사람 태반조직 DNA Topoisomerase Ⅰ에 관한 연구

이정복,권기량,임규,황병두 ( Jeong Bok Lee,Gi Ryang Kweon,Kyu Lim,Byung Doo Hwang ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.4

DNA topoisomerase I has been purified from human term placenta approximately 520-folds with a 10% yield. The enzyme shows a broad pH optimum from pH 6 to 9 and is heatlabile, being completely inactivated by heat treatment at 50℃ for 5 min. The enzyme is activated by K^+ and Na^+ approximately 20 and 8-folds respectively. Magnesium ion (Mg^(2+)) is the most potent activator, the activity being 20 and 40-folds activated at 2 mM and 10 mM respectively. But copper ion (Cu^(2+)) is a potent inhibitor. In the presence of Mg^(2+) and K^+, the enzyme is inhibited by physiologic concentration of ATP and GTP. Inhibitory mechanism of ATP is considered to be a inhibition of readoptation of active enzyme conformation and that of GTP is to be a inhibition of the prior step of DNA rejoining. The molecular weight is around 68,000. Camptothecin and 10-hydroxycamptothecin inhibit this enzyme, and the inhibitory action of 10-hydroxycamptothecin is potentiated by Mg^(2+) and K^+. DNA fragmentation by 10-hydroxycamptothecin is more potent than that by camptothecin in the DNA cleavage assay. Heparin and Cu^(2+) inhibit the prior step of DNA rejoining by this enzyme. From the above results, the inhibitory action mechanism of ATP, GTP, heparin, Cu^(2+) and the possible role of Mg^(2+) and K^+ for potentiating the inhibitory action of 10-hydroxycamptothecin, and the development the anticancer drug against DNA topoisomerase I as a target enzyme are discussed.

3차원 공동배양 모델에서 만성 저산소증이 별아교세포와 RBE4세포에 미치는 영향

반성배(Seong Bae Ban),권기량(Gi Ryang Kweon),김현(Hyun Kim) 대한해부학회 2007 Anatomy & Cell Biology Vol.40 No.2

뇌혈관장벽(Blood-brain barrier)은 뇌의 미세혈관의 내피세포와 별아교세포의 세포돌기(astrocytic process)에 의해 형성되는 선택적인 장벽으로서 중추 신경의 미세환경을 조절하여 신경세포의 기능을 유지하는데 매우 중요한 기능을 한다. 뇌혈관장벽을 형성하는 혈관내피세포와 별아교세포와의 상호 작용과 만성 저산소 환경에서의 반응을 증명하고자 본 연구를 시행하였다. 본 실험을 위하여 일차 배양된 별아교세포와 RBE4 세포를 사용하여 3차원 collagen gel 배양 및 3차원 transwell model을 구성하였다. 이 세포들을 confocal microscope으로 관찰하였고, western blotting study를 이용하여 별아교세포의 증식과 분화를 확인하였다. 3차원 collagen gel을 6일 동안 배양한 결과, 별아교세포의 세포돌기는 RBE4 세포로 길게 뻗어 있었다. Transwell model에서 별아교세포와 RBE4 세포를 공동 배양한 경우나 만성 저산소 환경에서 배양한 경우, 별아교세포의 분화와 증식이 뚜렷하게 증가하였다. 또한 transwell model에서 별아교세포와 RBE4 세포를 공동 배양한 경우나 만성 저산소 환경에서 배양한 경우 별아교세포의 세포돌기의 수가 증가하였으며, RBE4 세포로 유인되었다. 본 실험은 RBE4 세포가 별아교세포를 유도하여 뇌혈관장벽의 형성을 증가시킨다는 것을 제시하였으며, 3차원 collagen gel model이나 transwell model에서 만성 저산소 환경이 이러한 반응을 더욱 촉진시킨다는 연구 결과는 뇌혈관장벽에 대한 연구뿐만 아니라 세포돌기의 변화를 관찰하는 여러 연구에서 유용하게 사용될 수 있을 것이다. Chronic sublethal hypoxia induces brain adaptations associated with changes in neurovascular behavior. Changes to the neurovasculature also influence the formation of the brain-blood barrier (BBB). In this study, I investigated the influence of chronic sublethal hypoxia on astrocytes, using the coculture transwell model of primary cultured astrocytes and RBE4 (brain endothelial) cells. Using a 3D collagen gel model, cytoplasmic processes of astrocytes extended to clumps of endothelial cells. The numbers of astrocytes increased in cocultured and chronic hypoxic environments in the transwell model. Western blotting showed increased production of glial fibrillar acidic protein (GFAP) and proliferating cellular nuclear antigen (PCNA) in chronic hypoxia. I also confirmed the influence of hypoxia on the behavior of astrocytes in this model, using confocal microscopy. The numbers of cytoplasmic processes of astrocytes within the membrane increased in z sections. These data support the idea that chronic hypoxia might induce alterations in the formation of the BBB as part of the adaptation of the brain to chronic hypoxia. These transwell and 3D collagen gel models will probably be useful for functional as well as morphological experiments.

일차배양 Sertoli Cell에서 Testosterone에 의한 세포성 핵암유전자 발현에 관한 연구

임규,유지헌,김계영,권기량,황병두,Lim, Kyu,Yoo, Ji-Hun,Kim, Kye-Young,Kweon, Gi-Ryang,Hwang, Byung-Doo 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.1

일차배양 Sertoli cell에서 testosterone에 의한 c-myc 등 세포성 핵암유전자 발현 및 그 조절기전을 검색하여 다음과 같은 결과를 얻었다. Steady state에서 발현되는 Sertoli cell 세포성 핵암유전자는 c-fos, c-myc, c-jun이었으며 c-myb은 검출되지 않았고 이때 mRNA 크기는 각각 2.2 kb, 2.4 kb, 2.7 kb에 해당되었다. Testosterone에 의한 c-myc 및 c-jun mRNA의 발현은 시간경과에 따라 증가하였으며 유도되는 시간은 모두 16시간에 최고치에 달하여 유사하였다. c-Myc은 testosterone 농도에 비례해서 그리고 cycloheximide 처리로 각각 전사가 증가되었으나 actinomycin D 처리로 억제되었다. 연령에 따른 Sertoli cell에서의 c-myc mRNA 발현에 대한 testosterone의 영향은 steady state에서는 8일령이 가장 높았으며, testosterone에 의한 유도는 8일령, 14일령에서는 나타났으나 28일령에서는 유도되지 않았다. 이상의 결과로 미루어 보아 testosterone에 의해 유도된 c-myc mRNA가 testosterone에 의해 조절되는 Sertoli cell의 다른 유전자 발현의 조절에 관련되어 있을 것이라 시사된다. The expression of the nuclear protooncogenes have been associated with the cell proliferation and differentiation into changes in nuclear function. To evaluate the possibility that the nuclear protooncogenes play roles testosterone-dependent gene regulation, the effects of testosterone on the expression of the nuclear protooncogenes have been investigated in primary Sertoli cell cultures. c-Myc, c-jun and c-fos were expressed in steady state of primary Sertoli cells but c-myb was not detected. Testosterone increased c-myc and c-jun mRNA with maximal stimulation reached in 16 h. The induction of c-myc was dependent on the concentrations of testosterone. The testsoterone induced c-myc mRNA level was also increased in the cells treated with cycloheximide, but reduced by actinomycin D pretreatment. Even in the absence of testosterone, c-myc mRNA was clearly detectable in Sertoli cells from 8-days-old rats, but hardly detectable in cells from 14 and 28 days of age. Testosterone stimulated c-myc expression in the Sertoli cells from 8 and 14-days-old rats. These results suggest that transient expression of c-myc may play some roles to an intermediate step required for testosterone-dependent regulation of other genes expression of Sertoli cells.

일차배양 Sertoli cell 에서 Testosterone 에 의한 세포성 핵암유전자 발현에 관한 연구

임규,유지헌,김계영,권기량,황병두 ( Kyu Lim,Ji Hun Yoo,Kye Young Kim,Gi Ryang Kweon,Byung Doo Hwang ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

The expression of the nuclear protooncogenes have been associated with the cell proliferation and differentiation into changes in nuclear function. To evaluate the possibility that the nuclear protooncogenes play roles testosterone-dependent gene regulation, the effects of testosterone on the expression of the nuclear protooncogenes have been investigated in primary Sertoli cell cultures. c-Myc, c-jun and c-fos were expressed in steady state of primary Sertoli cells but c-myb was not detected. Testosterone increased c-myc and c-jun mRNA with maximal stimulation reached in 16 h. The induction of c-myc was dependent on the concentrations of testosterone. The testsoterone induced c-myc mRNA level was also increased in the cells treated with cycloheximide, but reduced by actinomycin D pretreatment. Even in the absence of testosterone, c-myc mRNA was clearly detectable in Sertoli cells from 8-days-old rats, but hardly detectable in cells from 14 and 28 days of age. Testosterone stimulated c-myc expression in the Sertoli cells from 8 and 14-days-old rats. These results suggest that transient expression of c-myc may play some roles to an intermediate step required for testosterone-dependent regulation of other genes expression of Sertoli cells.

오메가-3 지방산에 의한 COX-2/MMPs/VEGF 억제에 따른 대장암세포의 종양 형성 및 침윤 억제

신소연(Soyeon Shin),김용조(Yong-Jo Kim),한승현(Seung-Hyeon Han),프라산타(Prashanta Silwal),허준영(Jun-Young Heo),전영주(Young-Joo Jeon),박승길(Seung-Kiel Park),권기량(Gi-Ryang Kweon),박종일(Jong-Il Park),임규(Kyu Lim) 한국생명과학회 2017 생명과학회지 Vol.27 No.9

대장암은 미국 등 서양 국가뿐만 아니라 국내에서도 2번째로 많이 발병이 되는 암으로 알려져 있다. 역학조사에 의하면 오메가-3를 많이 섭취한 인종에서 대장암 발생빈도가 감소하고 최근 오메가-3는 수종의 암에 대해 항암작용을 나타낸다고 한다. 이에 본 연구에서는 대장암에서 DHA의 항침윤, 항혈관 신생 및 항종양 형성능 억제의 기전을 규명하여 다음과 같은 결과를 얻었다. DHA는 인체 대장암 세포주 HT29 의 증식을 농도 의존적으로 억제하였으나 AA는 거의 영향이 없었다. FACS 분석에서 DHA 처리했을 때 Sub G1 phase의 세포가 DHA의 농도 의존적으로 증가 하였다. DHA 처리 후 cleaved PARP가 증가하고, uncelaved caspase-3가 감소 하였다. HT29 세포의 침윤능은 DHA 처리에 의해 억제 되었다. DHA 처리 후 MMP-9 및 MMP-2 mRNA양이 감소 되었을 뿐만 아니라 그 promoter의 reporter 활성도 억제하였으며 VEGF promoter 활성도 DHA에 의해 억제 되었다. NF-kB promoter 활성 및 핵으로의 이동도 DHA에 의해 억제 되었다. In vivo 동물실험에서 생쥐 대장암 세포주인 MCA38에 대한 Fat-1 transgenic mice에서의 종양 형성능은 현저히 억제 되었다. 면역형광염색법을 이용한 Fat-1 transgenic mice의 종양 조직에서의 TUNEL 양성세포는 wild type mice에 비해 현저히 증가하였으나 CD31의 형광강도는 감소 되었다. 이상의 결과로 오메가-3는 대장암 세포에서 NF-kB 억제에 따른 COX-2, MMP-2 및 MMP-9 등 matrix matalloproteinase의 억제를 통한 침윤능의 억제, VEGF 억제를 통한 혈관신생의 억제등 복합적 기전에 의해 항암작용을 나타내리라 생각되며, 따라서 오메가-3는 대장암의 예방 및 치료에 유용하게 사용될 수 있으리라 생각된다. Epidemiology studies have reported a reduced incidence of colon cancer among populations that consume a large quantity of ω3-polyunsaturated fatty acids (ω3-PUFAs) of marine origin. Herein, we demonstrated a mechanism of anticancer action of ω3-PUFAs, showing that they suppressed invasion and tumorigenicity in colon cancer cells. Docosahexaenoic acids (DHA) inhibited the cell growth of HT29 cells. This action likely involved apoptosis, given that the DHA treatment increased the cleaved form of PARP and sub G1 cells. Moreover, the invasiveness of HT29 cells was inhibited following DHA treatment, whereas arachidonic acid (AA) had no effect. The levels of Matrix-metalloproteinase-9(MMP-9) and MMP-2 mRNA decreased after DHA pretreatment. DHA treatment inhibited MMP-9 and MMP-2 promoter activities and reduced VEGF promoter activity. DHA pretreatment also inhibited the activities of prostaglandin-2 (PGE2)-induced MMPs and the VEGF promoter. Cyclooxygenase-2(COX-2) overexpression increased the activity of MMPs and that of the Vascular endotherial growth factor (VEGF) promoter in HT29 cells, and DHA inhibited NF-kB and COX-2 promoter reporter activities. As shown by in vivo experiments, when mouse colon cancer cells (MCA38) were implanted into Fat-1 and wild-type mice, both the tumoral size and volume were dramatically inhibited in Fat-1 transgenic mice. Furthermore, TUNEL-positive cells increased in tumors from Fat-1 mice compared with wild mice. In immunohistochemistry, the intensity of CD31 in Fat-1 tumors was weaker. These findings suggest that ω3-PUFAs may inhibit tumorigenicity and angiogenesis as well as cancer cell invasion by suppression of COX-2, MMPs and VEGF via the reduction of NF-kB in colon cancer.

인체 구강암 세포주에서 Docosahexaenoic acid에 의한 세포독성 기전

홍태화(Tae-hwa Hong),김훈(Hoon Kim),신소연(Soyeon Shin),Kaipeng Jing,정소연(Soyeon Jeong),임현(Hyun Lim),윤동혁(Donghyuk Yun),정기은(Ki-Eun Jeong),이명렬(Myung-Ryul Lee),박종일(Jong-Il Park),권기량(Gi-Ryang Kweon),박승길(Seung Kiel 한국생명과학회 2013 생명과학회지 Vol.23 No.5

오메가-3 지방산은 많은 암에서 세포독성을 나타낸다고 보고 되어 왔으나 구강암에 대한 연구는 전혀 없다. 이에 본 연구에서는 구강암세포에서 오메가-3 지방산 중 DHA의 세포독성 기전을 규명하여 다음과 같은 결과를 얻었다. DHA는 구강암 세포주 SCC-4 및 SCC-9의 증식을 농도 의존적으로 억제하였으며, FACS 분석, TUNEL assay 및 PARP cleavage 등에 의해 자가사멸을 유도함이 확인 되었다. 또한 DHA는 LC-3II 단백증가, GFP-LC-3 dot 형성 및 autophagic flux assay 등에 의해 자가포식도 유도됨이 규명되었다. SCC-9 세포에서 AMPK의 인산화는 DHA 에 의해 증가 하였으나, p-AKT^Thr308, p-AKT^Ser473 및 mTOR단백양은 감소하였다. 이상의 결과로 DHA는 구강암세포에서 AMPK 활성증가 및 AKT 억제에 통한 mTOR 신호경로 차단에 따른 자가사멸 및 자가포식에 의해 세포독성을 나타낼 수 있음을 시사하며, 따라서 DHA는 구강암의 예방 및 치료에 유용하게 사용될 수 있으리라 생각된다. In the United States, about 40,000 new cases of oral cancer are diagnosed each year and nearly 7,800 patients died from it in 2012. Omega-3 polyunsaturated fatty acids have been found to have anticancer effects in a variety of cancer cell lines and animal models, but their effect in oral cancer remains unclear. This study was designed to examine the effect of docosahexaenoic acid (DHA, a kind of omega- 3 fatty acid) on oral cancer cells and the molecular mechanism of its action. We found that exposure of squamous cell carcinoma-4 (SCC-4) and squamous cell carcinoma-9 (SCC-9) human oral cancer cells to DHA induced growth inhibition in a dose- and time-dependent manner. Meanwhile, in addition to the elevated levels of apoptotic markers, such as cleaved PARP, subG1 portion and TUNEL-positive nuclei, DHA led to autophagic vesicle formation and an increase in autophagic flux, indicating the involvement of both apoptosis and autophagy in the inhibitory effects of DHA on oral cancer cells. Further experiments revealed that the apoptosis and autophagy induced by DHA were linked to inhibition of mammalian target of rapamycin (mTOR) signaling by AKT inhibition and AMP-activated protein kinase (AMPK) activation in SCC-9 cells. Together, our results suggest that DHA induces apoptosis- and autophagy-associated cell death through the AMPK/AKT/mTOR signaling pathway in oral cancer cells. Thus, utilization of omega-3 fatty acids may represent a promising therapeutic approach for chemoprevention and treatment of human oral cancer.

백서 재생간의 DNA topoisomerase Ⅰ 에 관한 연구

권기량,임규,황병두 충남대학교 의과대학 지역사회의학연구소 1991 충남의대잡지 Vol.18 No.1

The study was performed to evaluate changes of activity of DNA topoisomerase I that controls and modifies the topological state of DNA according to postoperative days after partial hepatectomy. The results of this experimental study were summarized as follows : 1. The liver mass doubles during the first 48 hours, and the rate of regeneration is greatest between first and second days after partial hepatectomy. 2. 25% of enzyme activity was increased in the first 12 hours in comparison with that of control after partial hepatectomy. The second days showed maximal increment. 200% in comparison with that of control. Later, enzyme activity was decreased till the seventh days after partial hepatectomy. 3. DNA Topo I was inhibited very slightly be camptothecin but 50% of enzyme activity was inhibited at 50μM of 10-hydroxycamptothecin. 4. In the case of spermine. 0.1mM. Complex formation was highest as 0.1mM spermine. 5. The enzyme shows a pH optimum around 6.4, but enzyme activity is seen within a broad range between pH 5.8 and 9.0. 6. The enzyme is completely inactivated by heat treatment for 5 minutes at 50℃. 7. 2mM Mg^2+ activates the enzyme activity over 3-folds. Cu^2+, Zn^2+ completely inhibit it and Mn^2+, Co^2+, and Se^2+ inhibit upto 40%, 40% and 60% at 2mM respectively. 8. There was no sighificant difference between resection and regeneration liver. These results suggested that the role of DNA topoisomerase I is related to 2nd peak of DNA synthesis in regeneration of liver after partial hepatectomy.

사람 대장조직 DNA Topoisomerase I 에 관한 연구

오수정,권기량,임규,황병두 충남대학교 의과대학 지역사회의학연구소 1991 충남의대잡지 Vol.18 No.2

DNA topoisomerase I was prepared from normal mucosa and cancer tissue of human colon and the properties were compared. Specific activity of the enzyme from normal mucosa was 257.2 unit/mg protein/ min and that from cancer tissue was 403 unit/mg protein/min. Both enzymes showed a broad pH optimum from pH 5.8 to pH 7.4 and arc heat-labile, being completely inactivated by heat treatment at 55℃ for 5 minutes. The enzyme from normal mucosa was activated 0.2 M K^+ and 0.15 M Na^+ approximately 20 and 4 folds and that from cancer tissue was activated approximately 40 and 10 folds, respectively. Mg^2+ was the most potent activator, the enzyme from normal mucosa being 5, 20, and 60 folds activated at 2 mM, 10 mM, and 20 mM, respectively and the enzyme from cancer tissue being 2.5, 13, 26 folds activated, respectively. Both enzymes were inhibited by 2 mM ATP and 5 mM GTP in the presence of Mg^2+ and K^+ , respectively. ddATP and ddGTP were the potent inhibitors of these enzymes. The enzvate front nurmal mucosa was activated by spermine, spermidine, and histone H3 and that from cancer tissue was activated by histone H1. The enzyme from normal mucosa yeas completely inactivated by alkaline phosphatase treatment whereas the enzyme activity from cancer tissue was indifferent. The molecular weight of both enzymes were 150 KD. Camptothecin and 10-OH-camptothecin variably inhibited DNA relaxation according to tile enzyme preparation and there was no DNA fragmentation in the cleavage assay. From the above results, the possible role of phosphorylation for regulation of this enzyme and the application of camptothecin to colon cancer were discussed.

가토뇌 Microtubule의 효소적 Carboxylmethylation이 Microtubule 기능 및 성상에 미치는 영향

곽명헌,권기량,임규,황병두 충남대학교 의과대학 지역사회의학연구소 1991 충남의대잡지 Vol.18 No.2

Microtubule purfied from rabbit brain by 2 cycles of polymerization and depolymerization in the presence of 2 mM GTP and 20% glycerol was carboxyl methylated by a purified protein methylase Ⅱ of rabbit brain, and changes in the properties of microtubule by carboxylmethylation were inverstigated. Tubulin and microtubule associated proteins were endogenously and exogenously carboxelmethvlated by protein methylase Ⅱ in a rate of 28.6 and 71.7 pmoles of methyl group incorporated into 1 mg protein for 15 minutes, respectively. Microtubule was found to be a better substrate than histone for this enzyme. Microtubule carboxylmethylation by protein methylase Ⅱ was activated 8%, 16% , 6%, 15% and 34% by 2 mM GDP, 2 mM Mg^2+, 2 mM Ca^2+, 40 μM colchicine and 40 μM podophyllotoxin, respectively. The binding of colchicine and Ca^2+ to microtubule were slightly enhanced by carboxylmethlated of microtubule. Polymerization kinetics of carhoxvlmethylated and noncarhuxylmethylated microtubule were similar. The inhibitory action of colrhicine and Ca^2+ on the microtubule polymerization was potentiated by carboxylnethylation of microtubule. These results were discussed with regard to the possible role of protein carboxylmethylation by protein methylase Ⅱ in the regulation of microtubule function.

Tubulin Polymerization에 대한 Podophyllotoxin의 영향

이성민,권기량,임규,황병두 충남대학교 의과대학 지역사회의학연구소 1991 충남의대잡지 Vol.18 No.1

Although the inhibition of microtubule assembly by podophyllotoxin is well established, the mediatory effect of some microtubule associated proteins for inhibition by podophyllotoxin is not clear. The microtubule(MADBP^+) was prepared from rabbit brain through 2 cycles of polymerization-depolymerization and the tubulin without DNA binding protein(MADBP^-) was prepared from MADBP^+ using double strand DNA-cellulose column chromatography and the properties of these tubulin preparation were compared with those of MADBP^+ being sigmoidal and MADBP^- being hyperbolic. MADBP^+ polymerization could he inhibited by podophyllotoxin, whereas MADBP^- polymerization could not. In the various concentration of podophyllotoain, there was little difference in colchicine binding activity between MADBP^- and MADBP^-. In the 2μM concentration of podophyllotoxin, the colchicine binding to MADBP^+ and MADBP^- were competitively inhibited with same extension. From the above results, the possible existence of some proteins which mediates the inhibitory effect of podophyllotoxin for tubulin polymerization was discussed.