• Proceedings of the PSK Conference
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• 2003.10b
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• pp.176.2-176.2
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• 2003

Transcription factors (TF) can bind tightly to specific DNA lesions formed by some anticancer agents. The formation these TF:(drug-modified DNA) complex may disrupt expression of genes critical for cell survival, and it was proved to be one of biochemical mechanisms of anticancer activity. Based on this model, we have designed programmable DNA Alkylating agents that can also attract TF, especially nuclear receptors. As a model compound, we designed drug molecules, RA-mustard and Am580-mustard, that enable to bind both retinoic acid receptor (RAR) and DNA by using molecular modeling techniques, and synthesized them by connecting chlorambucil and ligand for RAR with a linker unit. (omitted)

• Animal cells and systems
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• v.14 no.2
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• pp.73-81
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• 2010

In this study, monoclonal antibodies against lysosomal acid phosphatase (LAP) of a salamander, Hynobius leechii, were used to determine the spatial and temporal expression of the LAP in the regenerating limbs. The Western blot and immunohistochemical analysis in the limb regeneration revealed that LAP was highly expressed at the dedifferentiation stage, especially in the wound epidermis and dedifferentiating limb tissues such as muscle and cartilage. With RA treatment, the LAP expression became upregulated in terms of both level and duration in the wound epidermis, blastemal cell and dedifferentiating limb tissues. In addition, in situ activity staining of LAP showed a similar result to that of immunohistochemistry. Thus, the activity profile of LAP activity coincides well with the expression profile of LAP during the dedifferentiation period. Furthermore, to examine the effects of lysosomal enzymes including LAP on salamander limb regeneration, lysosome extract was microinjected into limb regenerates. Interestingly, when the lysosome extract was microinjected into limb regenerates with a low dose of RA($50\;{\mu}g/g$ body wt.), skeletal pattern duplication occurred frequently in the proximodistal and transverse axes. Therefore, lysosomal enzymes might cause the regenerative environment and RA plays dual roles in the modification of positional value as well as evocation of extensive dedifferentiation for pattern duplication. In conclusion, these results support the hypothesis that dedifferentiation is a crucial event in the process of limb regeneration and RA-evoked pattern duplication, and lysosomal enzymes may play important role(s) in this process.

• The Korean Journal of Zoology
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• v.39 no.1
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• pp.65-74
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• 1996

Treatment of regenerating amphibian limbs with retinoic acid (RA) is known to induce paftern duplication, which is closely related to the extent of dedifferentiation. In the present study, the activities of trypsin- and chymotrypsin-like proteases are examined to delineate a possible role in the process of dedifferentiation in the regenerating limbs of urodeles, the Korean salamander (Hynobius leechii) and the Mexican axolod (Ambystoma mexicanum). Specifically, we were interested to know if there is any correlation between trypsin- and chymotrypsin-like protease activities and the state of dedifferentiation which is augmented by RA treatment. We were also interested in expoloring if there is any species-specific difference in the profile of enzyme activities during limb regeneration. The results showed that the activities of these two enzymes reached a peak level at dedifferentiation stage, and RA treatment caused elevation of their activities, especially in the case of trypsin-like protease. The increase of trypsin-like protease activity after RA treatment was pronounced in the Korean salamander, which might reflect a species-specific responsiveness to RA. The present results imply that trypsin and chymotrypsin or similar proteases may play an active role in the process of dedifferentiation in regenerating limbs, and that trypsin or trypsin-like eryrymes might be involved in the RA-evoked enhancement of dedifferentiation which precedes overt pattern duplication.

• Proceedings of the SCSK Conference
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• 2003.09b
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• pp.508-518
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• 2003

SZ95 cell is an immortalized human sebaceous gland cell line that shows the morphologic, phenotypic and functional characteristics of normal human sebocytes. Sebocytes may play crucial parts in the pathophysiologic processes and disorders of the pilosebaceous unit. The secretory activity of the sebaceous gland is remarkably species-specific and acne is an exclusively human disease. Thus, this SZ95 cells offer possibilities for investigations on the physiology of the sebaceous gland and its role in sebum-associated skin disease such as acne. In this study, we investigated the effects of 13-cis-retinoic acid (13-cis-RA) and spironolactone, frequently used as therapeutic agents of acne, on the lipid synthesis and proliferation of human sebocytes. Cell proliferation was determined by MTT assay and cytoplasmic lipid droplets was shown by Oil-red a staining. Total lipid levels were biochemically estimated by the sulfo-phospho-vanilline reagent. 13-cis-RA and spironolactone significantly inhibited proliferation and lipid levels in a dose-dependent manner. Combined treatment with testosterone and 13-cis-RA or spironolactone resulted in a lower total lipid levels than that with androgen alone. These observations indicate that 13-cis-RA and spironolactone are potent inhibitors of both cell proliferation and lipid synthesis in human sebocytes. We will provide experimental evidence that this human sebocyte cell line serves as an adequate tool for evaluating the anti-lipogenic activity of various compounds potentially useful for the bioactive cosmeceutical ingredients on acne skin, and studying the intracellular biochemical markers depending on the types of compounds from various sources.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.867-870
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• 2001

All-trans-retinoic acid (RA) plays essential roles in the regulation of differentiation and proliferation. It has been proved that RA is effective in the treatments of epithelial and hematologic malignancies. However, in spite of its pronounced effects, the clinical applications of RA are limited due to the retinoid acute resistance. Although RA induces complete remission in a high proportion of the patients of acute promyelocytic leukemia (APL), the cancer was relapsed in many patients after a brief remission in spite of a continued RA treatment. Patients who relapsed from remission that was initially induced by RA had clinically resistant to further RA treatment. That is, specific cytochrome P450 enzymes in the liver were induced by the continuous oral administration of RA, thereby accelerating the metabolism of RA. To overcome this problem, biodegradable microspheres were proposed by us, previously. And, several microsphere formulations for RA delivery have been prepared and studied on their effectiveness. Recently, poly(D,L-lactide) (PDLLA) microsphere formulation was optimized, And, from the animal studies by using a mouse and a rat, it have appeared to be effective on both the inhibition of tumor growth and chemoprevention of a carcinogenesis. In this study, subacute toxicities of the PDLLA microsphere formulation have been investigated as a preclinical test. For the test, the microspheres was injected subcutaneously into the back site of rats, and body weight change, clinical signs, hematological changes, blood biochemistry were evaluated. As a result, severe toxicities such as mortality were observed at the dose of 100mg RA/kg, and toxicities were not observed at the dose of 50mg RA/kg, which is the effective dose against carcinogenesis. Bone fracture, observed at several rats, might be inhibited by treating them with anti-inflammatory drugs.

• Journal of Life Science
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• v.25 no.6
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• pp.703-708
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• 2015

Hox genes encode a highly conserved family of homeodomain-containing transcription factors controlling vertebrate pattern formation along the anteroposterior body axis during embryogenesis. Retinoic acid (RA) is a key morphogen in embryogenesis and a critical regulator of both adult and embryonic cellular activity. Specifically, RA regulates Hox gene expression in mouse- or human-derived embryonic carcinoma (EC) cells. Histone modification has been reported to play a pivotal role in the process of RA-induced gene expression and cell differentiation. As histone modification is thought to play an essential role in RA-induced Hox gene expression, we examined RA-induced initiation of collinear expression of Hox genes and the corresponding histone modifications in F9 murine embryonic teratocarcinoma (EC) cells. Hox expression patterns and histone modifications were analyzed by semiquantitative RT-PCR, RNA-sequencing, and chromatin immuno-precipitation (ChIP)-PCR analyses. The Hoxc4 gene (D0) was initiated earlier than the Hoxc5 to –c10 genes (D3) upon RA treatment (day 0 [D0], day 1 [D1], and day 3 [D3]). The Hox nonexpressing D0 sample had a strong repressive marker, H3K27me3, than the D1 and D3 samples. In the D1 and D3 samples, reduced enrichment of the H3K27me3 marker was observed in the whole cluster. The active H3K4me3 marker was closely associated with the collinear expression of Hoxc genes. Thus, the Hoxc4 gene (D1) and all Hoxc genes (D3) expressed H3K4me3 upon transcription activation. In conclusion, these data indicated that removing H3K27me3 and acquiring H3K4me3 regulated RA-induced Hoxc gene collinearity in F9 cells.

• The Korean Journal of Zoology
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• v.37 no.1
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• pp.40-48
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• 1994

세포분화에 따른 Small GTP-binding protein의 역할을 밝히기 위하여 Retinoic acid(RA)와 dibutyryl cyclic AMP(dbcAMP)로 분화를 유도한 F9 기형암종세포의 형태적인 변화와 함께 Small GTP-binding protein의 분포를 조사하였다. RA와 dbcAMP를 처리한 세포는 분화유도 5일경(초기 분화 단계)에 분명한 세포의 경계를 보이기 시작하여 7일경(분화 후기 단계)에는 거의 모든 세포가 등근 분화된 형태로 전환되었다. 이 분화과정 동안 세포막에는 많은 microvilli와 lamellopodia 같은 구조물이 나타났다. 아울러 초기 분화 단계에 많은 량의 laminin이 발현되었으며 분화 후기에 microtubule의 재분포가 관찰되었다. 세종류의 Small GTP-binding protein(25 23, 21 KD)이 F9 세포의 막성분과 세포질에서 관찰되었으며 분화가 진행됨에 따라서 세단백질 모두 증가되는 양상을 보였다 이러한 결과는 Small GTP-binding protein이 F9 세포의 분화에 특별한 기능을 가지고 있음을 시사해 주고 있다.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.102-102
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• 2003

Embryonic stem (ES) cells proliferate extensively in the undifferentiated state and have the potential to differentiate into a variety of cell types in response to various environmental cues. The generation of functional dopaminergic neurons from ES cells is promising for cell replacement therapy to treat Parkinson's disease. We compared the in vitro differentiation potential of pluripotent human embryonic stem (hES, MB03) cells induced with basic fibroblast growth factor (bFGF) or retinoic acid (RA). Both types of treatment resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)- $\alpha$ during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression, compared to control (P<0.05). In contrast, no effect was observed on the rate of mature or glutamic acid decarboxylase-positive neurons. Immunostaining and HPLC analyses revealed the higher levels of TH (20.3%) and dopamine in bFGF and TGF-$\alpha$ treated hES cells than in RA or BDNF treated hES cells. The results indicate that TGF-$\alpha$ may be successfully used in the bFGF induction protocol to yield higher numbers of functional dopaminergic neurons from hES cells.

• Tuberculosis and Respiratory Diseases
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• v.82 no.1
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• pp.42-52
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• 2019

Background: Transforming growth factor ${\beta}$ (TGF-${\beta}$), retinoic acid (RA), p38 mitogen-activated protein kinase (MAPK), and MEK signaling play critical roles in cell differentiation, proliferation, and apoptosis. We investigated the effect of RA and the role of these signaling molecules on the phosphorylation of Smad2/3 (p-Smad2/3) induced by TGF-${\beta}1$. Methods: A549 epithelial cells and CCD-11Lu fibroblasts were incubated and stimulated with or without all-trans RA (ATRA) and TGF-${\beta}1$ and with MAPK or MEK inhibitors. The levels of p-Smad2/3 were analyzed by western blotting. For animal models, we studied three experimental mouse groups: control, bleomycin, and bleomycin+ATRA group. Changes in histopathology, lung injury score, and levels of TGF-${\beta}1$ and Smad3 were evaluated at 1 and 3 weeks. Results: When A549 cells were pre-stimulated with TGF-${\beta}1$ prior to RA treatment, RA completely inhibited the p-Smad2/3. However, when A549 cells were pre-treated with RA prior to TGF-${\beta}1$ stimulation, RA did not completely suppress the p-Smad2/3. When A549 cells were pre-treated with MAPK inhibitor, TGF-${\beta}1$ failed to phosphorylate Smad2/3. In fibroblasts, p38 MAPK inhibitor suppressed TGF-${\beta}1$-induced p-Smad2. In a bleomycin-induced lung injury mouse model, RA decreased the expression of TGF-${\beta}1$ and Smad3 at 1 and 3 weeks. Conclusion: RA had inhibitory effects on the phosphorylation of Smad induced by TGF-${\beta}1$ in vitro, and RA also decreased the expression of TGF-${\beta}1$ at 1 and 3 weeks in vivo. Furthermore, pre-treatment with a MAPK inhibitor showed a preventative effect on TGF-${\beta}1$/Smad phosphorylation in epithelial cells. As a result, a combination of RA and MAPK inhibitors may suppress the TGF-${\beta}1$-induced lung injury and fibrosis.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.193-202
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• 2011

Objective: We found previously that $interferon$ $regulatory$ factor ($Irf$)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of $Irf-1$ in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of $Irf-1$ and the mouse oocyte maturation. Methods: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured $in$ $vitro$ for 16 hours in the presence of varying concentrations of RA (0-10 ${\mu}M$). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 ${\mu}M$). With 100 nM RA treatment, lowest level of $Irf-1$ mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-${\alpha}$, macrophage inflammatory protein-$1{\beta}$, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion: We concluded that the maturation of oocytes and $Irf-1$ expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes $in$ $vitro$ by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for $in$ $vitro$ oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.