were treated with the $CHCl_3$ fraction, the BuOH fraction, the EtOAc fraction and the $H_2O$ fraction, DNA ladder, chromatin condensation and increase of sub-G1 hypodiploid cells were observed. Furthermore, the $CHCl_3$ fraction and the BuOH fraction reduced Bc1-2 mRNA level, whereas Bax mRNA level was increased. These results suggest that the inhibitory effect of C. asiaticum on the growth of the HL-60 cell might be mediated through the induction of apoptosis via the down-regulation of Bc1-2. Taken together, components of C. asiaticum might have a therapeutic potential for the treatment of human leukemia.