일차배양 마우스 태아 세포의 Glutathione - S - transferases 활동도와 Benzo ( a ) Pyrene 대사에 관한 연구

임인경,한병돈,이기호,윤택구 ( In Kyoung Lim,Byoung Don Han,Kee Ho Lee,Taik Koo Yun ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.1

We examined the effect of BHA on the activities of glutathione-S-transferase(GST) and the metabolism of benzo(α)pyrene in the primary culture of embryonic fibroblasts derived from the NIH(GP) mice. Optimum pH of GST was 6.5 with 1.0 mM of 1-chloro-2, 4-dinitrobenzene, 1.0 mM of reduced glutathione, and 50-100 ㎍ of cytosol proteins in 0.1 M potassium phosphate buffer. Optimum pH of GST with 0.5 mM 1,2-epoxy-3 (p-nitrophenoxy) propane, 10 mM of GSH and 50-100 ㎍ of cytosol proteins in 0.1 M potassium phosphate buffer was found to be 5.8. The activities of GST catalyzing CDNB were increased on the 5th day of the culture and maintained on the 7th day. The culture medium containing 100 μM BHA significantly increased the activities of GST with CDNB on the 7th day as compared with those of the control. The fractions of the conjugated B(α)P metabolites in both of the control and the BHA media were increased on the 7th day as compared with those on the 3rd day of the culture, after incubation with 4 μM ³H-B(α)P (500 mCi/m ㏖) for 24 hours. However, the binding levels of B(α)P to the cellular DNA, RNA and proteins were not changed depending on the culture days and the culture medium. BHA had no transplacental effect on the activities of glutathione-S-transferases and the metabolism of B(α)P in our experiment.

Diethylnitrosamine/Nodularin Induced Hepatocarcinogenesis in Fischer 344 Male Rat : Molecular Mechanism of Tumor Promotion by Testosterone and TGF-β1 During Hepatocarcinogenesis

임인경 가톨릭중앙의료원 가톨릭암센터 1998 암심포지움 Vol.- No.2

DEN/nodularin 투여한 웅성 쥐 간발암모델에서 nodularin 투여로 형성된 증시성결절은 GST-p (+) 이형성을 보이는 변형된 간세포이면서 활발한 세포 증식능을 보이고 있었다. 이때, 결절의 주변조직은 결절 안의 간세포에 비여 더욱 활발하게 증식하다가, nodularin 투여 중지 후에 급격한 변화를 나타 냄으로써 결절 내의 간세포 증식만이 관찰되었다. 한편, 간세포고사를 유도할 수 있는 TGF-β 1의 발현은 증식성결절 내에 국한 되어 있으면서, 주변조직에는TGF-β 1의 수용체 발현이 활발하였다. 이와 같은 현상은 증식성 결절의 선택적 성장유도와 동시에 주변조직의 선택적 세포고사를 유도하기에 아주 적절한 조건을 형성하고 있는 것이다.

성장이 활발한 세포에서 c - fos 암 유전자의 발현

임인경,이기호,이도종,윤택구,한병돈,유주현 ( In Kyoung Lim,Kee Ho Lee,Do Jong Lee,Taik Koo Yun,Byoung Don Han,Ju Hyun Yu ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.2

We describe here the expression of c-fos oncogene in the growth-stimulated cells, e.g., mouse prenatal tissues, FCS treated NIH-3T3 fibroblast and A549 human lung cancer cells. Total cellular RNA was isolated from A/J and C57BL/6J mouse embryos at the 13th and 17th day of pregnancy. RNAs isolated from mouse prenatal tissues were strongly hybridized with c-fos. NIH-3T3 mouse fibroblast and A549 lung cancer cells were stimulated by the 15% FCS after two to three days of serum depletion. During the serum stimulation, c-fos expression was detected at time intervals by the dot blot analysis. c-fos induction was detected from 30` after serum stimulation in NIH-3T3 cells, which was turned off after 2 hrs. On the other hand, c-fos induction in the A549 lung cancer cells was independent of the serum stimulation.

Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2

임인경,Yun Yeong Lee,류민숙,Hong Seok Kim,Masami Suganuma,Kye-Yong Song 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.3

The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC)  and PKC1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKC accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS104 via PKC1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKC were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKC expression and increased epi-dermal and hair follicle cell proliferation. Thus, PKC downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKC degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKC expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21WAF1 gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.

마우스 태아 일차 배양 세포내 고분자 화합물과 Benzo ( a ) pyrene 의 결합에 관하여

임인경,이왕식,윤택구 ( In Kyoung Lim,Wang Sik Lee,Taik Koo Yun ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.1

We describe here the binding of benzo(a)pyrene to the cellular macromolecules of mouse embryonic fibroblasts in primary culture. On the 3rd day of the culture, DME medium containing 10% FCS was removed and the cells were incubated with fresh medium containing 1μM, 2 μM and 4μM of benzo(a)pyrene for 24 hours. Cell growh was inhibited more than 20% after the first 48 hours and more than 50% after 96 hours. B(a)P cytotoxicity was not so severe with the 24 hour treatment. ³H-B(a)P was applied to the cell monolayers and more than 90% remained in the culture medium after a 24 hr incubation and about 2 to 10% was loosely bound to the cell surface. The ³H-B(a)P tightly bound to the cellular macromolecules was less than 0.5%. The contents of cellular DNA, RNA and protein were not changed with the treatment of 2 to 8 μM of B(a)P for 24 hours. Specific binding of ³H-B(a)P to DNA was linearly increased depending on the concentration of B(a)P lower than 2 μM, and it was saturated at 24μ μmoles/㎎ with higher than 2 μM of B(a)P. Specific activity of ³H-B(a)P-protein adduct was also lineraly increased with 2 μM of B(a)P, but it was not saturated up to 8 μM of B(a)P.

백서 간장 암세포의 원형질막 단백질에 대한 연구

林仁京,黃德秀,金允洙 연세대학교 의과대학 1982 연세의대논문집 Vol.15 No.2

c-fos Oncogene Expression in the Growth-Stimulated Cells

임인경,이기호,이도종,윤택구,한병돈,유주현,Lim, In-Kyoung,Lee, Kee-Ho,Lee, Do-Jong,Yun, Taik-Koo,Han, Byoung-Don,Yu, Ju-Hyun Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.2

마우스 태생조직과 혈청 투여로 세포 성장을 유도시킨 NIH 3T3 마우스 섬유아세포 및 A549 사람 폐암 세포주에서 c-fos 유전자의 발현을 조사하였다. 태령 13 일과 17일된 A/J, C57BL/6J 마우스 태아, 태반 및 태아 외막과 2-3 일간 성장을 정지시킨후 15% 혈청 투여로 세포성장을 유도한 NIH 3T3 섬유아세포와 A549 폐암 세포주에서 세포 총 RNA를 분리하였다. 마우스 태아 및 관련 장기는 태령에 관계없이 c-fos의 강한 발현을 나타내였으나 NIH 3T3 세포에서는 혈청 첨가 30분 후에 c-fos 유전자 발현이 유도되었다가 2시간 이후는 대조군 수준으로 감소하였다. 한편, A549 암세포에서는 혈청 첨가에 관계없이 c-fos 유전자의 발현이 활발하였다. We describe here the expression of c-fos oncogene in the growth-stimulated cells, e.g., mouse prenatal tissues, FCS treated NIH-3T3 fibroblast and A549 human lung cancer cells. Total cellular RNA was isolated from A/J and C57BL/6J mouse embryos at the 13th and 17th day of pregnancy. RNAs isolated from mouse prenatal tissues were strongly hybridized with c-fos. NIH-3T3 mouse fibroblast and A549 lung cancer cells were stimulated by the 15% FCS after two to three days of serum depletion. During the serum stimulation, c-fos expression was detected at time intervals by the dot blot analysis. c-fos induction was detected from 30' after serum stimulation in NIH-3T3 cells, which was turned off after 2 hrs. On the other hand, c-fos induction in the A549 lung cancer cells was independent of the serum stimulation.

Carcinogenesis of Oral Cancer

임인경 대한두경부종양학회 1997 대한두경부종양학회 학술대회 논문집 Vol.1997 No.2

Studies on the Activities of Glutathione-S-Transferases and the Metabolism of Benzo($\alpha$)pyrene in the Primary Culture of Mouse Embryonic Fibroblast.

임인경,한병돈,이기호,윤택구,Lim, In-Kyoung,Han, Byoung-Don,Lee, Kee-Ho,Yun, Taik-Koo 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

NIH(GP) 마우스 태아 세포 일차배양계에서 GST활성도와 B($\alpha$)P 대사에 미치는 BHA의 영향에 관한 실험을 수행하였다. 1-Chloro-2,4-dinitrobenzene (CDNB)을 기질로 하였을 때 GST의 최적 pH는 6.5 이었으며, 이때 반응액은 1.0 mM CDNB, 1.0 mM GSH 그러고 $50-100\;{\mu}g$의 세포질 단백으로 조성 하였다. 1,2-Epoxy-3-(p-nitrophenoxy)propane (alkyl epoxide)를 기질로 하였을 때 GST의 최적 pH는 5.8이었으며, 이때 반응액은 0.5 mM alkyl epoxide, 10 mM GSH, $50-100\;{\mu}g$의 세포잘 단백으로 조성하였다. CDNB를 기질로 하는 GST 활성도는 배양 5일에 의미있게 증가되었으며 그 활성도는 배양 7일까지 유지되었다. BHA배지에서 7 일간 배양된 세포내 GST 활성도는 대조군에 비하여 의미있게 증가하였다. $4\;{\mu}M$ $^3H-B(\alpha)P$ (500 mCi/m mol) 첨가하여 24시간 배양한 후 회수한 완전 배지와 BHA배지에서 분리되는 수용성, B(a)P 대사산물의 분획은 배양 3일에 비하여 배양 7일에 현저히 증가하였으며, 이때 BHA 첨가로 인한 영향은 없었다. B($\alpha$)P-DNA, B($\alpha$)P-RNA 그리고 B($\alpha$)P-protein adducts 양은 배양 일수나 배양액의 조건에 의하여 차이가 없었다. We examined the effect of BRA on the activities of glutathione-S-transferase(GST) and the metabolism of benzo($\alpha$)pyrene in the primary culture of embryonic fibroblasts derived from the NIH(GP) mice. Optimum pR of GST was 6.5 with 1.0 mM of 1-chloro-2, 4-dinitrobenzene, 1.0 mM of reduced glutathione, and $50-100\;{\mu}g$ of cytosol proteins in 0.1 M potassium phosphate buffer. Optimum pH of GST with 0.5 mM 1,2-epoxy-3 (p-nitrophenoxy) propane, 10 mM of GSH and $50-100\;{\mu}g$ of cytosol proteins in 0.1 M potassium phosphate buffer was found to be 5.8. The activities of GST catalyzing CDNB were increased on the 5th day of the culture and maintained on the 7th day. The culture medium containing 100\;${\mu}M$ BRA significantly increased the activities of GST with CDNB on the 7th day as compared with those of the control. The fractions of the conjugated B($\alpha$)P metabolites in both of the control and the BRA media were increased on the 7th day as compared with those on the 3rd day of the culture, after incubation with $4\;{\mu}M$ $^3H-B(\alpha)P$ (500 mCi/m mol) for 24 hours. However, the binding levels of B($\alpha$)P to the cellular DNA, RNA and proteins were not changed depending on the culture days and the culture medium. BHA had no trans-placental effect on the activities of glutathione-S-transferases and the metabolism of B($\alpha$)P in our experiment.

Binding of Benzo(a)pyrene to the Cellular Macromolecules of Mouse Embryonic Fibroblasts in Primary Culture.

임인경,이왕식,윤택구,Lim, In-Kyoung,Lee, Wang-Sik,Yun, Taik-Koo 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

마우스 [NIH (GP)] 태아세포 일차 배양계에 투여된 Benzo(a)pyrene과 세포내 고분자 화합물의 결합량 측정을 시도하였다. $0.5\;{\mu}M\;-8.0\;{\mu}M$의 $^3H$-B(a)P 첨가 배지로 24시간 배양한 후, $^3H$-B(a)P의 세포내외 분포도를 조사한 결과, 가해준 $^3H$-B(a)P의 90% 이상은 배지내에 잔류되어 있었고, 2-10%는 세포 표면에 가볍게 부착되었으며 (alcohol soluble fraction) 0.5%미만은 세포내 고분자 화합물에 결함되었다(alcohol insoluble fraction). B(a)P 24시간 처리군의 세포내 DNA, RNA 단백철 농도는 대조군에 비하여 변화가 없었다. 이때 DNA에 결합되어 냐타나는 $^3H$-B(a)P의 비활성도는 첨가된 B(a)P 농도에 비례하여 증가하다가 $2\;{\mu}M$이상 투여시 $24\;{\mu\mu}moles/mg$으로 포화됨을 확인하였다. 단백질과의 결합량은 B(a)p $2\;{\mu}m$ 까지는 농도에 비례증가하였으며, $8\;{\mu}m$까지 B(a)P와 단백질 결합이 계속 증가하는 것을 확인 하였다(r=0.99). We describe here the binding of benzo(a)pyrene to the cellular macromolecules of mouse embryonic fibroblasts in primary culture. On the 3rd day of the culture, DME medium containing 10% FCS was removed and the cells were incubated with fresh medium containing $1\;{\mu}M$, $2\;{\mu}M$ and $4\;{\mu}M$ of benzo(a)pyrene for 24 hours. Cell growh was inhibited more than 20% after the first 48 hours and more than 50% after 96 hours. B(a)p cytotoxicity was not so severe with the 24 hour treatment. $^3H$-B(a)P was applied to the cell monolayers and more than 90% remained in the culture medium after a 24 hr incubation and about 2 to 10% was loosely bound to the cell surface. The $^3H$-B(a)P tightly bound to the cellular macromolecules was less than 0.5%. The contents of cellular DNA, RNA and protein were not changed with the treatment of 2 to $8\;{\mu}M$ of B(a)P for 24 hours. Specific binding of $^3H$-B(a)P to DNA was linearly increased depending on the concentration of B(a)p lower than $2\;{\mu}M$, and it was saturated at $24\;{\mu\mu}moles/mg$ with higher than $2\;{\mu}m$ of B(a)p. Specific activity of $^3H$-B(a)P-protein-protein adduct was also lineraly increased with $2\;{\mu}m$ of B(a)p, but it was not saturated up to $8\;{\mu}M$ of B(a)p.