Transfected NIH 3T3 Cells Research Articles

Coexpression of luxA and luxB genes of Vibrio fischeri in NIH3T3 mammalian cells and evaluation of its bioluminescence activities

Expression of bacterial luciferase enzyme (lux) in eukaryotic cells would provide a new bioreporter system for in vivo imaging and diagnostics technology. In spite of this, until now only a few efforts have been made to express bacterial luciferase enzyme in eukaryotic cells. We attempted to synthesize an expression construct of luxA and luxB genes from Vibrio fischeri. The luxA and luxB genes were cloned into the MCS of pTZ57R via the 5' kpnI, BamHI and BamHI, EcoRI restriction sites to generate pTZ57R/luxA and pTZ57R/luxB respectively, then newly synthesized constructs were cleaved with the same enzymes and respectively cloned into the pcDNA3.1(+) (Hyg) and pcDNA3.1(+) (Neo) expression vectors to create pcDNA3.1(+) (Hyg)/luxA and pcDNA3.1(+) (neo)/luxB. Recombinant constructs were cotransfected to the NIH3T3 cell line. Gene expression was confirmed by reverse transcription-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; in addition, bioluminescence characteristics of transfected NIH3T3 cell lines were evaluated by decanal supplement. In conclusion, in the current research, separate vector systems were constructed, which are composed of bacterial luciferase genes (luxA and luxB) that accordingly have not already been reported. These results hold promise toward the potential development of an autonomous light-generating lux reporter system in eukaryotic cells.

Nanomagnetic Gene Transfection for Non-Viral Gene Delivery in NIH 3T3 Mouse Embryonic Fibroblasts.

The objective of this work was to examine the potential of oscillating nanomagnetic gene transfection systems (magnefect-nano™) for improving the transfection efficiency of NIH3T3 mouse embryonic fibroblasts (MEFs) in comparison to other non-viral transfection techniques—static magnetofection™ and the cationic lipid agent, Lipofectamine 2000™. Magnetic nanoparticles (MNPs) associated with the plasmid coding for green fluorescent protein (GFP) were used to transfect NIH3T3 cells. The magnefect-nano system was evaluated for transfection efficiency, and any potential associated effects on cell viability were investigated. MNPs associated with the plasmid coding for GFP were efficiently delivered into NIH3T3 cells, and the magnefect-nano system significantly enhanced overall transfection efficiency in comparison to lipid-mediated gene delivery. MNP dosage used in this work was not found to affect the cell viability and/or morphology of the cells. Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect NIH3T3 cells and direct reporter gene delivery, highlighting the wide potential of nanomagnetic gene transfection in gene therapy.

C.O.2 DNM2 mutations cause multiple mtDNA deletions in muscle: A novel disorder of mtDNA maintenance

Centronuclear myopathy (CNM) is a congenital myopathy characterised by delayed motor milestones, progressive facial weakness, ptosis, ophthalmoplegia and centrally-located myonuclei caused by autosomal dominant mutations in the dynamin-2 gene, DNM2. Dnm2 is a large GTPase protein, one of three classical dynamins that is ubiquitously expressed, and key to regulating cytoskeleton and membrane trafficking within cells. Recently, a DNM2 (KI-Dnm2R465W) animal model was shown to develop phenotypic and morphological abnormalities similar to those observed in the human disease, with abnormal central accumulation of mitochondria in the muscle fibres of the heterozygous mice. Furthermore, mutations in OPA1 and MFN2 – also dynamin-like GTPase proteins which are involved in mitochondrial membrane fusion – have recently been shown to be associated with impaired mitochondrial DNA (mtDNA) stability, inferring a crucial role for these proteins in regulating mitochondrial networks and mtDNA maintenance. We investigated five patients with dominant DNM2 mutations. In addition to the typical features of CNM, the muscle biopsies demonstrated variable amount of cytochrome c oxidase (COX)-deficient muscle fibres indicative of mitochondrial dysfunction. We show here that these focal mitochondrial biochemical defects are due to clonally-expanded mtDNA deletions, characteristic of mtDNA maintenance abnormality. Confocal microscopy studies of patient fibroblasts reveal a quantitative disruption of the dynamic mitochondrial network associated with the p.R369W DNM2 mutation, which interestingly corresponded with the most severe mitochondrial defect in muscle. Similar results were observed in HeLa cells following Dnm2 siRNA knockdown, whilst transfection of NIH3T3 cells with mutant p.R369W Dnm2 led to a significant decrease in mitochondrial objects compared to controls. Together, our data suggest an important and emerging role for Dnm2 in mtDNA maintenance and stability.

Abstract 1786: Characterization of the inhibitory capacity of EMD1214063, a novel small molecule inhibitor of the MET hepatocyte growth factor receptor on a panel of MET mutated variants

Abstract The receptor tyrosine kinase MET is a prime target in clinical oncology because of its aberrant activation in a broad spectrum of malignancies. Here, we tested the anti tumor activity of a novel MET inhibitor, EMD1214063, on cancer cells overexpressing MET and in eight transfected NIH3T3 cell lines expressing different activating mutants of the MET kinase. Our results demonstrate a dose-dependent decrease in MET autophosphorylation in both MET overexpressing cell lines (IC50 of 20nM and 30nM for GTL-16 and H1993, respectively) and in six of the eight cell lines transfected with MET activating mutants (IC50 5-18nM). Blockade of MET by EMD1214063 resulted in reduced downstream activation of AKT, ERK and PLCγ. In contrast, lack of MET inhibition by EMD1214063 in the resistant Y1248H and L1213V MET mutants was associated with high levels of AKT, ERK and PLCγ activation. In all sensitive mutants, EMD1214063 attenuated MET-dependent cellular proliferation and significantly altered cell cycle distribution profiles (increase in G1 and concomitant decrease of S phase). Furthermore, EMD1214063 strongly affected MET-driven biological functions, such as cellular morphology, motility and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we established tumor transplantation models exploiting NIH3T3 cells expressing the H1112L or L1213V MET mutants, respectively sensitive and resistant, to in vitro treatment with EMD1214063. Animals bearing H1112L or L1213V tumors were randomized for the treatment with EMD1214063 (50mg/kg/day) or vehicle only. Remarkably, 5 days of treatment with EMD1214063 induced complete regression of sensitive H1112L tumors, while tumor growth remained unaffected in mice injected with resistant L1213V tumor cells and in vehicle-treated mice. Taken together, these results underscore the concordance between in vitro and in vivo effects of EMD1214063 and strongly support its efficacy in inhibiting MET-driven tumor cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1786. doi:1538-7445.AM2012-1786

RET: A Multi-Faceted Gene in Human Cancer

Receptor tyrosine kinases (RTK) are transmembrane (TM) proteins featuring an intracellular domain containing the tyrosine kinase (TK) enzyme. RTKs are often involved in cancer formation [13]. Notable examples are epidermal growth factor receptor (EGFR/ HER1) and anaplastic lymphoma kinase (ALK) in non-small cell lung carcinoma (NSCLC) [4], KIT in gastrointestinal stromal tumors (GIST) [5], FLT3 in acute myeloid leukemia (AML) [6], and HER2/ ERBB2/neu in breast cancer [7]. In some cases, cancer cells up-regulate expression of the RTK (as an example HER2 in breast cancer), its cognate growth factor or both, in other cases, structural alterations such as chromosomal rearrangements leading to the RTK recombination to heterologous genes (as an example EML4-ALK in lung adenocarcinoma) or point mutations (as EGFR, KIT or FLT3 mutations in NSCLC, GIST, or AML, respectively), lead to unchecked kinase and oncogenic activity [1-3]. This notion has stimulated the search for agents, such as monoclonal antibodies against the RTK extracellular domain (like trastuzumab for HER2 or cetuximab for EGFR) or ATP-competitive small molecule protein kinase inhibitors (PKIs) (like gefitinib and erlotinib for EGFR or crizotinib for ALK), to combat cancers driven by oncogenic RTKs [1-3]. The RET RTK was originally identified as an oncogene activated by a rearrangement occurred in vitro during transfection of NIH3T3 cells with human lymphoma DNA [8]. RET protein belongs to a cell-surface complex able to bind glial-derived neurotrophic factor (GDNF) ligands (GDNF, neurturin, artemin, and persephin) in conjunction with co-receptors of the GDNF receptor α family, designated GFRα 1-4 [9]. Binding to the ligand-co-receptor complex leads to RET dimerization and kinase activation. RET expression is tightly regulated during development and in the adulthood is limited to specific tissues, including neural crest-derived cells. RET is essential for the development of the enteric neurvous system and kidney, and germline loss-of-function mutations in RET cause Hirschsprung disease (aganglionic megacolon) and congenital anomalies of the kidney or lower urinary tract [10,11]. RET gene maps to chromosome 10q11.2. Fig. 1 shows that it is splitted in 21 coding exons. Exons 1-10 code for the extracellular region; exon 11 codes for the COOH-terminal part of the extracellular region, the TM domain, and the intracellular juxtamembrane domain. Finally, exons 12-21 code for the intracellular domain. An alternative splicing at exon 19 determine the synthesis of three RET protein isoforms with different C-terminal tails. In RET9 (1072 aa), exon 19 is unspliced; in RET51 (1114 aa), exon 19 is spliced to exon 20; in RET43 (1106 aa), exon 19 is spliced to to exon 21 [12-15]. RET9 and RET51 are the most abundant and well characterized isoforms (Fig. 1). RET protein features an extracellular portion (RET-EC) that

Germline RET sequence variation I852M and occult medullary thyroid cancer: harmless polymorphism or causative mutation?

Rearranged during transfection (RET) gene analysis, widely used to identify carriers at risk of medullary thyroid cancer (MTC), occasionally uncovers novel sequence 'variants of unknown clinical significance' including RET I852M. This study aimed to clarify whether RET I852M represents a harmless polymorphism or a pathogenic mutation. Clinical investigation supported by functional characterization of I852M mutant cells in vitro. Genotype-phenotype correlation including five kindreds from a three-generational Caucasian I852M RET family. A node-negative occult MTC was found in the 64-year-old index patient who had increased basal and stimulated peak calcitonin levels of 190 and 13 307 ng/l, respectively. Her 4-year-old grandson had no histopathological evidence of C-cell disease although his serum calcitonin levels had increased within 5 months from 3·2 to 6·3 ng/l basally and from 17·2 to 24·5 ng/l after pentagastrin stimulation. His mother and two 11- and 1·5-year-old siblings, also carrying the gene, had normal basal and stimulated calcitonin levels and hence did not undergo surgery. Functional characterization of transfected NIH3T3 cells in vitro (cell proliferation rate; cell viability; anchorage-independent cell growth; cell migration; and invasion) indicated that I852M mutant cells have transforming and migratory activities similar to American Thyroid Association (ATA) class A V804M mutants. I852M mutants demonstrated a weaker proliferative potential than fast-proliferating ATA class C C634R mutants and revealed a weaker migratory activity compared with aggressively growing ATA class D A883F mutants. I852M sequence variations represent genuine RET mutations, falling into ATA class A of weakly activating RET germline mutations.

Regulatory Roles of Heterogeneous Nuclear Ribonucleoprotein M and Nova-1 Protein in Alternative Splicing of Dopamine D2 Receptor Pre-mRNA

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.

Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.

Abstract 5007: WT1 promotes tumor growth and vascularization by regulating matrix metalloproteinase 9 in Ewing's sarcoma xenografts

Abstract WT1, a zinc-finger transcription factor, is expressed in tumors of varied origins including sarcomas. High level WT1 expression often correlates with poor patient survival. The role of WT1 in sarcoma biology and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. The aim of this study is to investigate the effect of WT1 expression on sarcoma growth and angiogenesis. We transfected the WT1-null Ewing's sarcoma cell line SK-ES-1 with one of two major WT1 isoforms, and used shRNA to silence WT1 expression in the WT1-positive Ewing's sarcoma cell line MHH-ES. Cells were injected into the flanks of immune deficient mice and tumor growth was monitored. Tumors arising fromWT1-expressing SK-ES-1 cells grew faster than control, while tumors arising from WT1-silenced MHH-ES cells grew more slowly than control. Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31 and the pericyte marker αSMA. In xenografted SK-ES-1 cells, quantification of CD31 positive area in WT1 expressing tumors showed 60 -70 % more compared to the controls. In contrast, CD31 expression is abolished in tumors arising from WT1-silenced MHH-ES cells. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays showed that WT1 can upregulate MMP9 promoter activity in transiently transfected NIH3T3 cells. Also, immunohistochemical analysis showed increased MMP9 expression in WT1-expressing tumors. Finally, imunohistochemical analysis of primary Ewing's sarcoma samples showed correlated expression of WT1, VEGF, and MMP9. Taken together, our data suggest that WT1 promotes tumor growth and angiogenesis, perhaps via upregulation of MMP9. Increased tumor angiogenesis may lead to more aggressive tumors and a worse outcome for patients, explaining the effect of WT1 on prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2011-5007

Mannose Trimming Is Required for Delivery of a Glycoprotein from EDEM1 to XTP3-B and to Late Endoplasmic Reticulum-associated Degradation Steps

Although the trimming of α1,2-mannose residues from precursor N-linked oligosaccharides is an essential step in the delivery of misfolded glycoproteins to endoplasmic reticulum (ER)-associated degradation (ERAD), the exact role of this trimming is unclear. EDEM1 was initially suggested to bind N-glycans after mannose trimming, a role presently ascribed to the lectins OS9 and XTP3-B, because of their in vitro affinities for trimmed oligosaccharides. We have shown before that ER mannosidase I (ERManI) is required for the trimming and concentrates together with the ERAD substrate and ERAD machinery in the pericentriolar ER-derived quality control compartment (ERQC). Inhibition of mannose trimming prevents substrate accumulation in the ERQC. Here, we show that the mannosidase inhibitor kifunensine or ERManI knockdown do not affect binding of an ERAD substrate glycoprotein to EDEM1. In contrast, substrate association with XTP3-B and with the E3 ubiquitin ligases HRD1 and SCF(Fbs2) was inhibited. Consistently, whereas the ERAD substrate partially colocalized upon proteasomal inhibition with EDEM1, HRD1, and Fbs2 at the ERQC, colocalization was repressed by mannosidase inhibition in the case of the E3 ligases but not for EDEM1. Interestingly, association and colocalization of the substrate with Derlin-1 was independent of mannose trimming. The HRD1 adaptor protein SEL1L had been suggested to play a role in N-glycan-dependent substrate delivery to OS9 and XTP3-B. However, substrate association with XTP3-B was still dependent on mannose trimming upon SEL1L knockdown. Our results suggest that mannose trimming enables delivery of a substrate glycoprotein from EDEM1 to late ERAD steps through association with XTP3-B.

Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

The Effect of Charge-Reversal Amphiphile Spacer Composition on DNA and siRNA Delivery

A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic chains are described for delivery of DNA and siRNA. Among them, the amphiphiles possessing a glycine spacer (e.g., B-GlyGly) showed effective DNA transfection in CHO and NIH 3T3 cells, as well as siRNA gene knockdown in HepG2 and UASMC cells. Ethidium bromide quenching assays revealed that DNA was released the fastest from the lipoplex of B-GlyGly in the presence of esterase. Also, X-ray diffraction results indicated that the DNA was located between the adjacent lipid bilayers in the lipoplex of B-GlyGly. These distinct features appear to be required for high transfection activity.

PH‐Domain‐driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor (GEF) that is essential for the synaptic clustering of gephyrin and GABAA receptors in selected regions of the mammalian central nervous system. It has been previously proposed that Cb regulates gephyrin clustering by activating Cdc42, and thus acts as a signal transducer in a membrane activation process which labels postsynaptic membrane domains for inhibitory synapse formation. Here, we dissected the functional roles of the Dbl-homology (DH) and pleckstrin homology (PH) domains of the constitutively active splice variant Cb II by substituting conserved amino acid residues that are required for GEF activity towards Cdc42 and phosphoinositide binding, respectively. A Cb II mutant lacking any detectable GEF activity towards Cdc42 was still fully active in inducing gephyrin scaffold formation, both in transfected NIH-3T3 cells and in cultured hippocampal neurons. Furthermore, mice with a forebrain-specific inactivation of the Cdc42 gene displayed normal densities of gephyrin and GABA(A) receptor clusters in the hippocampus. In contrast, substitution of Cb II PH-domain residues essential for phosphoinositide binding abolished gephyrin recruitment to synaptic sites. Our results provide evidence that the formation of gephyrin scaffolds at inhibitory synapses requires an intact Cb II PH-domain but is Cdc42-independent.

Acid-transforming polypeptide micelles for targeted nonviral gene delivery

Efficient delivery of therapeutic genes requires overcoming key extracellular and intracellular barriers. These include stability during circulation, internalization by target cells, facilitated endosomal escape, and localization of genes in destined intracellular compartments (e.g., nucleus). Micelles that transform their structure in the mildly acidic endosome and release their cargo genes into the cytoplasm were synthesized by self-assembling DNA with PEG-conjugated poly(ketalized serine) [PEG-poly(kSer)]. It was confirmed that, upon acid-hydrolysis of ketal linkages, poly(kSer) converts to neutral and naturally occurring poly(serine), destabilizing PEG-poly(kSer)/DNA micelles. In vitro studies demonstrated that PEG-poly(kSer) micelles were able to transfect NIH 3T3 cells more efficiently than both PEG-poly(Lys)/DNA micelles and poly-L-lysine/DNA polyplexes through efficient DNA dissociation in the cytoplasm. In addition, the core of PEG-poly(kSer)/DNA micelles were cross-linked via acid-cleavable amine-bearing branches, and the resulting cross-linked PEG-poly(kSer)/DNA micelles showed improved transfection capability in the presence of serum. Conjugation of folic acids (FAs) at the PEG termini of the acid-transforming micelles resulted in selectively increased cellular internalization and transfection of FA receptor-expressing HeLa cells over NIH 3T3 cells, implicating the possibility of cancer-targeted gene delivery using FA-PEG-poly(kSer)/DNA micelles. This study demonstrates that the acid-transforming PEG-poly(kSer)/DNA micelles are promising nonviral vectors for stimuli-responsive, efficient, biocompatible, and targeted gene delivery.

Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells

The design of appropriate gene delivery systems is essential for the successful application of gene therapy to clinical medicine. Cationic lipid-mediated delivery is a viable alternative to viral vector-mediated gene delivery in applications where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells such as neurons is often reported to be of low efficiency, due to the presumed inability of the DNA to translocate to the nucleus. Lipid-mediated delivery of RNA is an attractive alternative to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we report a comparative investigation of cationic lipid-mediated delivery of RNA versus DNA vectors encoding the reporter gene green fluorescent protein (GFP) in Chinese Hamster Ovary (CHO) and NIH3T3 cells following chemical inhibition of proliferation, and in primary mixed neuronal cell cultures. Using optimized formulations and transfection procedures, we assess gene expression by flow cytometry to specifically address some of the advantages and disadvantages of lipid-mediated RNA and DNA gene transfer. Despite inhibition of cell proliferation, over 45% of CHO cells express GFP after lipid-mediated transfection with RNA vectors. Transfection efficiency of DNA encoding GFP in proliferation-inhibited CHO cells was less than 5%. Detectable expression after RNA transfection occurs at least 3 h earlier than after DNA transfection, but DNA transfection eventually produces a mean level of per cell GFP expression (as assayed by flow cytometry) that is higher than after RNA transfection. Transfection of proliferation-inhibited NIH3T3 cells and primary mixed neuronal cultures produced similar results, with RNA encoded GFP expression in 2–4 times the number of cells as after DNA encoded GFP expression. These results demonstrate the increased efficiency of RNA transfection relative to DNA transfection in non-dividing cells. We used firefly luciferase encoded by RNA and DNA vectors to investigate the time course of gene expression after delivery of RNA or DNA to primary neuronal cortical cells. Delivery of mRNA resulted in rapid onset (within 1 h) of luciferase expression after transfection, a peak in expression 5–7 h after transfection, and a return to baseline within 12 h after transfection. After DNA delivery significant luciferase activity did not appear until 7 h after transfection, but peak luciferase expression was always at least one order of magnitude higher than after RNA delivery. The peak expression after luciferase-expressing DNA delivery occurred 36–48 h after transfection and remained at a significant level for at least one week before dropping to baseline. This observation is consistent with our in vivo delivery results, which are shown as well. RNA delivery may therefore be more suitable for short-term transient gene expression due to rapid onset, shorter duration of expression and greater efficiency, particularly in non-dividing cells. Higher mean levels of expression per cell obtained following DNA delivery and the longer duration of expression confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression.

Coaxial electrohydrodynamic spraying of plasmid DNA/polyethylenimine (PEI) polyplexes for enhanced nonviral gene delivery

DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method to produce DNA/PEI polyplexes in a more controlled manner. Both pGFP/PEI and pSEAP/PEI polyplexes were produced by EHDS with a coaxial needle setup. The size of the polyplexes was determined using dynamic light scattering, and their ability to transfect NIH 3T3 cells was observed by using an inverted fluorescence microscope (pGFP) or quantified by measuring the activity level of alkaline phosphatase (pSEAP). At nitrogen to phosphate ratio (N/P) of 6.7, the polyplexes produced by coaxial EHDS had delivery efficiencies up to 2.6 times higher than those produced by bulk mixing. The N/P ratio and the structure of the EHDS used to make the polyplexes were crucial factors in determining the delivery efficiency.

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real-time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real-time. Two heat parameters were considered in combination with sham- and 100 mT-exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult.

In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Phosphorylation of Serine 271 on 5-Lipoxygenase and Its Role in Nuclear Export

The enzyme 5-lipoxygenase (5-LO) initiates the biosynthesis of leukotrienes, inflammatory mediators involved in immune diseases and defense. The subcellular localization of 5-LO is regulated, with nuclear import commonly leading to increased leukotriene production. We report here that 5-LO is constitutively phosphorylated on Ser-271 in transfected NIH 3T3 cells. This residue is nested in a classical nuclear export sequence, and phosphorylated Ser-271 5-LO was exclusively found in the nucleus by immunofluorescence and by fractionation techniques. Mutation of Ser-271 to Ala allowed nuclear export of 5-LO that was blocked by the specific nuclear export inhibitor leptomycin b, suggesting that phosphorylation of Ser-271 serves to interfere with exportin-1-mediated nuclear export. Consistent with previous reports that purified 5-LO can be phosphorylated on Ser-271 in vitro by MAPK-activated protein kinase 2, the nuclear export of 5-LO was increased by either treatment with the p38 inhibitor SB 203,580 or co-expression of a kinase-deficient p38 MAPK. Nuclear export of 5-LO can also be induced by KN-93, an inhibitor of Ca2+/calmodulin-dependent kinase II, and the effects of SB 203,580 plus KN-93 are additive. Finally, HeLa cells, which lack nuclear 5-LO, also lack constitutive phosphorylation of Ser-271. Taken together, these results indicate that the phosphorylation of Ser-271 serves to inhibit the nuclear export of 5-LO. This action works in concert with nuclear import, which is regulated by phosphorylation on Ser-523, to determine the subcellular distribution of 5-LO, which in turn regulates leukotriene biosynthesis.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells

We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.