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Bone morphogenetic protein 4 (BMP4), a member of the TGF [Beta] superfamily, has been implicated in the dorsoventral specification of both mesoderm and ectoderm. High levels of BMP4 signaling appear to specify ventral lineages, while lower levels are causally associated with the development of dorsal lineages. We have previously identified a homeobox-containing transcription factor (PV.1) which is a likely mediator of the ventralizing effects of BMP4 in the mesoderm. Here we provide evidence that PV.1 also functions downstream of BMP4 in the patterning of ectoderm, specifying epidermal and suppressing neural gene expression. PV.1 is expressed in the prospective neuroectoderm at the time of ectodermal fate determination. BMP4 and xSmad1 (a downstream effector of BMP4) induce PV.1 in uncommitted ectoderm and the dominant negative form of the BMP4 receptor (DN-BR) blocks PV.1 expression. In animal pole explants PV.1 counteracts the neuralizing effects of chordin and the DN-BR and restores them to their original epidermal fate. To address the physiological significance of these observations we employed an animal cap transplantation system and demonstrated that overexpression of PV.1 in the prospective neuroectoderm specifically blocks neurogenesis in intact embryos. Thus, PV.1 plays an important role in the ventralization of both mesoderm and ectoderm. We have previously shown that PV.1 is also preferentially expressed in the ventral endoderm, suggesting that this transcription factor may be involved in the ventralization of all three germ layers. Key Words: BMP4; homeobox gene; neural induction.

Animal pole tissue size which is associated with ventral mesoderm influences erythrocyte differentiation in Xenopus embryo. The differentiation of the erythrocytes in the ventral mesoderm in combination culture is initiated and enhanced by the animal pole tissues. The stimulation is characterized by different sizes and regions of the animal pole tissue. Ventral mesoderm explants are associated with the various regions in the animal pole tissue involved in the stimulation.

In adult vertebrates, fibroblast growth factor (FGF) synergizes with many hematopoietic cytokines to stimulate the proliferation of hematopoietic progenitors. In vertebrate development, the FGF signaling pathway is important in the formation of some derivatives of ventroposterior mesoderm. However, the function of FGF in the specification of the embryonic erythropoietic lineage has remained unclear. Here we address the role of FGF in the specification of the erythropoietic lineage in the Xenopus embryo. We report that ventral injection of embryonic FGF (eFGF) mRNA at as little as 10 pg at the four-cell stage suppresses ventral blood island (VBI) formation, whereas expression of the dominant negative form of the FGF receptor in the lateral mesoderm, where physiologically no blood tissue is formed, results in a dramatic expansion of the VBI. Similar results were observed in isolated ventral marginal zones and animal caps. Bone morphogenetic protein-4 (BMP-4) is known to induce erythropoiesis in the Xenopus embryo. Therefore, we examined how the BMP-4 and FGF signaling pathways might interact in the decision of ventral mesoderm to form blood. We observed that eFGF inhibits BMP-4-induced erythropoiesis by differentially regulating expression of the BMP-4 downstream effectors GATA-2 and PV.1. GATA-2, which stimulates erythropoiesis, is suppressed by FGF. PV.1, which we demonstrate to inhibit blood development, is enhanced by FGF. Additionally, PV.1 and GATA-2 negatively regulate transcription of each other. Thus, BMP-4 induces two transcription factors which have opposing effects on blood development. The FGF and BMP-4 signaling pathways interact to regulate the specification of the erythropoietic lineage.

Abstract: Introduction: In China Herba Epimedii is one of the most common herbs that could be prescribed for treating osteoporosis. It is known to increase the overall mineral content, therefore, to promote bone formation and to increase lumbar bone mineral density (BMD). The present study was aimed at investigating the effect of flavonoids of Herba Epimedii (HEF) on osteogenesis in human MSCs. Methods: The human bone marrow-derived MSCs (BM-MSCs) were isolated and their osteogenic differentiation was evaluated by their alkaline phosphatase (ALP) activities and level of mineralization. After treating with total flavonoids during osteogenic differentiation process, differential mRNA expression was examined by RT-PCR. Results: The total time needed for osteogenic differentiation of BM-MSCs was significantly shortened by adding HEF. Up-regulation of mRNA expression by HEF was observed for several marker genes and osteogenic regulators. HEF was also found to inhibit osteoclastogenesis of MSCs by enhancing the ratio OPG/RANKL. Conclusions: Our study demonstrated that the HEF could improve osteogenic differentiation and inhibit the osteoclast differentiation of BM-MSCs concurrently. [Copyright &y& Elsevier]

Summary: Our previous study has suggested an oncogenic role of eIF-5A2 in ovarian tumorigenesis. Abnormalities of eIF-5A2, however, in colorectal carcinoma are unclear. In this study, amplification and overexpression of eIF-5A2 in colorectal carcinoma were studied by fluorescence in situ hybridization and immunohistochemistry using colorectal carcinoma tissue microarrays, including 139 primary colorectal carcinomas and their adjacent normal mucosa, 22 paired premalignant adenomas, and 42 metastatic tumors. The immunohistochemistry results showed that overexpression of EIF-5A2 was detected in none of normal epithelial mucosa, 35.3% of colorectal adenomas, 53.2% of primary colorectal carcinomas, and 67.6% of metastases. Amplification of eIF-5A2 was detected in 15.8% (16/101) of informative colorectal carcinomas, and most of them showed overexpression of EIF-5A2. In primary colorectal carcinomas, the frequency of EIF-5A2 overexpression was significantly higher in colorectal carcinomas with lymphovascular invasion (61.2%) than that in colorectal carcinomas without lymphovascular invasion (36.6%, P < .05). In addition, significant positive associations were found between EIF-5A2 overexpression and the tumors'' later pN and pM stages, as well as increased tumor cell proliferation (P < .05). These findings suggest that overexpression of EIF-5A2 in colorectal carcinomas may be important in the acquisition of a metastatic phenotype and plays an important role in colorectal carcinoma development and progression. [Copyright &y& Elsevier]

Background & Aims: FHL2 (4-1/2 LIM protein 2) is an adapter and modifier in protein interactions that is expressed mainly in the heart and ovary. It functions in a cell type- or promoter-specific manner. The aims of this study were to examine its expression in gastrointestinal cancers and to determine its role in cell differentiation and tumorigenesis. Methods: FHL2 expression in cancerous and normal gastrointestinal cells was detected by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. The effect of FHL2 suppression by both antisense and siRNA methods on cell differentiation and growth were evaluated in vitro and in vivo. Results: FHL2 expression was up-regulated in gastrointestinal cancer, compared with matched normal tissues. Stable transfection of gastric cancer cell line, AGS, and colon cancer cell line, Lovo, with antisense FHL2 induced lengthened or shuttle-shape morphologic changes with long or dendritic-like cytoplasmic processes and decreased the nuclear:cytoplasmic ratio. FHL2 antisense induced expressions of carinoembryonic antigen and E-cadherin and the maturation of F-actin. Furthermore, FHL2 antisense inhibited the transcriptions of some oncogenes including cox-2, survivin, c-jun, and hTERT, and suppressed the promoter activity of activator protein-1 and hTERT. Suppression of FHL2 inhibited serum-dependent, anchorage-dependent and -independent cell growth, and suppressed de novo tumor formation in nude mice xenograft. Conclusions: Suppression of FHL2 induces cell differentiation and inhibits tumorigenesis. Antisense or siRNA methods targeting FHL2 is a promising strategy for treatment of gastrointestinal cancers. [Copyright &y& Elsevier]

Background & Aims: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of methylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression. Methods: XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis. Results: Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers. Conclusions: A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at −2nd, −1st, and +3rd positions are functionally more important in its transcriptional regulation. [Copyright &y& Elsevier]

Background & Aims: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is a novel tumor suppressor and interferon (IFN)-stimulated gene. All-trans retinoic acid (ATRA) exerts an antiproliferative effect on tumor cells through up-regulation of IFN regulatory factor 1 (IRF-1) and the downstream IFN-stimulated genes. The aim of this study was to determine the effect and mechanism of ATRA on XAF1 expression and the role of XAF1 in ATRA-induced growth inhibition in colon cancer. Methods: Gene expression is detected by reverse-transcription polymerase chain reaction and immunoblotting. The transcription activity of XAF1 promoter is examined by luciferase reporter assay. The activity of IFN regulatory factor binding element (IRF-E) is assessed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Cell growth is evaluated by both in vitro and in vivo in nude mice xenografts. Results: IFN-alfa stimulates XAF1 promoter activity in the colon cancer cells Lovo and SW1116 dose-dependently. An IRF-1 binding element (IRF-E–XAF1) is found in the -30 to -38 nucleotide region upstream of the ATG initiator codon of the XAF1 gene. Site-directed mutagenesis of IRF-E–XAF1 abrogates native and IFN-induced promoter activity and binding capacity. ATRA induces XAF1 expression both in vitro and in vivo through interaction with IRF-E–XAF1. Overexpression of XAF1 increases cell susceptibility to ATRA-induced growth suppression both in vitro and in vivo. Furthermore, the effect of ATRA on XAF1 expression is independent of the promoter methylation and the subcellular distribution of XIAP. Conclusions: XAF1 participates in ATRA-induced growth suppression through IRF-1–mediated transcriptional regulation. [Copyright &y& Elsevier]

Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Because the microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, we evaluated the effect of garlic on the expression of the MTP gene in vitro in cell lines and in vivo in rats. Fresh garlic extract (FGE) reduced MTP mRNA levels in both the human hepatoma HepG2 and intestinal carcinoma Caco-2 cells in dose-dependent fashion; significant reductions were detected with 3 g/L FGE. Maximal 72 and 59% reductions, respectively, were observed with 6 g/L FGE. To evaluate the in vivo effect of garlic on MTP gene expression, rats were given a single oral dose of fresh garlic homogenate (FGH), with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats fed FGH had significantly (46% of the control) lower intestinal MTP mRNA levels compared with the control rats, whereas hepatic MTP mRNA levels were not affected. These results suggest a new mechanism for the hypolipidemic effect of fresh garlic. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation. [ABSTRACT FROM AUTHOR]

Angiogenesis has been found as an attractive target for drug therapy as it is necessary for tumor growth. Accumulating evidences show that microRNAs (miRNAs), which are a group of highly conserved, single-stranded, short non-coding RNAs, play important roles through directly targeting angiogenic factors and protein kinases. The purpose of this study is to investigate the role of miR-195 in breast cancer development and angiogenesis through targeting IRS1. We show that miR-195 is inversely related with Insulin receptor substrate 1 (IRS1) in both breast cancer cells and breast cancer tissues. Induction of miR-195 could suppress IRS1 protein expression through binding to its 3′UTR regions either by transfection with miR-195 oligo or by infection with lentivirus encoding miR-195 gene. Moreover, re-expression of IRS1 reverses miR-195-mediated repression of tumor cell growth and miR-195 inhibits tumor angiogenesis through suppressing IRS1-VEGF axis. These data suggest that miR-195 mimics are potential therapeutic agents for breast cancer diagnose. [ABSTRACT FROM AUTHOR]

Paclitaxel is a main ingredient in the combination chemotherapy treatment of advanced human cervical squamous cell carcinomas. We investigated the roles and underlying molecular mechanisms of PinX1 in cervical squamous cell carcinomas (CSCC) cells response to paclitaxel and its clinical significances. The expression dynamics of PinX1 was first examined by immunohistochemistry in 122 advanced CSCC patients treated with cisplatin/paclitaxel chemotherapy. The expression of PinX1 was significantly associated with the effects of cisplatin/paclitaxel chemotherapy in advanced CSCCs ( P < 0.05). High expression of PinX1 correlated with CSCC’s response to cisplatin/paclitaxel chemotherapy, and was an independent predictor of shortened survival ( P < 0.05). A series of in vivo and in vitro assays were performed to elucidate the function of PinX1 on CSCC cells chemosensitivity to paclitaxel and underlying mechanisms. In CSCC cells, the levels of PinX1 were only associated with the cytotoxicity and sensitivity of paclitaxel, in which knockdown of PinX1 dramatically enhanced paclitaxel cytotoxicity, whereas the reestablishment of PinX1 levels substantially reduced the paclitaxel-induced killing effect. In addition, we identified that the ability of PinX1 to stabilize the tension between sister kinetochores and maintain the spindle assembly checkpoint was the main reason CSCC cells undergo apoptosis when treated with paclitaxel, and further studies demonstrated that shortened distance between sisters kinetochores by nocodazole confers upon PinX1-replenished cells a sensitivity to the death inducing paclitaxel effects. Furthermore, our study of CSCC cells xenografts in nude mice confirmed the role of PinX1 in paclitaxel sensitivity in vivo . Our data reveal that PinX1 could be used as a novel predictor for CSCC patient response to paclitaxel, and the role of PinX1-mediated paclitaxel sensitivity might represent a new direction for the development of a new generation of microtubule drugs. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) are members of non-coding RNAs. They are involved in diverse biological functions. MiRNAs are precisely regulated in a tissue- and developmental-specific manner, but dysregulated in many human diseases, in particular cancers. Transcriptional regulation, post-transcriptional regulation, as well as genetic alterations, are the three major mechanisms controlling the spatial and temporal expression of miRNAs. Emerging evidence now indicates that transcriptional and epigenetic regulations play major roles in miRNA expression. This review summarizes the current knowledge and discusses the future challenges. [Copyright &y& Elsevier]

Abstract: In this study we describe a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600, and sebacoyl chloride. 1H NMR, FT-IR, and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. The mPPS-FA/DNA nanoparticles exhibited low cytotoxicity as transfection of B16-F0, U87MG, CHO-1, and Ho-8910 cells produced >80% viability indicating low cytotoxicity of the polymer. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910, and A549 cells was investigated in vitro as compared to the lipid-based transfection agent Lipofectamine™2000 and Linear PEI 22kDa (L-PEI 22kDa). We found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1, and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be competitively blocked by free folic acid molecules. In contrast, in low FR expressing A549 cells, mPPS-FA showed similar low transfection efficiency as mPPS. Taken together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier. [Copyright &y& Elsevier]

Abstract: The function of EZH2 in tumorigenesis and liver metastasis of pancreatic cancer has never been elucidated in vivo. EZH2 was overexpressed in pancreatic carcinomas and its overexpression was associated with tumor differentiation and pT status. Suppression of EZH2 caused a significant growth inhibition of pancreatic cancer cells in vitro and markedly diminished their tumorigenicity in vivo. Knock-down of EZH2 inhibited liver metastasis of pancreatic cancer in vivo. EZH2 has a crucial role in tumor growth and liver metastasis of pancreatic cancer. [Copyright &y& Elsevier]

Abstract: Neuronal apoptosis sculpts the developing brain, and nearly all identified classes of neurons seem to be produced “in excess” during development. FoxO transcription factors regulate apoptosis in vitro in deprived of neurotrophins. It is unknown if FoxO3a is involved in the development of neurons. Here, we report a role of FoxO3a during neuronal development in zebrafish. By using in situ hybridization, we revealed that FoxO3a transcripts in zebrafish were gradually confined to regions of the central nervous system during embryonic development, including the forebrain, midbrain, midbrain–hindbrain boundary and hindbrain. By using FoxO3a morpholino antisense oligonucleotides, we observed that FoxO3a loss-of-function led to neural developmental defects, including increased neural apoptosis as detected by acridine orange and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. These defects could be partially rescued by the injection of FoxO3a mRNA. In this study, we found that FoxO3a loss-of-function resulted in the decreased expression of neuronal markers as determined by in situ hybridization and relative quantitative real-time PCR. Furthermore, the activation of FoxO3a was required for the maintenance of neuron survival but not necessary for the induction of neurogenesis. Our results indicated that FoxO3a might be essential for the maintenance of neural development in zebrafish. Therefore, this work provides novel evidence of FoxO3a in the embryonic neurodevelopment from zebrafish to other mammals. [ABSTRACT FROM AUTHOR]

Abstract: A physiologically stable gold compound, gold(III) meso-tetraphenylporphyrin (gold-1a), has been shown to be effective in inducing apoptosis and prolonging the survival of hepatocellular carcinoma (HCC)-bearing rats as well as inhibiting the tumor growth of mice bearing nasopharyngeal carcinoma (NPC), neuroblastoma and colon carcinoma. In this study, we showed that gold-1a prolonged the survival of NPC metastasis-bearing mice and inhibited intrahepatic and lung metastasis. Histologically, gold-1a markedly reduced tumor microvessel formation. Consistently, in in vitro studies, gold-1a inhibited migration and invasion of C666-1 human NPC cells. The data strongly support the use of gold(III) compounds to treat cancer metastasis. [Copyright &y& Elsevier]

Abstract: A number of microRNAs (miRNAs) that are evolutionarily conserved not beyond primate lineage have been identified. These primate-specific miRNAs (ps-miRNAs) may attribute to the difference between high-level primates and non-primate mammals or lower vertebrates. Despite of their importance, the genome-wide miRNA conservation patterns and the properties of these ps-miRNAs are largely elusive. In this study, we developed a robust classification system to assess the conservation pattern of all human mature miRNAs across 44 vertebrate genomes. By this comparative genomic analysis, a novel set of 269 ps-miRNAs were identified. We found that many ps-miRNAs were enriched in chromosome 19 and X, forming two main clusters hereafter referred as C19MC and CXMC, respectively. When comparing the seed of ps-miRNAs themselves or with non-ps-miRNAs, more than one half ps-miRNAs sharing common seeds were belonged to C19MC, 9 of which retained a unique seed that had been reported to be enriched in human embryonic stem cells (hESCs) specific miRNAs. Moreover, the most abundant ps-miRNA common seed was possessed by miR-548 family. Most ps-miRNAs had very low expression in adult tissues, which may be attributed to temporal and spatial specific transcript regulation. The ps-miRNAs with relatively high expression were mainly belonged to C19MC and CXMC, and preferentially expressed in hESCs and reproductive system. Sequence anatomy revealed that C19MC ps-miRNAs were highly conserved but not beyond primates and of great sequence similarity. Gene Ontology and KEGG pathway enrichment analyses of predicted target genes indicated that C19MC ps-miRNAs were strongly associated with developmental processes and various cancers. In conclusion, ps-miRNAs may play critical roles in differentiation and growth regulation during early development, especially in maintaining the pluripotency of hESCs. Results from this study may help explaining the differences between primates and lower vertebrates at genetic level. [Copyright &y& Elsevier]