Empty Vector (empty + vector)

Distribution by Scientific Domains


Selected Abstracts


Role of ataxia telangiectasia mutated in insulin signalling of muscle-derived cell lines and mouse soleus

ACTA PHYSIOLOGICA, Issue 4 2010
I. Jeong
Abstract Aim:, Ataxia telangiectasia mutated (ATM) reportedly plays a role in insulin-stimulated activation of Akt in some cell types but not in others. The role of ATM in insulin signalling has not been firmly resolved for skeletal muscle cells, for which Akt phosphorylation is a pivotal step in stimulation of glucose transport. Accordingly, our aim was to determine the role of ATM in insulin effects for cell lines derived from skeletal muscle and for skeletal muscle. Methods:, We examined insulin effects in L6 myotubes, mouse soleus, C2C12 myotubes and differentiated rhabdomyosarcoma (RD) cells in the presence and absence of a low concentration (1 ,m) of the ATM inhibitor KU55933. We also compared insulin signalling in C2C12 cells expressing shRNA against ATM and control cell lines (empty vector; cells expressing non-targeting shRNA). Results:, In L6 myotubes and mouse soleus muscle, KU55933 inhibited insulin-stimulated phosphorylation of the 160 kDa substrate of Akt (AS160) despite no effect on Akt. In contrast, KU55933 prevented insulin-stimulated Akt phosphorylation in C2C12 myotubes. Furthermore, C2C12 myotubes expressing shRNA against ATM displayed reduced insulin-stimulated Akt phosphorylation compared to controls. KU55933 also decreased insulin-stimulated Akt phosphorylation in differentiated RD cells. Conclusion:, These model-dependent differences in the role of ATM in insulin action demonstrate a role of ATM in insulin-stimulated phosphorylation of Akt (in C2C12 and RD cells) but also allow the elucidation of a novel, Akt-independent role of ATM (in L6 myotubes and mouse soleus, at the level of AS160) in insulin signalling. [source]


An SNF2 factor involved in mammalian development and cellular proliferation

DEVELOPMENTAL DYNAMICS, Issue 1 2001
Eric H. Raabe
Abstract Members of the SNF2 (Sucrose Non-Fermenter) family of chromatin-remodeling proteins function in processes ranging from DNA repair to transcription to methylation. Using differential display, we recently identified a novel member of the SNF2 family that is highly expressed at the mRNA level in proliferating cells and is down-regulated during apoptosis. We have named this gene PASG (Proliferation-Associated SNF2-like Gene). Northern blot analysis of adult mouse tissues shows PASG to be highly expressed in proliferating organs such as thymus, bone marrow, and testis and absent from nonproliferative tissues such as brain and heart. In situ hybridization analysis of mouse embryos shows that PASG is differentially expressed during development, with highest expression in developing face, limbs, skeletal muscle, heart, and tail. In vitro, PASG expression correlates with a shift from a quiescent to a proliferative state. Mice null for PASG (also known as LSH or Hells) are reported to die perinatally, although the mechanism for lethality is unclear (Geiman and Muegge, 2000). To test the hypothesis that PASG functions in cell proliferation, we compared 5-bromodeoxyuridine (BrdU) incorporation in C33A cells transiently transfected with PASG versus empty vector and found that PASG transfected cells showed a significant decrease in the amount of BrdU incorporation. These findings suggest that PASG plays a role in cell proliferation and may function in the development of multiple cell lineages during murine embryogenesis. © 2001 Wiley-Liss, Inc. [source]


TCR-, chains derived from peripheral ,, T cells can take part in ,, T-cell development

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2008
Nabil Bosco
Abstract Between 10 and 20% of the peripheral ,, T cells express cytoplasmic TCR-, proteins, but whether such TCR-, chains can partake in ,, T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3,-deficient mice with Pax5-TCR-, deficient proB cells expressing, via a retroviral vector, TCR-, chains from either peripheral ,, or ,, T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15×106 cells), contained few ,, T but no ,, T cells. In contrast, thymi from mice receiving proB cells containing ,, or ,, T-cell-derived TCR-, chains contained 80,130×106 cells, and showed a normal CD4, CD8 and ,, TCR expression pattern. However, regardless of the source of TCR-, chain, reconstituted mice rapidly showed signs of autoimmunity dying 5,15,wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-, chains from ,, T cells can efficiently take part in ,, T-cell development. The implications of these findings for ,, T-cell development will be discussed. [source]


Rab4 facilitates cyclic adenosine monophosphate,stimulated bile acid uptake and Na+ -taurocholate cotransporting polypeptide translocation,

HEPATOLOGY, Issue 5 2008
Christopher M. Schonhoff
Cyclic adenosine monophosphate (cAMP) stimulates hepatic bile acid uptake by translocating sodium-taurocholate (TC) cotransporting polypeptide (Ntcp) from an endosomal compartment to the plasma membrane. Rab4 is associated with early endosomes and involved in vesicular trafficking. This study was designed to determine the role of Rab4 in cAMP-induced TC uptake and Ntcp translocation. HuH-Ntcp cells transiently transfected with empty vector, guanosine triphosphate (GTP) locked dominant active Rab4 (Rab4(GTP)), or guanosine diphosphate (GDP) locked dominant inactive Rab4 (Rab4(GDP)) were used to study the role of Rab4. Neither Rab4(GTP) nor Rab4(GDP) affected either basal TC uptake or plasma membrane Ntcp level. However, cAMP-induced increases in TC uptake and Ntcp translocation were enhanced by Rab4(GTP) and inhibited by Rab4(GDP). In addition, cAMP increased GTP binding to endogenous Rab4 in a time-dependent, but phosphoinositide-3-kinase,independent manner. Conclusion: Taken together, these results suggest that cAMP-mediated phosphoinositide-3-kinase,independent activation of Rab4 facilitates Ntcp translocation in HuH-Ntcp cells. (HEPATOLOGY 2008.) [source]


EBAG9 is a tumor-promoting and prognostic factor for bladder cancer

INTERNATIONAL JOURNAL OF CANCER, Issue 4 2009
Jinpei Kumagai
Abstract Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast and prostate cancers, indicating that EBAG9 may contribute to tumor proliferation. In the present study, we assess the role of EBAG9 in bladder cancer. We generated human bladder cancer EJ cells stably expressing FLAG-tagged EBAG9 (EJ-EBAG9) or empty vector (EJ-vector), and investigated whether EBAG9 overexpression modulates cell growth and migration in vitro as well as the in vivo tumor formation of EJ transfectants in xenograft models of BALB/c nude mice. EBAG9 overexpression promoted EJ cell migration, while the effect of EBAG9 to cultured cell growth was rather minimal. Tumorigenic experiments in nude mice showed that the size of EJ-EBAG9-derived tumors was significantly larger than EJ-vector-derived tumors. Loss-of-function study for EBAG9 using small interfering RNA (siRNA) in xenografts with parental EJ cells showed that the intra-tumoral injection of EBAG9 siRNA markedly reduced the EJ tumor formation compared with control siRNA. Furthermore, immunohistochemical study for EBAG9 expression was performed in 60 pathological bladder cancer specimens. Intense and diffuse cytoplasmic immunostaining was observed in 45% of the bladder cancer cases. Positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients (p = 0.0001) and it was an independent prognostic predictor for disease-specific survival in multivariate analysis (p = 0.003). Our results indicate that EBAG9 would be a crucial regulator of tumor progression and a potential prognostic marker for bladder cancer. © 2008 Wiley-Liss, Inc. [source]


In vivo evaluation of the role of DNp73, protein in regulating the p53-dependent apoptotic pathway after treatment with cytotoxic drugs

INTERNATIONAL JOURNAL OF CANCER, Issue 3 2007
Maria Antonietta Sabatino
Abstract The amino terminus truncated p73 isoform, ,Np73,, shows dominant negative behavior toward TAp73 and wild-type p53, and has oncogenic potential. By contrast, we recently showed that in HCT116 clones forced expression of ,Np73, did not increase in vitro cellular resistance to anticancer agents. The purpose of this study was to characterize in vivo models and to investigate the functional interaction between the ,Np73, isoform and the p53 pathway. Human colon carcinoma HCT116 clones expressing inducible ,Np73, (HCT116/DN3, HCT116/DN14) and HCT116/8a (transfected with the mock empty vector), transplanted in immunodeficient nude mice, were used to study the antitumor activity of cis -diammine-dichloro-platinum (cDDP) (4 mg/kg, i.v., q7d × 3) and Doxorubicin (DX) (7.5 mg/kg, i.v., q7d × 3), with or without tetracycline-induced ,Np73, overexpression. ,Np73, expression was confirmed by RT-PCR, immunoblotting and immunohistochemical analysis. ,Np73, subcellular localization after DX treatment was checked by an immunofluorescence assay. Western blot was used to analyze p53, p21, Bax, Bcl-2 and p53AIP1 expression. ,Np73, overexpression did not modify the antitumor activity of either DX or cDDP in xenograft models. DX reduced ,Np73, protein expression, without affecting its nuclear localization. p53, p21, Bax and p53AIP1 protein expression increased and Bcl-2 decreased in HCT116 clone derived tumors 24 hr after DX exposure, independently of the presence of ,Np73,. Overexpression of ,Np73, does not affect tumor growth in vivo, does not increase the resistance of established tumors to anticancer agents and does not antagonize p53 apoptotic functions. © 2006 Wiley-Liss, Inc. [source]


Modulation of sulfur mustard induced cell death in human epidermal keratinocytes using IL-10 and TNF-,,,

JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2005
Aziz Qabar
Abstract We compared the effects of overexpressing a tightly regulated anti-inflammatory cytokine, interleukin 10 (IL-10), and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-,) on sulfur mustard induced cytotoxicity in human epidermal keratinocytes. Both cytokines were overexpressed when compared with the cells transfected with the empty vector as determined by quantitative ELISA. Cells overexpressing interleukin 10 suppressed the pro-inflammatory cytokines interleukin 8 and interleukin 6 following exposure to 50,300 ,M sulfur mustard. These cells exhibited delayed onset of sulfur mustard induced cell death. On the other hand, cells overexpressing tumor necrosis factor alpha induced a sustained elevation in both interleukin 6 and 8 expression following exposure to 50,300 ,M sulfur mustard. These cells were sensitized to the effects of sulfur mustard that resulted in an increased sulfur mustard induced cell death. Normal human epidermal keartinocytes treated with sulfur mustard exhibited elevated levels of tumor necrosis factor alpha expression and increased activity of nuclear factor kappa B. Gene array data indicated that cells overexpressing interleukin 10 induced several genes that are involved in growth promotion and cell-fate determination. We, therefore, identify IL-10 and TNF-, signal transduction pathways and their components as possible candidates for early therapeutic intervention against sulfur mustard induced cell injury. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:213,225, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20089 [source]


Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence, Genotype/Phenotype Correlation, and a Novel Non-UBA Domain Mutation (P364S) Associated With Increased NF-,B Signaling Without Loss of Ubiquitin Binding,,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2009
Sarah L Rea
Abstract Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-,B luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-,B activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-,B signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations. [source]


Overexpression of Lysyl Hydroxylase-2b Leads to Defective Collagen Fibrillogenesis and Matrix Mineralization,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2005
Suchaya Pornprasertsuk
Abstract Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. Introduction: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. Materials and Methods: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. Results: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. Conclusions: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone. [source]


Smad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3-E1 Cells,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002
Hideaki Sowa
Abstract Transforming growth factor (TGF) , is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-, signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-, on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-, inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L -azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. [source]


,-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

JOURNAL OF NEUROCHEMISTRY, Issue 5 2009
Nishani T. Hettiarachchi
Abstract Parkinson's disease (PD) is characterized in part by the presence of ,-synuclein (,-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the ,-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type ,-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of ,-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the ,-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of ,-synuclein. However, only WT ,-syn transfected cells displayed significantly impaired viability. Our findings suggest that ,-syn regulates Ca2+ entry pathways and, consequently, that abnormal ,-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis. [source]


The small heat shock protein Hsp27 protects cortical neurons against the toxic effects of ,-amyloid peptide

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2009
Michael King
Abstract Neurofibrillary tangles and amyloid plaques are considered to be hallmarks of Alzheimer's disease (AD), and the toxic effects of amyloid-, peptide (A,) lead to activation of stress-related signaling and neuronal loss. The small heat shock protein Hsp27 is reported to be increased in AD brains and to accumulate in plaques, but whether this represents a potentially protective response to stress or is part of the disease process is not known. We hypothesized that increased expression of Hsp27 in neurons can promote neuronal survival and stabilize the cytoskeleton in the face of A, exposure. By using neonatal rat cortical neurons, we investigated the potential role of Hsp27 in neuronal cultures in the presence or absence of A,. We initially tested whether a heat stress (HS) would be sufficient to induce endogenous Hsp27 expression. HS not only did not result in neuronal Hsp27 up-regulation but made the cells more vulnerable to A, exposure. We then used cDNA transfection to overexpress EGFP-Hsp27 (or the empty vector) in cultures and then assessed neuronal survival and growth. Transfected neurons appeared healthy and had robust neuritic outgrowth. A, treatment induced significant cell death by 48,72 hr in nontransfected and empty-vector-expressing cultures. In contrast, cultures expressing Hsp27 did not display significant apoptosis. Our results show that Hsp27-expressing neurons were selectively protected against the deleterious effects of A, treatment; neuronal degeneration was prevented, and A,-induced alterations in mitochondrial size were attenuated. We also demonstrate that Hsp27 expression can enhance neurite growth in cortical neurons compared with control vector-transfected cells. Overall, our study provides new evidence that Hsp27 can provide a protective influence in primary cortical neurons in the face of toxic concentrations of amyloid. © 2009 Wiley-Liss, Inc. [source]


Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA

JOURNAL OF VIRAL HEPATITIS, Issue 3 2005
X.-R. Ren
Summary., Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 ± 5.4% (P = 0.00003) and 55.8 ± 6.2% (P = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold (P = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection. [source]


Characterization of the caspase cascade in a cell culture model of SOD1-related familial amyotrophic lateral sclerosis: expression, activation and therapeutic effects of inhibition

NEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 5 2005
S. Sathasivam
There is increasing evidence that apoptosis or a similar programmed cell death pathway is the mechanism of cell death responsible for motor neurone degeneration in amyotrophic lateral sclerosis. Knowledge of the relative importance of different caspases in the cell death process is at present incomplete. In addition, there is little information on the critical point of the death pathway when the process of dying becomes irreversible. In this study, using the well-established NSC34 motor neurone-like cell line stably transfected with empty vector, normal or mutant human Cu-Zn superoxide dismutase (SOD1), we have characterized the activation of the caspase cascade in detail, revealing that the activation of caspases-9, -3 and -8 are important in motor neurone death and that the presence of mutant SOD1 causes increased activation of components of the apoptotic cascade under both basal culture conditions and following oxidative stress induced by serum withdrawal. Activation of the caspases identified in the cellular model has been confirmed in the G93A SOD1 transgenic mice. Furthermore, investigation of the effects of anti-apoptotic neuroprotective agents including specific caspase inhibitors, minocycline and nifedipine, have supported the importance of the mitochondrion-dependent apoptotic pathway in the death process and revealed that the upstream caspase cascade needs to be inhibited if useful neuro-protection is to be achieved. [source]


Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

THE JOURNAL OF GENE MEDICINE, Issue 5 2004
Naoki Kobayashi
Abstract Background The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. Methods Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. Results PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. Conclusions These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability. Copyright © 2004 John Wiley & Sons, Ltd. [source]


Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle

THE PLANT JOURNAL, Issue 2 2005
Craig B. Orchard
Summary The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25 -expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25 -expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25- expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25 -expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25 -expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable. [source]


Overexpression of EIF3S3 promotes cancer cell growth

THE PROSTATE, Issue 11 2006
Kimmo J. Savinainen
Abstract BACKGROUND Amplification and overexpression of EIF3S3 gene has been demonstrated in breast and prostate cancer. Here, our goal was to study the effect of EIF3S3 on cell growth. METHODS The effect of EIF3S3 on growth of NIH 3T3 murine fibroblasts as well as breast (SK-Br-3 and ZR-75-1) and prostate (PC-3 and LNCaP) cancer cell lines was examined by using transfection with inducible pTet-Off system and siRNAs. RESULTS NIH 3T3 cells with overexpression of EIF3S3 grew significantly faster than cells transfected with empty vector and survived longer when grown in soft agar. The EIF3S3 overexpression was associated with increased fraction of cells in S-phase and with phosphorylation of retinoblastoma (Rb) protein. siRNA treatment inhibited significantly (P,=,0.0022) the growth of all breast and prostate cancer cell lines studied. CONCLUSIONS The results suggest that EIF3S3 regulates cell growth and viability, and that overexpression of the gene may provide growth advantage to the cancer cells. Prostate © 2006 Wiley-Liss, Inc. [source]


Infiltration of tumor-reactive transforming growth factor-beta insensitive CD8+ T cells into the tumor parenchyma is associated with apoptosis and rejection of tumor cells,

THE PROSTATE, Issue 3 2006
Qiang Zhang
Abstract BACKGROUND TGF-, is a potent immunosuppressant. High levels of TGF-, produced by cancer cells have a negative inhibition effect on surrounding host immune cells and leads to evasion of the host immune surveillance and tumor progression. In the present study, we report a distinct ability of tumor reactive, TGF-,-insensitive CD8+ T cells to infiltrate into established tumors, secrete relevant cytokines, and induce apoptosis of tumor cells. METHODS CD8+ T cells were isolated from the spleens of C57BL/6 mice, which were primed with irradiated mouse prostate cancer cells, the TRAMP-C2 cells. After ex vivo expansion, these tumor reactive CD8+ cells were rendered TGF-,-insensitive by infection with a retroviral (MSCV)-mediated dominant negative TGF-, type II receptor (T,RIIDN). Control CD8+ cells consist of those transfected with the GFP-only empty vector and naïve CD8+ T cells. Recipient mice were challenged with a single injection of TRAMP-C2 cells 21 days before adoptive transfer of CD8+ T cells was performed. Forty days after the adoptive transfer, all animals were sacrificed. The presence of pulmonary metastases was evaluated pathologically. Serial slides of malignant tissues were used for immunofluorescent staining for different kinds of immune cell infiltration, cytokines, and apoptosis analysis. RESULTS Pulmonary metastases were either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-,-insensitive CD8+ T cells (3 out of 12) when compared to GFP controls (9 out of 12), and naïve CD8+ T cells (12 out of 12). Results of immunofluorescent studies demonstrated that only tumor-reactive TGF-,-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis when compared to CD4+ T cells, NK cells, and B cells. A large amount of cytokines such as perforin, nitric oxide, IFN-,, IL-2, TNF-, were secreted in tumor tissue treated with tumor-reactive TGF-,-insensitive CD8+ T cells. No immune cells infiltration and cytokine secretion were detected in tumor tissues treated with naïve T cells and GFP controls. CONCLUSIONS Our results demonstrate the mechanism of anti-tumor effect of tumor-reactive TGF-,-insensitive CD8+ T cells that adoptive transfer of these CD8+ T cells resulted in infiltration of these immune cells into the tumor parenchyma, secretion of relevant cytokines, and induction of apoptosis in tumor cells. These results support the concept that tumor-reactive TGF-,-insensitive CD8+ T cells may prove beneficial in the treatment of advanced cancer patients. © 2005 Wiley-Liss, Inc. [source]


Cadherin 11 promotes invasive behavior of fibroblast-like synoviocytes

ARTHRITIS & RHEUMATISM, Issue 5 2009
Hans P. Kiener
Objective To define the expression pattern of cadherin 11 in the destructive pannus tissue of patients with rheumatoid arthritis, and to determine whether cadherin 11 expression in fibroblast-like synoviocytes controls their invasive capacity. Methods Cadherin 11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin 11 in regulating the invasive behavior of fibroblast-like synoviocytes, we generated L cell clones expressing wild-type cadherin 11, mutant cadherin 11, and empty vector,transfected controls. The invasive capacity of L cell transfectants and cultured fibroblast-like synoviocytes treated with a blocking cadherin 11,Fc fusion protein or control immunoglobulin was determined in Matrigel invasion assays. Results Immunohistochemical analysis revealed that cadherin 11 is abundantly expressed in cells at the cartilage,pannus junction in rheumatoid synovitis. Assays to determine invasion demonstrated a 2-fold increased invasive capacity of cadherin 11,transfected L cells compared with L cells transfected with E-cadherin or control vector. The invasive behavior of L cells stably transfected with a cadherin 11 construct that lacked the juxtamembrane cytoplasmic domain was diminished to the level of vector control L cells. Furthermore, treatment with the cadherin 11,Fc fusion protein diminished the invasive capacity of fibroblast-like synoviocytes. Conclusion The results of these in vitro studies implicate a role for cadherin 11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast-like synoviocytes in chronic synovitis and rheumatoid arthritis. [source]


Mammary serine protease inhibitor inhibits epithelial growth factor-induced epithelial-mesenchymal transition of esophageal carcinoma cells

CANCER, Issue 1 2009
Zhen Cai PhD
Abstract BACKGROUND: By using proteomic technology, the authors previously observed the substantial down-regulation of mammary serine protease inhibitor (maspin) in esophageal squamous cell carcinoma and metastases. In the current study, they examined the effects of maspin re-expression in a maspin-null esophageal cancer cell line EC109 and also investigated the underlying mechanism. METHODS: A cell line with stable maspin expression was established. An epithelial growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) model was used to mimic some aspects of the metastatic process in vitro. The effects of maspin reintroduction on EGF-induced EMT and cell growth characteristics were evaluated. Comparative proteomic analysis of transfected cells versus parental cells was then performed to explore the potential mechanism. RESULTS: The introduction of maspin into EC109 cells was able to inhibit EGF-induced EMT and altered cell growth characteristics, including the serum dependence, proliferative response to EGF stimulation, and colony formation ability in soft agar, indicating a conversion from a malignant phenotype to a benign phenotype. Proteomic analysis revealed a significant down-regulation of a group of glycolytic enzymes in maspin-transfected cells. In addition, maspin-transfected cells expressed much lower levels of hypoxia-inducible factor 1, than parental cells or empty vector transfected cells. CONCLUSIONS: Maspin exhibited a metastasis-suppressive effect, which may be a consequence of the reversal of the malignant phenotype of EC109 cells. The switch of cellular metabolic phenotype to low glycolysis by the gain of maspin function may play a key role in the process. This finding provides additional evidence of the tumor metastasis-suppressive activity of maspin and may indicate a new direction for future studies of the mechanism of maspin. Cancer 2009. © 2008 American Cancer Society. [source]