Activation of LXRβ inhibits proliferation,promotes apoptosis,and increases chemosensitivity of gastric cancer cells by upregulating the expression of ATF4

Recently, great advances have been achieved in both surgery and chemotherapy for the treatment of gastric cancer, but there is still poor prognosis for this disease. The aim of this study is to investigate the role of liver X receptor β (LXRβ) in chemosensitivity of gastric cancer SGC7901 cells. From 171 patients with gastric cancer, the gastric cancer and paracancerous tissues were selected to measure the expression of LXRβ and ATF4. Gastric cancer cell lines were cultured and screened to figure out the proliferation and apoptosis of gastric cancer SGC7901 cells with the treatment of LXRβ agonist (GW3965), ATF4 short hairpin RNA (shRNA), and chemotherapy drug paclitaxel. The expression of apoptosis-related gene cleaved caspase-3 was detected by Western blot analysis. First, we found that the expressions of LXRβ and ATF4 in gastric cancer tissues and cells were significantly lower than those in their paracancerous tissues and gastric mucosal epithelial cells. In addition, activation of LXRβ and paclitaxel treatment suppressed proliferation of SGC7901 cells, and the expression of ATF4 was upregulated in a concentration-dependent manner. Furthermore, shRNA significantly inhibited the expression of ATF4 and blocked the chemosensitivity of SGC7901 cells to LXRβ activation. Our study demonstrates that the expression of LXRβ was low in gastric cancer. In addition, activation of LXRβ may inhibit the proliferation of gastric cancer cells, promote apoptosis, and increase chemosensitivity by upregulating the expression of ATF4.     

Expression of the human interferon-β gene cloned in phage M13 mp7

The single-stranded DNA phage, M13 mp7 was used in the construction of an expression vector containing the coding sequence for mature interferon-β (IFN-β). Two clones expressed a fused polypeptide showing the biological and physicochemical properties of IFN-β, despite the fact that the N-terminal amino acid sequence had been changed; 10⁶ I.U./l of culture were produced with a molecular weight of 20 000.     

DNA polymorphism of the human complementC8β gene: formal genetics and intragenic localization

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length humanC8β cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymesTaqI andBam HI. TheTaq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are 0.68 and 0.32, respectively. The second RFLP withBam HI is correlated with theTaq I variants: 3 kbBam HI; 4.9 kbTaq I and 3.3 kbBam HI; 2.8/2.1 kbTaqI. Both RFLPs could be mapped to the 3′ portion of theC8β gene. Based on the size of genomic restriction fragments, theC8β gene can be estimated to have a size of 32–36 kb. Because of the even frequency distribution, theC8β DNA polymorphisms may be useful in gene mapping and disease association studies.     

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-β signaling

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’ culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.     

Co-culture of bovine muscle satellite cells with preadipocytes increases PPARγ and C/EBPβ gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes

We hypothesized that preadipocyte differentiation would be depressed by differentiating myoblasts, whereas preadipocytes would promote adipogenic gene expression in myoblasts in a co-culture system. We also determined the effects of arginine, a biological precursor of nitric oxide, and/or trans-10, cis-12 conjugated linoleic acid (CLA) on adipogenic gene expression during differentiation of bovine preadipocytes and myoblasts. Bovine semimembranosus satellite cells (BSC) and subcutaneous preadipocytes were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's modified Eagle medium (DMEM) and 1% antibiotics during the 3-day proliferation period. After proliferation, BSC and preadipocytes were treated for 3 days with 3% horse serum/DMEM and 5% FBS/DMEM with antibiotics, respectively. Media also contained 100 μM oleic acid, 10 μg/ml insulin, 1 μg/ml pioglitazone and 1 μg/ml dexamethasone. Subsequently, the differentiating myoblasts and adipocytes were cultured in their respective media with 5 mM arginine and/or 40 μM trans-10, cis-12 CLA for 4 days. Finally, myoblasts and adipocytes were single- or co-cultured for 2 h singly or in combination. Arginine stimulated SCD gene expression, whereas CLA depressed SCD gene expression in adipocytes and myoblasts (P=.002). Co-culture of adipocytes and myoblasts elicited an increase in C/EBPβ and PPARγ gene expression in differentiated myoblasts (P≤.01) and an increase in GPR43 gene expression in adipocytes (P=.01). Expression of AMPKα and CPT1ß was unaffected by co-culture, although SCD gene expression tended (P=.12) to be depressed by co-culture. These experiments demonstrated that co-culture of adipocytes with myoblasts increased adipogenic gene expression in the myoblastic cells.     

Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels

MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels.     

Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.     

Effect of human interferon β gene transfer upon human glioma, transplanted into nude mouse brain, involves induced natural killer cells

We investigated the immunological responses induced by human interferon β (IFNβ) gene transfer in human gliomas produced in the brains of nude mice. A suspension of human glioma U251-SP cells was injected into the brains of nude mice. The IFNβ gene was transferred by intratumoral injection with cationic liposomes or cationic liposomes associated with anti-glioma monoclonal antibody (immunoliposomes). When intratumoral injection of liposomes or immunoliposomes containing the human IFNβ gene was performed every second day for a total of six injections, starting 7 days after tumor transplantation, complete disappearance of the tumor was observed in six of seven mice that had received liposomes and in all seven mice receiving immunoliposomes. In addition, experimental gliomas injected with immunoliposomes were much smaller than those injected with ordinary liposomes following delayed injections beginning 14 days after transplantation. An immunohistochemical study of the treated nude mouse brains revealed a remarkable induction of natural killer (NK) cells expressing asialoGM1 antigen. To investigate the significance of NK cells in the antitumor effect, we injected liposomes or immunoliposomes containing the human IFNβ gene into tumors in nude mice depleted of NK cells by irradiation and anti-asialoGM1 antibody administration. The antitumor effect of the liposomes or immunoliposomes was abolished. Subsequent subcutaneous glioma challenge of the nude mice after intracerebral tumor implantation and gene transfer resulted in no subcutaneous tumor growth. These results suggest that the induction of NK cells is important in the cytocidal effect of liposomes or immunoliposomes containing the human IFNβ gene upon experimental gliomas. Received: 10 February 1998 / Accepted: 1 September 1998     

Generation of a novel murine model of Aβ deposition based on the expression of human wild-type amyloid precursor protein gene

Mouse models of Alzheimer disease (AD) have been generated based on Amyloid-β Precursor Protein (AβPP) and the Presenilin (PSEN) gene mutations associated with familial AD (FAD). Such models have provided valuable insights into AD pathogenesis and represent an important research tool for the discovery of potential treatments. To model amyloid deposition in AD, we generated a new mouse line based on the presence of two copies of the genomic region encoding human wild-type AβPP as well as a mutation (L166P) in the murine Psen1. By ~6 months of age, these mice have begun to develop cerebral Aβ pathology with a significant increase in the levels of AβPP C-terminal fragments and Aβ42, as well as increase Aβ42/Aβ40 ratio. Since in the brain and other tissues of these mice, wild-type human AβPP mRNA and protein levels are comparable to those of endogenous AβPP, this model may allow studies about the role of AβPP isoforms in the pathogenesis of AD. This animal model may be suitable to test drugs aimed at inhibiting expression or altering splicing and processing of AβPP, without artifacts associated with the presence of mutations in AβPP or overexpression due to the use of exogenous promoters. These features of the new model are of critical importance in assessing the success of therapeutic interventions.     

Skeletal muscle-specific expression of human blood coagulation factor Ⅸ rescues factor Ⅸ deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

Generation of a novel murine model of Aβ deposition based on the expression of human wild-type amyloid precursor protein gene

Mouse models of Alzheimer disease (AD) have been generated based on Amyloid-β Precursor Protein (AβPP) and the Presenilin (PSEN) gene mutations associated with familial AD (FAD). Such models have provided valuable insights into AD pathogenesis and represent an important research tool for the discovery of potential treatments. To model amyloid deposition in AD, we generated a new mouse line based on the presence of two copies of the genomic region encoding human wild-type AβPP as well as a mutation (L166P) in the murine Psen1. By ~6 months of age, these mice have begun to develop cerebral Aβ pathology with a significant increase in the levels of AβPP C-terminal fragments and Aβ42, as well as increase Aβ42/Aβ40 ratio. Since in the brain and other tissues of these mice, wild-type human AβPP mRNA and protein levels are comparable to those of endogenous AβPP, this model may allow studies about the role of AβPP isoforms in the pathogenesis of AD. This animal model may be suitable to test drugs aimed at inhibiting expression or altering splicing and processing of AβPP, without artifacts associated with the presence of mutations in AβPP or overexpression due to the use of exogenous promoters. These features of the new model are of critical importance in assessing the success of therapeutic interventions.     

Function of the phosphatidylinositol transfer protein gene family: is phosphatidylinositol transfer the mechanism of action?

Phosphatidylinositol transfer proteins (PITPs) bind and facilitate the transport of phosphatidylinositol (PI) and phosphatidylcholine between membrane compartments. They are highly conserved proteins, are found in both unicellular and multicellular organisms, and can be present as a single domain or as part of a larger, multi-domain protein. The hallmark of PITP proteins is their ability to sequester PI in their hydrophobic pocket. Ablation or knockdown of specific isoforms in vivo has wide ranging effects such as defects in signal transduction via phospholipase C and phosphoinositide 3-kinase, membrane trafficking, stem cell viability, Drosophila phototransduction, neurite outgrowth, and cytokinesis. In this review, we identify the common mechanism underlying each of these phenotypes as the cooperation between PITP proteins and lipid kinases through the provision of PI for phosphorylation. We propose that recruitment and concentration of PITP proteins at specific membrane sites are required for PITP proteins to execute their function rather than lipid transfer.     

Genetic analysis of transgenome structure and size of chromosome-mediated gene transfer lines

The TK-selected chromosome-mediate gene transferlines were analysed using DNA dot blot method,G-11banding and in situ hybridization.The results showedthat CMGT can provide a wide variety of intermediatesize of the transgenome from greater than 80,000kb toless than 2,000kb.Some of transfectants are intergratedinto mouse chromosome which can be detected by G-11banding and in situ hybridization     

Retracted: Smad4 gene silencing enhances the chemosensitivity of human lymphoma cells to adriamycin via inhibition of the activation of transforming growth factor β signaling pathway

There are diverse investigations focused on the therapies of lymphoma. Our research was taken to identify the effects of lentiviral-mediated Smad4 gene silencing on chemosensitivity of human lymphoma cells to adriamycin (ADM) via transforming growth factor β (TGFβ) signaling pathway. Raji/ADM cells were cultured and infected with lentiviral particles Smad4-short hairpin (shRNA) and control-shRNA. Then, the messenger RNA (mRNA) and protein levels of TGFβ signaling pathway–related factors (Smad4, Smad3, cyclinE, cyclinD1, and p21) in Raji/ADM cells were determined. The effect of Smad4-shRNA on cell viability, invasion and migration, and apoptosis were also detected. Compared with the Raji group, increased mRNA and protein levels of Smad4, Smad3, cyclinE, cyclinD1, enhanced cell proliferation, migration and invasion as well as decreased mRNA, and protein levels of p21 and cell apoptosis rate were found in the Raji/ADM and control-shRNA groups. However, Smad4 gene silencing resulted in decreased mRNA and protein levels of Smad4, Smad3, cyclinE, and cyclinD1 along with inhibited cell proliferation, migration and invasion but increased expression of p21 together with cell apoptosis. Collectively, Smad4 gene silencing can inhibit the activation of TGFβ signaling pathway, thereby enhancing the chemosensitivity of human lymphoma cells to ADM.     

Contribution of gene conversion in the evolution of the human β-like globin gene family

Gene conversion is referred to as one of two types of mechanisms known to act on gene families, mainly to maintain their sequence homogeneity or, in certain cases, to produce sequence diversity. The concept of gene conversion was established 20 years ago by researchers working with fungi. A few years later, gene conversion was also observed in the human genome, i.e. the γ-globin locus. The aim of this article is to emphasize the role of genetic recombination, particularly of gene conversion, in the evolution of the human β-like globin genes and further to summarize its contribution to the convergent evolution of the fetal globin genes. Finally, this article attempts to re-examine the origin and spread of specific mutations of the β-globin cluster, such as the sickle cell or β-thalassemia mutations, on the basis of repeated gene conversion events. Received: 13 February 1997 / Accepted: 15 May 1998