Molecular identification of archaic bones as a native Korean black bear: implications for the ongoing bear restoration program.

The genetic investigation of the archeological or museum samples, including endangered species, provides vital information necessary to plan, implement, and revisit conservation strategies. In South Korea, the Asian black bear went almost extinct in wild by 2002, without leaving any authentic specimens representing the native population. Recently researchers found a set of animal bones in a natural cave in Mt. Taebaek (South Korea), suspected to be of a bear. In the present study, we undertook a molecular investigation and radiocarbon dating to establish the species' identity, phylogenetic position, and approximate age of the recovered specimen. The genetic investigation (CytB, COI, D-loop, SRY, and ZFX-ZFY) identified the sample as a male Asian black bear with close phylogenetic affinity with Northeast Asian bears. Radiocarbon dating estimated the bones to be aged 1800-1942 calAD. These findings indicate that the bone specimens found in the natural cave in Mt. Taebaek were from an individual that naturally inhabited South Korea long before the importing of farm bears (the 1980s) and initiation of wild population restoration (2004). The present study provides the first genetic information record of the native South Korean black bear. Our findings reaffirm the appropriateness of the ongoing bear restoration program in South Korea, with the reintroduction of individuals from North Korea and Russia.

Glycosaminoglycans and glycolipids as potential biomarkers in lung cancer.

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

Effects of estrogen receptor α and ß on the expression of visfatin and retinol-binding protein 4 in 3T3-L1 adipocytes.

The aim of this study was to investigate the effects of estrogen and estrogen receptor α (ERα) and ß (ERß) on the expression of visfatin and retinol-binding protein 4 (RBP4) by treating 3T3-L1 adipocytes with estradiol (E2), estrogen receptor agonists and antagonists. Mature adipocytes were exposed to E2, the ERα agonist, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), the ERß agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), E2 with the ERα antagonist, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP), and E2 with the ERß antagonist, (5R, 11R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol [(R,R)-THC], at various concentrations. To determine the effects of ER subtypes on the expression of adipokines, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot analyses were performed. E2 concentrations of 10-5 and 10-6 mol/l induced a statistically significant increase in the expression of RBP4 (P=0.012 and P=0.011, respectively). In the cells treated with 10-5 mol/l PPT, RBP4 expression significantly increased (P<0.05) in a dose-dependent manner. Treatment with the ERα antagonist, MPP (10-5 mol/l), and E2 suppressed the expression of RBP4 (P=0.032). However, the expression of RBP4 was not significantly altered when the cells were treated with the ERß agonist or antagonist. The expression of visfatin was not affected by different concentrations of E2 and ERs. 17ß-estradiol significantly increased the secretion of RBP4 and upregulated RBP4 expression via ERα but not ERß in 3T3-L1 adipocytes. RBP4 expression was regulated by estrogen in the 3T3-L1 adipocytes and this effect was selectively mediated by ERα.

Promoter hypermethylation of the CFTR gene and clinical/pathological features associated with non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: The exact role of the cystic fibrosis transmembrane conductance regulator (CFTR) in pathophysiology, and the mechanisms regulating its expression are poorly understood. The CFTR gene is known to be genetically or epigenetically associated with several cancers. In the present study, the methylation status of the promoter region of the CFTR gene and its expression in primary non-small cell lung cancer (NSCLC) were investigated. METHODS: The methylation status of the promoter region of the CFTR gene in NSCLC tissue was assessed by pyrosequencing and methylation-specific PCR. Expression of the CFTR gene was analysed by real-time PCR, and CFTR gene reactivation was investigated using 5-aza-2'-deoxycytidine. The correlation between methylation of the CFTR gene and the clinical features of the patients was assessed. RESULTS: Methylation of the CFTR gene in NSCLC was quantitatively high by pyrosequencing analysis and qualitatively frequent by methylation-specific PCR analysis. Expression of the CFTR gene was significantly lower in NSCLC compared with normal lung tissue. In addition, the demethylating agent 5-aza-2'-deoxycytidine increased CFTR gene expression. Methylation of the CFTR gene was significantly greater in squamous cell carcinomas than in adenocarcinomas. CFTR gene methylation was associated with significantly poorer survival in young patients, but not in elderly patients. CONCLUSIONS: These findings suggest that DNA methylation may be important for downregulation of CFTR gene expression in lung cancer. Promoter hypermethylation of the CFTR gene may be an important prognostic factor in younger patients with NSCLC.

Lysophosphatidic acid induces STAT3 phosphorylation and ovarian cancer cell motility: their inhibition by curcumin.

Lysophosphatidic acid (LPA) is a biolipid that stimulates tumor cell invasion and metastasis. In this report, we determined the role of signal transducers and activators of transcription 3 (STAT3) and the effect of a chemopreventive agent, curcumin, on LPA-induced ovarian cancer cell motility. LPA phosphorylated STAT3 in a dose-dependent manner. Treatment of cells with a JAK/STAT inhibitor, AG490, inhibited LPA-induced cell motility. In contrast, transfection of a constitutively active form of STAT3 induced ovarian cancer cell motility. LPA also stimulated interleukin (IL)-6 and IL-8 secretion, which results in STAT3 phosphorylation. Treatment of the cells with curcumin inhibited LPA-induced IL-6 and IL-8 secretion and STAT3 phosphorylation, leading to blocked ovarian cancer cell motility. Collectively, the present study shows the critical role of STAT3 in ovarian cancer cell motility and that this process can be prevented by curcumin.

Association of matrix metalloproteinase-3 with cardiogenic activity during Noggin-induced differentiation of mouse embryonic stem cells.

BACKGROUND: Despite the pluripotency of embryonic stem (ES) cells, their clinical applications have been hindered due to the lack of reliable differentiation methods. Recently, it was shown that Noggin could effectively induce cardiomyocyte differentiation by transient treatment of ES cells. METHODS: To determine how Noggin may induce cardiac differentiation, we compared differentially expressed genes during Noggin-induced differentiation of ES cells using microarray analysis. We found Matrix metalloproteinase-3 (Mmp-3) expression was highly up-regulated by Noggin treatment. To understand the role of Mmp-3 in the cardiac differentiation of ES cells, we inhibited Mmp-3 activity by treating with a specific Mmp-3 inhibitor during Noggin-induced cardiac differentiation of ES cells. We also analyzed the expression levels of cardiac markers and the ratio of spontaneously beating embryoid bodies (EBs) in the presence of the Mmp-3 inhibitor. RESULTS: We analyzed EB samples from zero, two, and four days with or without Noggin treatment, and found that the expression levels of 2 (0 day), 56 (2 days), and 805 (4 days) genes were altered with Noggin treatment. Up-regulation of Mmp-3 was closely associated with relative increases of cardiogenic, vasculogenic, and hematopoietic genes in EB treated with Noggin. By inhibiting Mmp-3 activity, we verified that Mmp-3 activation is partly responsible for both the expression of cardiac markers and the elevated ratio of spontaneously beating to non-beating EBs. CONCLUSIONS: The concurrent expression of Mmp-3 with many cardiogenic genes and the specific inhibition of Mmp-3 revealed a critical role for Mmp-3 in Noggin-induced cardiac differentiation of ES cells.

Depletion of embryonic stem cell signature by histone deacetylase inhibitor in NCCIT cells: involvement of Nanog suppression.

The embryonic stem cell-like gene expression signature has been shown to be associated with poorly differentiated aggressive human tumors and has attracted great attention as a potential target for future cancer therapies. Here, we investigate the potential of the embryonic stem cell signature as molecular target for the therapy and the strategy to suppress the embryonic stem cell signature. The core stemness gene Nanog is abnormally overexpressed in human embryonic carcinoma NCCIT cells showing gene expression profiles similar to embryonic stem cells. Down-regulation of the gene by either small interfering RNAs targeting Nanog or histone deacetylase inhibitor apicidin causes reversion of expression pattern of embryonic stem cell signature including Oct4, Sox2, and their target genes, leading to cell cycle arrest, inhibition of colony formation in soft agar, and induction of differentiation into all three germ layers. These effects are antagonized by reintroduction of Nanog. Interestingly, embryonic carcinoma cells (NCCIT, NTERA2, and P19) exhibit a higher sensitivity to apicidin in down-regulation of Nanog compared with embryonic stem cells. Furthermore, the down-regulation of Nanog expression by apicidin is mediated by a coordinated change in recruitment of epigenetic modulators and transcription factors to the promoter region. These findings indicate that overexpression of stemness gene Nanog in NCCIT cells is associated with maintaining stem cell-like phenotype and suggest that targeting Nanog might be an approach for improved therapy of poorly differentiated tumors.

Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells.

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.

Genome-wide combination profiling of copy number and methylation offers an approach for deciphering misregulation and development in cancer cells.

Copy number changes and DNA methylation alterations are crucial to gene regulation in mammals. Recently, a number of microarray studies have been based on copy number and DNA methylation alterations in order to find clinical biomarkers of carcinogenesis. In this study, we attempted to combine profiles of copy number and methylation patterns in four human cancer cell lines using BAC microarray-based approaches and we detected several clinically important genes which showed genetic and epigenetic relationships. Within the clones analyzed, many contained cancer-related genes involved in cell cycle regulation, cell division, signal transduction, tumor necrosis, cell differentiation, and cell proliferation. One clone included the FHIT gene, a well-known tumor suppressor gene involved in various human cancers. Our combined profiling techniques may provide a method by which to find new clinicopathologic cancer biomarkers, and support the idea that systematic characterization of the genetic and epigenetic events in cancers may rapidly become a reality.

Identification of origin of unknown derivative chromosomes by array-based comparative genomic hybridization using pre- and postnatal clinical samples.

Microarray-based comparative genomic hybridization (array CGH) is a high-resolution and comprehensive method for detecting both genome-wide and chromosome-specific copy-number imbalance. We have developed an array CGH analysis system (consisting of an array CGH chip plus its exclusive analysis software) for constitutional genetic diagnosis and have evaluated the suitability of our system for molecular diagnosis using pre- and postnatal clinical samples. In a blind study, each of the 264 sample karyotypes identified by array CGH analysis was consistent with that identified by traditional karyotype analysis--with one exception, case (47, XXX)--and we were able to identify origins, such as small supernumerary marker chromosomes, which cannot be determined by conventional cytogenetics. We also acquired very accurate, fast and reliable results using a diminutive amount of clinical samples. Taken together, the array CGH platform developed in this study is a rapid, powerful and sensitive technology for pre- and postnatal diagnosis using a very small amount of clinical sample.

Coordinated change of a ratio of methylated H3-lysine 4 or acetylated H3 to acetylated H4 and DNA methylation is associated with tissue-specific gene expression in cloned pig.

Various cell types in higher multicellular organisms are genetically homogenous, but are functionally and morphologically heterogeneous due to the differential expression of genes during development, which appears to be controlled by epigenetic mechanisms. However, the exact molecular mechanisms that govern the tissue-specific gene expression are poorly understood. Here, we show that dynamic changes in histone modifications and DNA methylation in the upstream coding region of a gene containing the transcription initiation site determine the tissue-specific gene expression pattern. The tissue-specific expression of the transgene correlated with DNA demethylation at specific CpG sites as well as significant changes in histone modifications from a low ratio of methylated H3- lysine 4 or acetylated H3-lysine 9, 14 to acetylated H4 to higher ratios. Based on the programmed status of transgene silenced in cloned mammalian ear-derived fibroblasts, the transgene could be reprogrammed by change of histone modification and DNA methylation by inhibiting both histone deacetylase and DNA methylation, resulting in high expression of the transgene. These findings indicate that dynamic change of histone modification and DNA methylation is potentially important in the establishment and maintenance of tissue-specific gene expression.

Histone deacetylase inhibitor apicidin induces cyclin E expression through Sp1 sites.

We show that a histone deacetylase (HDAC) inhibitor apicidin increases the transcriptional activity of cyclin E gene, which results in accumulation of cyclin E mRNA and protein in a time- and dose-dependent manner. Interestingly, apicidin induction of cyclin E gene is found to be mediated by Sp1- rather than E2F-binding sites in the cyclin E promoter, as evidenced by the fact that specific inhibition of Sp1 leads to a decrease in apicidin activation of cyclin E promoter activity and protein expression, but mutation of E2F-binding sites of cyclin E promoter region fails to inhibit the ability of apicidin to activate cyclin E transcription. In addition, this transcriptional activation of cyclin E by apicidin is associated with histone hyperacetylation of cyclin E promoter region containing Sp1-binding sites. Our results demonstrate that regulation of histone modification by an HDAC inhibitor apicidin contributes to induction of cyclin E expression and this effect is Sp1-dependent.

Cooperation of H2O2-mediated ERK activation with Smad pathway in TGF-beta1 induction of p21WAF1/Cip1.

Although it has been demonstrated that p21WAF1/Cip1 could be induced by transforming growth factor-beta1 (TGF-beta1) in a Smad-dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood. In this study, we investigated a possible role of hydrogen peroxide (H2O2)-ERK pathway in TGF-beta1 induction of p21WAF1/Cip1 in human keratinocytes HaCaT cells. Using pharmacological inhibitors specific for MAP kinase family members, we found that ERK, but not JNK or p38, is required for TGF-beta1 induction of p21WAF1/Cip1. ERK activation by TGF-beta1 was significantly attenuated by treatment with N-acetyl-l-cysteine or catalase, indicating that reactive oxygen species (ROS) generated by TGF-beta1, mainly H2O2, stimulates ERK signaling pathway to induce the p21WAF1/Cip1 expression. In support of this, TGF-beta1 stimulation caused an increase in intracellular ROS level, which was completely abolished by pretreatment with catalase. ERK activation does not appear to be associated with nuclear translocation of Smad-3, because ERK inhibition did not affect nuclear translocation of Smads by TGF-beta1, and H2O2 treatment alone did not cause nuclear translocation of Smad-3. On the other hand, ERK inhibition ablated the phosphorylation of Sp1 by TGF-beta1, which was accompanied with the disruption of interaction between Smad-3 and Sp1 as well as of the recruitment of Sp1 to the p21WAF1/Cip1 promoter induced by TGF-beta1, indicating that ERK signaling pathway might be necessary for their interaction. Taken together, these results suggest that activation of H2O2-mediated ERK signaling pathway is required for p21WAF1/Cip1 expression by TGF-beta1 and led us to propose a cooperative model whereby TGF-beta1-induced receptor activation stimulates not only a Smad pathway but also a parallel H2O2-mediated ERK pathway that acts as a key determinant for association between Smads and Sp1 transcription factor.

beta-Carboline alkaloid suppresses NF-kappaB transcriptional activity through inhibition of IKK signaling pathway.

Nuclear factor (NF)-kappaB transcription factors play an evolutionarily conserved and critical role in the triggering and coordination of both innate and adaptive immune responses. Therefore, there is intense interest in understanding the regulation of this transcription factor in the context of various diseases. Studies investigated the suppression mechanism of NF-kappaB signaling pathways by a beta-carboline alkaloid (C-1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. beta-Carboline alkaloid decreased the level of inducible nitric oxide sythase (iNOS) protein and NOS promoter activities in a concentration-dependent manner. This effect was accompanied by the reduction of NF-kappaB DNA binding activity as well as NF-kappaB nuclear translocation. In addition, beta-carboline alkaloid reduced the degradation and phosphorylation of IkappaB, and attenuated IKK activity in LPS-stimulated RAW 264.7 cells. Taken together, these results indicate that beta-carboline alkaloid has the capability to suppress NF-kappaB signaling pathway through inhibition of IKK activity in LPS-stimulated RAW 264.7 cells.

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 macrophages by sesquiterpene lactones.

The molecular mechanism underlying the suppression of lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production was investigated in RAW 264.7 macrophages treated with sesquiterpene lactones, zaluzanin-C and estafiatone, isolated from Ainsliaea. Zaluzanin-C and estafiatone decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 macrophages with an IC50 of about 6.61 microM and 3.80 microM, respectively. In addition, these compounds inhibited the synthesis of PGE(2) in LPS/IFN-gamma-treated RAW 264.7 macrophages. Furthermore, treatment with zaluzanin-C and estafiatone resulted in a decrease in inducible No Synthase (iNOS) and Cyclooxygenase-2 (COX-2) protein and mRNA expression levels. Zaluzanin-C and estafiatone inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by parallel reduction of phosphorylation and degradation of inhibitor of kappaB (IkB). In addition, these effects were completely blocked by treatment with cysteine, indicating that the inhibitory effect of zaluzanin-C and estafiatone might be mediated by alkylation of either NF-kappaB itself or an upstream molecule of NF-kappaB. These results demonstrate that the suppression of NF-kappaB activation by zaluzanin-C and estafiatone might be attributed to inhibition of nuclear translocation of NF-kappaB resulting from blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signaling pathways.

Protein profiles of bovine placenta derived from somatic cell nuclear transfer.

Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT.