Establishment of a Human Embryonic Germ Cell Line and Comparison with Mouse and Human Embryonic Stem Cells

Hyun Soo Yoon,김철근,Jong Hyuk Park,Sun Jong Kim,Jung Bok Lee,Ji Min Song,Sung Il Roh 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly estab-lished, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differ-entiation over a year of continuous culture. They are karyotypically normal and express high levels of alka-line phosphatase, Oct-4, and several cell-surface mark-ers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.

TM6, a Novel Nuclear Matrix Attachment Region, Enhances Its Flanking Gene Expression through Influencing Their Chromatin Structure

Lusha Ji,Cheng-Chao Zheng,Rui Xu,Longtao Lu,Jiedao Zhang,Guodong Yang,Jinguang Huang,Changai Wu 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Nuclear matrix attachment regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase II-binding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two to-bacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription.

MicroRNA-409-3p Inhibits Migration and Invasion of Bladder Cancer Cells via Targeting c-Met

Xin Xu,Liping Xie,Hong Chen,Yiwei Lin,Zhenghui Hu,Yeqing Mao,Jian Wu,Xianglai Xu,Yi Zhu,Shiqi Li,Xiangyi Zheng 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

There is increasing evidence suggesting that dysregulation of certain microRNAs (miRNAs) may con-tribute to tumor progression and metastasis. Previous studies have shown that miR-409-3p is dysregulated in some malignancies, but its role in bladder cancer is still unknown. Here, we find that miR-409-3p is down-regulated in human bladder cancer tissues and cell lines. Enforced expression of miR-409-3p in bladder cancer cells significantly reduced their migration and invasion without affecting cell viability. Bioinformatics analysis identified the pro-metastatic gene c-Met as a potential miR-409-3p target. Further studies indicated that miR-409-3p suppressed the expression of c-Met by binding to its 3-untranslated region. Silencing of c-Met by small interfering RNAs phenocopied the effects of miR-409-3p overexpression, whereas restoration of c-Met in bladder cancer cells bladder cancer cells overexpressing miR-409-3p, partially reversed the suppressive effects of miR-409-3p. We further showed that MMP2 and MMP9 may be downstream effector proteins of miR-409-3p. These findings indicate that miR-409-3p could be a potential tumor suppressor in bladder cancer.

Epithelial-Mesenchymal Transition and Migration of Prostate Cancer Stem Cells Is Driven by Cancer-Associated Fibroblasts in an HIF-1alpha/beta-Catenin-Dependent Pathway

Yong Luo,Ling Lan,Yong-Guang Jiang,Jia-Hui Zhao,Ming-Chuan Li,Neng-Bao Wei,Yun-Hua Lin 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Although cancer stem cells (CSCs) play a crucial role in seeding the initiation of tumor progression, they do not always possess the same potent ability as tumor metastasis. Thus, precisely how migrating CSCs occur, still remains unclear. In the present study, we first comparatively analyzed a series of prostate CSCs, which exhibited a dynamically increasing and disseminating ability in nude mice. We observed that the transcriptional activity of HIF-1 and -catenin became gradually elevated in these stem cells and their epithelial-mesenchymal transition (EMT) characteristic altered from an epithelial type to a mesenchymal type. Next, we further used cancer-associated fibroblasts (CAFs), which were cultured from surgically re-sected tissues of prostate cancer (PCa) to stimulate prostate CSCs. Similar results were reconfirmed and showed that the protein levels of both HIF-1 and -catenin were markedly improved. In addition, the EMT phenotype displayed a homogenous mesenchymal type, accompanied with increased aggressive potency in vitro. Most importantly, the aforementioned promoting effect of CAFs on prostate CSCs was completely repressed after “silencing” the activity of -catenin by transfection of stem cells with ShRNA. Taken together, our observations suggest that prostate migrating CSCs, with a mesenchymal phenotype, could be triggered by CAFs in a HIF-1alpha/beta-catenin-depen-dent signaling pathway.

Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase

차형진,최관용,장도수,김연길,홍비학,우재성,김경태 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

Proteins have evolved to compensate for detrimental mu-tations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isome-rase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Pre-vious results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 Å resolution, respectively, and compared with previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a second-site suppressor.

Autophagy: A Critical Regulator of Cellular Metabolism and Homeostasis

Stefan W. Ryter,Suzanne M. Cloonan,Augustine M. K. Choi 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

Autophagy is a dynamic process by which cytosolic mate-rial, including organelles, proteins, and pathogens, are sequestered into membrane vesicles called autopha-gosomes, and then delivered to the lysosome for degra-dation. By recycling cellular components, this process provides a mechanism for adaptation to starvation. The regulation of autophagy by nutrient signals involves a com- plex network of proteins that include mammalian target of rapamycin, the class III phosphatidylinositol-3 kinase/Be-clin 1 complex, and two ubiquitin-like conjugation systems. Additionally, autophagy, which can be induced by multiple forms of chemical and physical stress, including endo-plasmic reticulum stress, and hypoxia, plays an integral role in the mammalian stress response. Recent studies indicate that, in addition to bulk assimilation of cytosol, autophagy may proceed through selective pathways that target distinct cargoes to autophagosomes. The principle homeostatic functions of autophagy include the selective clearance of aggregated protein to preserve proteostasis, and the selective removal of dysfunctional mitochondria (mitophagy). Additionally, autophagy plays a central role in innate and adaptive immunity, with diverse functions such as regulation of inflammatory responses, antigen presen-tation, and pathogen clearance. Autophagy can preserve cellular function in a wide variety of tissue injury and disease states, however, maladaptive or pro-pathogenic outcomes have also been described. Among the many diseases where autophagy may play a role in-clude proteo-pathies which involve aberrant accumulation of proteins (e.g., neurodegenerative disorders), infectious diseases, and metabolic disorders such as diabetes and metabolic syndrome. Targeting the autophagy pathway and its regu-latory components may eventually lead to the develop-ment of therapeutics.

EBP50 Phosphorylation by Cdc2/Cyclin B Kinase Affects Actin Cytoskeleton Reorganization and Regulates Functions of Human Breast Cancer Cell Line MDA-MB-231

Chaoyuan Sun,Junqi He,Junfang Zheng,Shan Cheng,Duiping Feng 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

The actin cytoskeleton plays an important role in cell shape determination, adhesion and cell cycle progression. Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+-H+ exchanger regulatory factor 1 (NHERF1), associates with actin cytoskeleton and is related to cell cycle progression. Its Ser279 and Ser301 residues are phosphorylated by cyclin-dependent kinase 2 (cdc2)/cyclin B during the mitosis phase. However, the biological significance of EBP50 phosphorylation mediated by cdc2/cyclin B is not clear. In the present study, MDA-MB-231 cells with low levels of endogenous EBP50 protein were stably trans-fected with constructs of EBP50 wild type (WT), phospho-deficient (serine 279 and serine 301 mutated to alanine-S279A/S301A) or phospho-mimetic (serine 279 and serine 301 mutated to aspartic acid-S279D/S301D) mutants. Subsequently, multiple phenotypes of these cells were characterized. Failure of cdc2/cyclin B-mediated EBP50 phosphorylation in cells expressing S279A/S301A (AA cells) significantly increased F-actin content, enhanced the adherence of cells to the extracellular matrix, altered cell morphology and caused defects in cytokinesis, as reflected in the formation of giant cells with heteroploid DNA and multinucleation or giant nuclei. Furthermore, knockdown of EBP50 expres-sion in AA cells rescued cell defects such as the cytokinesis failure and abnormal cell morphology. EBP50 S279A/ S301A had a weaker binding affinity with actin than EBP50 S279D/S301D, which might explain the increase of F-actin content in the AA cells. The present results suggest that cdc2/cyclin B-mediated EBP50 phos-phorylation may play a role in the regulation of various cell functions by affecting actin cytoskeleton reorgan-ization.

Nuclear FAK: a New Mode of Gene Regulation from Cellular Adhesions

Ssang-Taek Steve Lim 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

Focal adhesion kinase (FAK) is a protein tyrosine kinase (PTK) crucial in regulation of cell migration and prolifera-tion. In addition to its canonical roles as a cytoplasmic kinase downstream of integrin and growth factor receptor signaling, recent studies revealed new aspects of FAK action in the nucleus. Nuclear FAK promotes p53 and GATA4 degradation via ubiquitination, resulting in enhan-ced cell proliferation and reduced inflammatory responses. FAK can also serve as a co-transcriptional regulator that alters a gene transcriptional activity. These findings established a new paradigm of FAK signaling from cellular adhesions to the nucleus. Although physiological stimuli for controlling FAK nuclear localization have not been completely characterized, FAK shuttles from focal adhesions to the nucleus to directly convey extracellular signals. Interestingly, nuclear translocation of FAK becomes pro-minent in kinase-inhibited conditions such as in de-adhe-sion and pharmacological FAK inhibition, while a small fraction of nuclear FAK is observed a normal growth condition. In this review, roles of nuclear FAK in regulating transcription factors will be discussed. Furthermore, a potential use of a pharmacological FAK inhibitor to target nuclear FAK function in diseases such as inflammation will be emphasized.

Bone Morphogenetic Protein 9 Overexpression Reduces Osteosarcoma Cell Migration and Invasion

Zilan Lv,Ya-guang Weng,Dandan Yang,Jie Li,Min Hu,Min Luo,Xiaoqin Zhan,Peipei Song,Chen Liu,Huili Bai,Baolin Li,Yang Yang,Yingying Chen,Qiong Shi 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Transforming growth factor- (TGF-) is known to pro-mote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF- family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the ex-pression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.

Development of an Efficient Agrobacterium-Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

안율균,전종성,윤무경 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expres-sed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst non-transgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.