Dominant Negative Gene Research Articles

Gene therapy: Recent advances and applications in gastroenterology and hepatology.

The advantages and disadvantages of viral and non-viral vectors for gene delivery are reviewed. Advances in systems for introduction of new gene expression are described, including self-deleting retroviral transfer vectors, chimeric viruses and chimeric oligonucleotides. Systems for inhibition of gene expression are also discussed including antisense oligonucleotides, ribozymes and dominant-negative genes. Examples of the use of these systems in animal models and clinical trials for gastrointestinal disorders are discussed.

Involvement of H-rasin Erythroid Differentiation of TF1 and Human Umbilical Cord Blood CD34+++Cells

ABSTRACT: To investigate the role of therasgene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-rasgene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-rasgene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-rascDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-rasin vitro. The mRNA levels of β- and γ-, but not α-, globin genes were significantly higher in H-rastransduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-rastransduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34+++cells which are highly enriched for hematopoietic stem / progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-rasin erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34+++cells transduced with the H-rascDNA, compared to that from mock transduced cells. These data suggest a possible role forrasin erythroid differentiation.

Protection from reoxygenation injury by inhibition of rac1.

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

TNF-α and IL-1 Upregulate Membrane-Bound and Soluble E-Selectin through a Common Pathway

Background: Endothelial cell adhesion molecules such as E-selectin promote the capture of neutrophils (PMN) in the microcirculation and initiate the inflammatory response. In contrast, when “shed” into the microcirculation, soluble E-selectin can bind PMN in the blood stream, reducing the number available for adhesion to injured tissue. These experiments were designed to better characterize the molecular response to cytokines and the balance between cell surface (bound) and soluble (unbound) E-selectin. Methods: Cultured human umbilical veins, exposed to human recombinant TNF-α or IL-1 (10 pg/ml), were analyzed for E-selectin mRNA induction (Northern blot), E-selectin cell surface expression (flow cytometry), and sE-selectin release (ELISA). Transcriptional regulation was analyzed via Raf kinase dominant negative gene transfection. Results: E-selectin mRNA expression was markedly increased at 2 h and sustained through 8 h. No further induction was noted at 12 h. Upregulation of cell surface E-selectin was noted (mean fluorescence) as early as 2 h for TNF-α (baseline, 12.28 ± 1.32; TNF-α, 23.03 ± 1.81) or 4 h for IL-1 (baseline, 12.28 ± 1.32; IL-1, 70.00 ± 3.04) with maximum expression at 6 h (TNF-α, 118.8±15; IL-1, 94.11 ± 9.34). Expression returned to baseline levels by 24 h. Soluble E-selectin (ng/ml) assays demonstrated later increases beginning at 12 h (TNF-α, 0.313 ± 0.077; IL-1, 0.159 ± 0.075) and continuing through 24 h (TNF-α, 0.340 ± 0.062; IL-1, 0.157 ± 0.030). Transfection of endothelial cells with Raf kinase 301 dominant negative gene resulted in proportionate decreases in the peak expression in both surface (bound) E-selectin (TNF-α, 51.7%; IL-1, 29.6%) and sE-selectin (TNF-α, 49.2%; IL-1, 34.5%). Conclusion: The temporal sequence of late decreases in cell surface E-selectin accompanied by increases in soluble E-selectin indicates that the source of E-selectin in the microcirculation is shed receptors rather than synthesis of a different type of receptor. Enhancement of such “shedding” may decrease PMN adhesion to injured tissue and have therapeutic potential.

Helix-loop-helix proteins in Schwann cells: a study of regulation and subcellular localization of Ids, REB, and E12/47 during embryonic and postnatal development.

Although basic helix-loop-helix (bHLH) proteins play an important role in transcriptional control in many cell types, the role of HLH proteins in Schwann cells has yet to be assessed. In this study, we have analyzed the expression of the dominant negative HLH genes, Id1 to Id4 and the class A gene REB, during Schwann cell development. We found that mRNA derived from these genes was present in the Schwann cell lineage throughout development including embryonic precursors and mature cells. The mRNA levels were not significantly regulated during development. Nevertheless, by using antibodies against the four different Id proteins, we found clear regulation of some of these genes at the protein level, in particular Id 2, 4, and REB, both in amount and nuclear/cytoplasmic localization. All these proteins are found in the nuclei of Schwann cell precursors but are not seen in nuclei of Schwann cells of newborn nerves. We observed extensive overlap in Id expression, especially in Schwann cell precursors that co-expressed all four Id proteins and REB. We also showed that Id 1 and 2 were up-regulated as Schwann cells progressed through the cell cycle. These data indicate that HLH transcription factors act as regulators of Schwann cell development and point to the existence of as yet unidentified cell type-specific bHLH proteins in these cells.

The 19th Annual Meeting of the European Lipoprotein Club

The European Lipoprotein Club met September 9 to 12, 1996, in Tutzing, Germany. There were 99 participants from 13 European countries and the United States. Dr Jonathan C. Fox from the University of Pennsylvania, Philadelphia, opened the meeting with a state-of-the-art lecture entitled “Cardiovascular Gene Therapy: Current Concepts.” Gene therapy constitutes a novel therapeutic approach to selected cardiovascular diseases and is designed to augment traditional techniques of diagnosis and treatment by targeting the molecular mechanisms of disease. Dr Fox first presented the general strategies driving the development of gene therapy. In the case of deleterious gene mutations causing disease, the defective gene can be replaced with a normal copy. In a pathophysiologic process that can be alleviated through augmented gene function, a beneficial gene can be overexpressed or a mutated “designer” gene, which is either more active or in some way more beneficial than its natural precursor, can be expressed. Normal genes may also confer therapeutic benefits by virtue of their expression in a novel environment. The expression of harmful genes can also be inhibited by antisense oligonucleotides, antisense RNA expression vectors, ribozymes, or triplex DNA. Dominant negative genes can be expressed, eg, encoding a protein that binds to regulatory …

Class II Haplotype Differentially Regulates Immune Response in HgCl2-Treated Mice

One of the most striking features of exposure to low doses of mercury in mice is the high-titer haplotype-linked anti-nucleolar (ANoA) autoantibody response. Mice of H-2shaplotype have been high responders, while H-2bmice have been low. This pattern has been attributed to the class II molecule itself, but the poor response of F1crosses between high and low responders raised the possibility that the anti-fibrillarin specificity was actually due to a closely linked dominant negative gene. To test the role of class II explicitly, F1crosses between congenic B6.SJL (H-2s) and C57BL/6 (H-2b) mice with a targeted deletion of I-AbAβwere generated, creating mice heterozygous for all MHC loci, but expressing only I-As. In comparison with B6.SJL, no diminution of ANoA titers was found, proving that I-Asitself was responsible for susceptibility and I-Abfor downregulation. Unlike I-A, expression of the I-E class II molecule could not downregulate the response in an otherwise susceptible mouse. These results suggest a complicated role for class II in the regulation of a novel, environmentally induced autoimmune response.

Myelin mutants: model systems for the study of normal and abnormal myelination.

Spontaneous mutations that perturb myelination occur in a range of species including man, and together with engineered mutations have been used to study disease, normal myelination and axon/glial inter-relationships. Only a minority of the currently defined mutations have an apparently simple pathogenesis due to lack of a functional protein. Mutations in the myelin basic protein gene lead to a lack of protein, resulting in changes in the structure of myelin, which can be rescued by transgenic complementation. The pathogenesis of autosomal dominant and X-linked mutations affecting either oligodendrocytes or Schwann cells is more complex. Point mutations may act in a dominant negative manner and gene dosage is clearly linked to phenotypic change. Mutations in regulatory genes, such as those encoding transcription factors, can also disturb myelination by selected cell types. Other less-well studied and unexpected consequences of myelin mutations, such as seizures in mutations affecting genes expressed in Schwann cells and axonal changes associated with dysmyelination, are also considered. With the major developments in gene mapping and cloning it is now relevant to study mutations in a variety of species with the real prospect of defining their molecular basis. Examples are given of unusual, but potentially useful, uncharacterized mutations in dog and bovine.

Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.

Transformants that expressed either the wild-type rasG gene, an activated rasG-G12T gene, or a dominant negative rasG-S17N gene, all under the control of the folate-repressible discoidin (dis1gamma) promoter, were isolated. All three transformants expressed high levels of Ras protein which were reduced by growth in the presence of folate. All three transformants grew slowly, and the reduction in growth rate correlated with the amount of RasG protein produced, suggesting that RasG is important in regulating cell growth. The pVEII-rasG transformant containing the wild-type rasG gene developed normally despite the presence of high levels of RasG throughout development. This result indicates that the down regulation of rasG that normally occurs during aggregation of wild-type strains is not essential for the differentiation process. Dictyostelium transformants expressing the dominant negative rasG-S17N gene also differentiated normally. Dictyostelium transformants that overexpressed the activated rasG-G12T gene did not aggregate. The defect occurred very early in development, since the expression of car1 and pde, genes that are normally induced soon after the initiation of development, was repressed. However, when the transformant cells were pulsed with cyclic AMP, expression of both genes returned to wild-type levels. The transformants exhibited chemotaxis to cyclic AMP, and development was synergized by mixing with wild-type cells. Furthermore, cells that were pulsed with cyclic AMP for 4 h before being induced to differentiate by plating on filters produced small, but otherwise normal, fruiting bodies. These results suggest that the rasG-G12T transformants are defective in cyclic AMP production and that RasG - GTP blocks development by interfering with the initial generation of cyclic AMP pulses.

The transactivation potential of a c-Myc N-terminal region (residues 92-143) is regulated by growth factor/Ras signaling.

The colony stimulating factor-1 receptor (CSF-1R) affects mitogenic growth and gene expression in NIH 3T3 cells through signaling pathways that require the products of the c-ras and c-myc proto-oncogenes. In this work we tested the hypothesis that there is direct communication between the Ras and Myc pathways. In transient transfection assays Ras increased by 5-fold transcriptional transactivation by chimeric c-Myc-Gal4 proteins. A constitutive active form of the CSF-1R also stimulated this activity and co-expression of a dominant negative ras gene ablated receptor stimulation. Deletion analysis of the c-Myc N-terminal region demonstrated that amino acid residues between positions 92 and 143 are the targets for Ras action. Transactivation by chimeric Myc proteins that were stably expressed could be transiently enhanced by either CSF-1 or serum, with peak activity occurring 2 h after mitogen stimulation. The steady-state levels of the chimeric c-Myc transactivators were increased following stimulation with CSF-1 or serum, but this increase in steady-state protein level did not strictly correlate with the increase in transactivation activity. Thus, Ras signaling may directly affect the activity of the c-Myc N-terminal region.

Do Products of the myc Proto-Oncogene Play a Role in Transcriptional Regulation of the Prothymosin α Gene?

The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the intact human prothymosin alpha gene or reporter constructs that mimic its structure. Rather, they suggest that the human prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes.

Ras Proteins Regulate Multiple Mitogenic Pathways in A10 Vascular Smooth Muscle Cells

We examined the role of ras proteins in mitogenesis and proliferation of A10 smooth muscle cells. An assay was developed in which a dominant negative ras gene could be transiently expressed in vascular smooth muscle cells. As opposed to cells transfected with a control plasmid, those transfected with a dominant negative ras expression plasmid exhibited a decrease in thymidine uptake in response to serum, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor and thrombin stimulation. In addition, expression of dominant negative ras blocked smooth muscle cell proliferation as assessed by the number of surviving colonies after transfection and neomycin selection. Our data is the first to show that ras is essential for smooth muscle cell proliferation and is a mediator in multiple smooth muscle cell mitogenic signalling pathways.

Intermediate filament molecular biology

Epidermal keratin intermediate filaments appear to have a structural function. The functions of other intermediate filaments are being elucidated using a combination of molecular genetic methods, including the expression of dominant negative mutant proteins and gene targeting. The differential expression of intermediate filament genes is regulated by both the accessibility of multiple regulatory elements and the activity or level of multiple positive and negative transcription factors.

T<SUB>3</SUB> Binding Properties of Nuclear Fibroblast Receptors from Patients Homozygous and Heterozygous for A Dominant Negative Mutation of C-erbAβ

This study represents the first measurements of T3 binding to nuclear receptors in fibroblasts from a homozygous child with a dominant negative thyroid hormone receptor gene. Previously demonstrated in this child and kindred was a three base pair deletion in the c-erbAβ thyroid hormone receptor gene resulting in the loss of threonine codon 332. In this study using cultured skin fibroblasts from this homozygous child and his heterozygous father there was normal binding affinity and a two-fold less binding capacity in comparison to control fibroblasts. These data are consistent with loss of β-receptor T3-binding activity, but the presence of normally functioning α-receptors in vivo.

A dominant positive and negative selectable gene for use in mammalian cells.

We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells.

A dominant positive and negative selectable gene for use in mammalian cells.

A dominant positive and negative selectable gene for use in mammalian cells.