• 생명과학회지
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• 제5권1호
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• pp.1-7
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• 1995

Mammary orgamoids(ductal and endbud fragments) were cultured in a complete hormone medium(CHM) with 10%FBS, estradiol, progesterone, hydrocortisone, insulin, and prolactin, Several types of colonies were observed: stellate(14$$\pm$5.5%), duct(41$\pm$5.6%), web(35$\pm$3.6%), squamous(6$\pm$2.1%), and lobuloduct(4$\pm$1.2%), Squamous colony was typical squamous metaplasia(SM) with several layers of squamous epithlia and keratin pearls. At the immunocytochemical study, casein proteins were predominantly localized near the apical surfaces of the cells or in the lumina of ductal or lobuloductal colonies. To inhibit the formation of SM, we treated organoids with all-trans retinoic acid(RA) from 10$^{-6}$ to 10$^{-17}$ M in CHM. Formation of SN was completely inhibited at 10$^{-9}$M RA in CHM. The frequency of lobuloductal colony formation was increased with the augmentation of RA concentration.

• Imaging Science in Dentistry
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• 제33권2호
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• pp.97-105
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• 2003

Purpose : To evaluate the effect of all-trans-retinoic acid (ATRA) on the radiosensitivity of normal human oral keratinocyte (NHOK). Materials and methods: Relative cell survival fraction including SF2 (survival fraction at 2 Gy) was calculated on the basis of colony formation assay. Data were fitted to the linear-quadratic model to establish the survival curve and calculate α and β values. Using flow cytometry at 1, 2, 3, 4, and 5 days after exposure to 2 and 10 Gy irradiation, cell cycle arrest and apoptosis were analysed. To understand the molecular mechanism of the radiosensitization of ATRA on NHOK, proteins related with apoptosis and cell cycle arrest were investigated by Western blot analysis. Results: Treatment with ATRA resulted in a significant decrease of SF2 value for NHOK from 0.63 to 0.27, and increased α and β value, indicating that ATRA increased radiosensitivity of NHOK. ATRA increased LDH significantly, but increasing irradiation dose decreased LDH, suggesting that the radiosensitizing effect of ATRA is not directly related with increasing cell necrosis by ATRA. ATRA did not induce appotosis but increased G2 arrest after 10 Gy irradiation, implying that the increased radiosensitivity of NHOK may be due to a decrease in mitosis casued by increasing G2 arrest. ATRA inhibited the reduction of p53 at 3 days after l0Gy irradiation and increased p21 at 1 day after 10 Gy irradiation. Further study is required to determine the precise relationship between this effect and the radiosensitizing effect of A TRA. Conclusion: These results suggested that ATRA increase radiosensitivity by inhibiting mitosis caused by increasing G2 arrest.

• Journal of Photoscience
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• 제9권2호
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• pp.518-520
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• 2002

Photodynamic therapy (PDT) is a treatment modality based on photochemical reaction and the resultant cytotoxic reactive oxygen species. The platelet thrombus formation leading to stasis observed in vivo during PDT is called vascular shut down (VSD) effect. To investigate the mechanism of the VSD effect, we observed Human Umblical Vein Endothelial Cell (HUVEC) injury induced by photochemical reaction. We observed cell retraction and blebbing after PDT. It seems that the injury was not fetal and only morphological change. Then, the cytoplasm was stained by Calcein-AM and subendothelial area was evaluated from fluorescence microscopy. The rate of subendothelial area after PDT increased significantly. Second, we investigated interaction between neutrophils and HUVEC. Human promyelocytic leukemia cells (HL-60) were differentiated into neutrophil by incubation with all-trans retinoic acid. Calcein-AM labeled neutrophil adhesion to HUVEC was evaluated from fluorescence microscopy. PDT-induced neutrophil adhesion to HUVEC depended more on the exposure of subendothlial area than on neutrophil activation. This result suggests that there is a certain interaction between neutrophil and HUVEC during PDT.

• 한국식품영양과학회지
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• 제24권3호
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• pp.470-486
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• 1995

유선에 존재하는 유선 상피 모세포(mammary epithelial stem cells)의 존재 증거, 정상 조직에서 이들의역할, flow cytometry 및 면역 염색법에 의한 세포 분리, 세포 기질 단백질을 이용한 삼차원적 세포 배양에서의 증식 등을 요약한다. 유선의 실질 조직에 상피 모세포가 존재한다는 것은 여러 형태의 이식 실험에서 설명되었고 또 모세포의 표현형적 특징들은 여러 가지의 monoclonal antibodies에 의해 논증되었다. 이들 연구의 결과들은 유선의 모세포군이 end bud와 유선의 기저층(basal layer)에 존재한다고 제시하고 있다. 이들을 분리, 동정하기 위해 FITC-PNA와 PE-Thy-1.1 항체와 같은 세포 표지자를 이용하여 유선 상피 세포를 4군으로 나눌 수 있었다. FITC-PNA에만 양성 반응을 보인 PNA+ 세포군, PE-Thy-1.1에만 양성 반응을 보인 Thy-1.1+ 세포군, 이들 두 표지자에 양성 반응을 보인 B+ 세포군, 그리고 양쪽에 음성 반응을 보인 B- 세포군이었는데 이들을 flow cytometry로 분리하고 생체에 이식 실험을 하였을 때 PNA+ 세포군이 유선 모세포들을 가장 많이 가진 것으로 확인되었다. 그리고 유선 상피세포로 이루어진 유선 조직 절편(organoids) 이들 상피세포군을 세포외기질 단백질체인 Matrigel 내에서 배양한 결과 a) stellate, b) duct, c) web, d)squamous, e) lobuloduct 등 5종류의 다세포 구조물이 생성됨을 확인하였다. 이들 중 편평상피화생의 구조물은 정상적인 유선 조직에서는 나타나지 않는 구조물인데 all-trans retinoic acid를 처리하였을 때 배지의 조정에 따라 다소 차이는 있으나 대부분 이들 편평상피화생의 생성이 억제됨을 확인하였다. 이상의 결과로 보아 본 연구에 이용된 생체 이식법 및 삼차원적 세포외기질 세포 배양법이 상피세포의 성장, 분화 및 모세포 연구에 유용하게 이용될 수 있으리라 사료된다.

• 대한화장품학회지
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• 제29권2호
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• pp.79-104
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• 2003

Introducing to the market place, safe and effective product is an important responsibility of clinical investigators as well as regulatory agencies in all developed countries. Products claiming to improve skin wrinkles are no exceptions. To date, Renova(R) (all-trans retinoic acid), Avage(R) (tazarotene), and Botox(R) (botulinum toxin) are the only agents FDA approved to ameliorate wrinkles associated with photoaged skin in the USA. For all three, clinical evaluation of wrinkle severity was the primary endpoint required for the approval process. No sophisticated instrument measurements of wrinkles were required, nor used in the pivotal studies. The Division of Dermatologic & Dental Products of the US FDA (Director, Jonathan Wilkin, MD) is not against the use of mechanical instruments in assessing wrinkle severity. Its position on this issue however, remains that any such device must be grounded in patients' or product users' perspective, which means that the evaluation instrument must be clinically relevant and clinically perceptible. Sophisticated devices that can detect minimal improvement, but imperceptible to the users are considered useless in the eyes of the US FDA. Two instruments that have been tried in some antiwrinkle studies in the USA are silicone replicas and Primos. Despite their sophistications, they have clear limitations; thus have never replaced clinical evaluations in these studies. At most, they have served as secondary measures to provide corroborative data on the clinical efficacy of antiwrinkle products. For the foreseeable future, at least in the USA, careful clinical assessment of wrinkles will continue to serve as the critical benchmark to determine whether an antiwrinkle product has enough efficacy to benefit its users. We must not lose sight of the fact that sophisticated devices are only to serve in generating supportive evidence, and not the primary evidence, in any clinical studies.

• Biomolecules & Therapeutics
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• 제22권2호
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• pp.114-121
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• 2014

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

• Journal of Pharmaceutical Investigation
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• 제35권1호
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• pp.33-38
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• 2005

Recently increasing attention has been focused on solid lipid nanoparticles (SLN) as a parenteral drug carrier due to its numerous advantages that can come from both polymeric particle and fat emulsions, together with the possibility of controlled release and increasing drug stability. Lipophilic drugs such as paclitaxel, cyclosporin A, and all-trans retinoic acid have been successfully entrapped in SLN but the incorporation of hydrophilic drugs in SLN is very limited because of their very low affinity to the lipid. Therefore, as a new approach to improve the loading of hydrophilic drugs, a w/o/w emulsion technique has been developed. The primary objective of the current study was to improve the loading efficiency of a model hydrophilic drug, glycine (Log P = -3.44) into SLN. The proposed preparation process is as follows: A heated aqueous phase consisting of 0.1 ml of glycine solution in water (100 mg/ml), and poloxamer 188 (5 mg) were then added to a molten oil phase containing precirol (100 mg) and lecithin (5 mg). This mixture was dispersed by sonicator, leading to a w/o emulsion. A double emulsion (w/o/w) was formed after the addition of 2% poloxamer solution to the above dispersed system. After cooling the double emulsion, solid lipid nanosuspensions were successfully formed. The lipid nanoparticles had the mean particle size of 441.25 nm, and the average zeta potential of -20.98 mV. The drug loading efficiency was measured to be 8.54% and the drug loading amount was measured to be 0.92%. The w/o/w emulsion method showed an increased loading efficiency compared to conventional o/w emulsion method.

• BMB Reports
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• 제35권4호
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• pp.377-383
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• 2002

Arsenic trioxide ($As_O_3$) was recently demonstrated to be an effective inducer of apoptosis in patients with relapsed acute promyelocytic leukemia (APL) as well as patients with APL in whom all-trans-retinoic acid and conventional chemotherapy failed. Chronic myelogenous leukemia cells are highly resistant to chemotherapeutic drugs. To determine if $As_O_3$ might be useful for the treatment of chronic myelogenous leukemia, we examined the ability of $As_O_3$ to induce apoptosis in K562 cells. In vitro cytotoxicity of $As_O_3$ was evaluated in K562 cells by a MTT assay: the $IC_50$ value for $As_O_3$ was determined to be $10\;{\mu}m$. When analyzed by agarose gel electorphoresis, the DNA fragments became evident after incubation of the cells with $20\;{\mu}m$ $As_O_3$ for 24 h. We also found morphological changes and chromatin condensation of the cells undergoing apoptosis. Activation of caspase-3 was observed 6 h after treatment with $20\;{\mu}m$ $As_O_3$ by a Western blot analysis. Next, we examined the MAP kinase-signaling pathway of $As_O_3$-induced apoptosis in K562 cells. $As_O_3$ at $10\;{\mu}m$ strongly induced the activation of p38, inhibited $As_O_3$ induced apoptotic cell death. These results suggest that $As_O_3$ is able to induce the apoptotic activity in K562 cells, and its apoptotic mechanism may be associated with the activation of p38.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10875-10878
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• 2015

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.

• The Korean Journal of Physiology and Pharmacology
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• 제5권1호
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• pp.79-86
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• 2001

Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukemia. Dendritic cells (DCs) are important antigen-presenting cells in the development of antileukemic T-cell responses. In this study, we investigated the strategies to overcome ATRA resistance of APL cells by inducing the differentiation of DCs from human leukemic cell lines for the developtment of adoptive immunotherapy. CD83 was used as a mature DC marker in this study and the expression of CD83 mRNA was determined by RT-PCR method. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 with phorbol 12-myristate 13-acetate (PMA) resulted in the expression of myeloid-related DC phenotypes, while treatment of RPMI 7666 with fms-like tyrosine kinase 3 ligand (Flt3-ligand, FL) and treatment of NC-37 with PMA and FL led to the expression of lymphoid-related DC phenotypes. In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes could be generated from HL-60, NC-37 and RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used to generate antileukemic T cells in vitro for adoptive immunotherapy.