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At least 775 records · Page 43Linked to original sources
PubMed1997

A proliferation switch for genetically modified cells.

Receptor dimerization is the key signaling event for many cytokines, including erythropoietin. A system has been recently developed that permits intracellular protein dimerization to be reversibly activated in response to a lipid-soluble dimeric form of the drug FK506, called FK1012. FK1012 is used as a pharmacological mediator of dimerization to bring together FK506 binding domains, taken from the endogenous protein FKBP12. In experiments reported herein, FK1012-induced dimerization of a fusion protein containing the intracellular portion of the erythropoietin receptor allowed cells normally dependent on interleukin 3 to proliferate in its absence. FK506 competitively reversed the proliferative effect of FK1012 but had no influence on the proliferative effect of interleukin 3. Signaling pathways activated by FK1012 mimicked those activated by erythropoietin, because both JAK2 and STAT5 were phosphorylated in response to FK1012. This approach may provide a means to specifically and reversibly stimulate the proliferation of genetically modified cell populations in vitro or in vivo.

Animals↗
PubMed2005

Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.

BACKGROUND: Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling. METHODS AND RESULTS: High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication. CONCLUSIONS: Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.

Animals↗
PubMed1999

Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12.

Bone marrow-derived dendritic cells (BM-DCs) retrovirally transduced with genes encoding murine interleukin (IL)-12 stably expressed bioactive IL-12 protein at high levels. Intratumoral injection with IL-12 gene-modified BM-DCs resulted in regression of day 7 established weakly immunogenic tumors (MCA205, B16, and D122). This antitumor effect was substantially better than that of IL-12-transduced syngeneic fibroblasts or nontransduced BM-DCs. Furthermore, intratumoral injection with IL-12-transduced dendritic cells (DCs) induced specific TH1-type responses to the tumor in regional lymph nodes and spleen at levels greater than those of IL-12-transduced fibroblasts or nontransduced BM-DCs. Trafficking studies confirmed that intratumorally injected IL-12-transduced DCs, but not fibroblasts, could migrate to the draining lymph node to the same extent as nontransduced BM-DCs. This strategy designed to deliver genetically modified DCs to tumor sites is associated with systemic and therapeutic antitumor immunity and is an alternative approach to those that use delivery of DCs loaded with tumor antigen. These results support the clinical application of IL-12 gene-modified DCs in patients with cancer.

Animals↗
PubMed2003

Design of a genetically modified soybean protein preventing hypertension based on an anti-hypertensive peptide derived from ovalbumin.

Food proteins can be a source of various bioactive peptides including such possessing anti-hypertensive activity. While most orally active anti-hypertensive peptides derived from food proteins inhibit the angiotensin I-converting enzyme (ACE), ovokinin (2-7) (RADHPF), a peptide isolated from a chymotryptic digest of ovalbumin, has been shown to induce nitric oxide-dependent vasorelaxation in an isolated mesenteric artery as well as anti-hypertensive effect after oral administration in spontaneously hypertensive rats (SHRs). Rational amino acid replacement lead to the ovokinin (2-7) analog, RPLKPW, which had the highest anti-hypertensive activity among the tested peptides. Furthermore, oral administration (0.1 mg/kg) of the peptide lowered the blood pressure of SHR but not of normotensive Wistar-Kyoto (WKY) rats. In order to develop a novel use of this potent anti-hypertensive peptide for prevention of hypertension, RPLKPW has been genetically introduced into the homologous sequences in soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The recombinant RPLKPW-incorporated alpha' subunit expressed in E. coil has been shown to exert anti-hypertensive activity after oral administration in SHR. Thus, RPLKPW-incorporated alpha' subunit is the first example of a genetically modified food protein possessing physiological activity based on a bioactive peptide.

Animals↗
PubMed2006

Intrafamilial phenotypic variability in families with RDS mutations: exclusion of ROM1 as a genetic modifier for those with retinitis pigmentosa.

OBJECTIVES: To identify suspected RDS mutations in families in which different people have been identified with either generalised retinal dystrophy or macular dystrophy. METHODS: Two families with a retinal dystrophy were extensively phenotyped and blood was taken for mutation analysis of the RDS (all) and ROM1 (retinitis pigmentosa patients only) genes. RESULTS: A novel p.Trp94X mutation in RDS was found in all three affected members of a two-generation family that was associated with retinitis pigmentosa in the son, pattern dystrophy in the daughter and fundus flavimaculatus in the mother. In the second family, the proband with retinitis pigmentosa carried a p.Arg220Trp mutation. The mother, who was unavailable for mutation screening, had adult vitelliform macular dystrophy. No ROM1 mutations were found in those with retinitis pigmentosa in either family. CONCLUSION: Mutations in RDS can be associated with an intrafamilial variation in retinal disease. The phenotypes range from Stargardt-like macular dystrophy to classic retinitis pigmentosa. CLINICAL RELEVANCE: Intrafamilial phenotypic variation may be due to the presence of environmental or genetic modifying factors. The presence of a modifying-sequence change in the coding region of ROM1 for two people with retinitis pigmentosa from two families with intrafamilial variation in RDS mutation phenotype has been excluded in this study.

Adult↗
PubMed2005

In vivo analysis of a genetically modified adenoviral vector targeted to human CD40 using a novel transient transgenic model.

BACKGROUND: Retargeting is necessary to overcome the limitations of adenovirus (Ad)-based gene therapy vectors. To this end, we previously constructed an adenovirus with the fiber knob domain replaced by a fibritin trimerization motif fused to the CD40 ligand (Ad5Luc.FF/CD40L). We demonstrated the utility of this fiber replacement strategy for targeting CD40 (hCD40) on human dendritic cells in vitro. The in vivo targeting capacity of this virus, however, is unknown, and there is a limited repertoire of animal models that present hCD40 at an accessible site. Therefore, a new animal model for evaluating CD40-targeted vectors is required. METHODS: We constructed a recombinant adenovirus that expresses hCD40 under transcriptional control of the flt-1 promoter (AdflthCD40). Expression of hCD40 was validated both in vitro and in transgenic mice expressing the human coxsackie adenovirus receptor (hCAR mice). We then evaluated the targeting efficiency of Ad5Luc.FF/CD40L to hCD40 expressed in the pulmonary vasculature of the hCAR mice. RESULTS: Infection of flt-1-positive cells with AdflthCD40 resulted in abundant hCD40 expression in vitro, which could subsequently be targeted by Ad5Luc.FF/CD40L. In vivo administration of AdflthCD40 to hCAR mice resulted in hCD40 expression in the pulmonary vasculature, which was successfully targeted with systemically administered Ad5Luc.FF/CD40L. CONCLUSIONS: This is the first data showing that genetically modified Ad5Luc.FF/CD40L can successfully target hCD40 in vivo. Our data also establishes the utility of transcriptionally targeted, Ad-mediated transient expression of human target molecules in the pulmonary vasculature of hCAR mice as models for in vivo analysis of targeted gene therapy vectors.

Animals↗
PubMed2007

New mortalin and histidyl tRNA synthetase isoforms point out a pitfall in proteomic analysis of Egr1 genetically modified mice.

Egr1 (Zif268) is an immediate early gene encoding an inducible transcription factor involved in synaptic plasticity and several forms of memory in rodents. Using 2-DE and MS, we compared proteomes of hippocampal subregions and cortex in Egr1-deficient and wild-type littermates. Two significant differences were identified: a shift in the pI of the molecular chaperone mortalin (mtHsp70/PBP74/Grp75) and the apparent disappearance of histidyl tRNA synthetase (HisRS). We found that the pI shift for mortalin in Egr1-deficient mice was caused by a difference in protein sequence: D626G. Using cDNA sequencing, we demonstrated for both mortalin and HisRS that protein differences were not due to a lack of Egr1 but to DNA polymorphism between the C57Bl/6J and 129/Sv strains used to generate the Egr1-deficient mice. Our results show that mortalin and HisRS genes, which map closely to the Egr1 locus, have conserved the 129/Sv haplotype despite numerous back-crossing of the null mice progeny with C57Bl/6J animals. This demonstrates that allelic differences between mouse strains can introduce variations in differential proteomic analyses of genetically modified organisms. Finally, we report the identification of new isoforms of HisRS and mortalin (mot-3) encoded by the 129/Sv haplotype.

Amino Acid Sequence↗
PubMed2001

Roles of macrophages in measles virus infection of genetically modified mice.

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.

Animals↗
PubMed2005

Cardiac and intestinal natriuretic peptides: insights from genetically modified mice.

Since the original discovery of atrial natriuretic peptide (ANP) more than two decades ago, the application of gene targeting technology in mice has provided new insights into the diverse physiological functions of natriuretic peptides and their membrane guanylyl cyclase (GC) receptors. Disruption of the genes for ANP or its receptor, GC-A, demonstrated that this system is not only essential for the maintenance of normal blood pressure and volume, but in addition exerts local antihypertrophic effects in the heart. Disruption of the genes encoding B-type (BNP) or C-type natriuretic peptides (CNP) or the CNP-receptor, GC-B, demonstrated that these "natriuretic" peptides are in fact unlikely to physiologically regulate renal sodium excretion but instead exert important autocrine/paracrine cGMP-mediated effects on cellular proliferation and differentiation in various tissues. Notably, the intestinal peptide uroguanylin, which activates a third guanylyl cyclase receptor (GC-C), exerts diuretic/natriuretic activity and links the intestine and kidney in an endocrine way to modulate renal function in response to oral salt load. Reviewed here is the physiology of cardiac and intestinal natriuretic peptides and their guanylyl cyclase receptors, with special focus on the information gained to date from genetically modified mice.

Animals↗
PubMed2001

Genetically modified dendritic cells in cancer therapy: implications for transfusion medicine.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) identified in various tissues, including the skin (Langerhans cells), lymph nodes (interdigitating and follicular DCs), spleen, and thymus. Properties of DCs include the ability to (1) capture, process, and present foreign antigens; (2) migrate to lymphoid-rich tissue; and (3) stimulate innate and adaptive antigen-specific immune responses. Until recently, the ability to study DCs has been limited by their absence in most culture systems. It is now known that specific cytokines can be used to expand DCs to numbers sufficient for their in vitro evaluation and for their use in human immunotherapy trials. Human DCs can be derived from hematopoietic progenitors (CD34+-derived DCs) or from adherent peripheral blood monocytes (monocyte-derived DCs). Cultured DCs can be recognized by a typical veiled morphologic appearance and expression of surface markers that include major histocompatibility complex class II, CD86/B7.2, CD80/B7.1, CD83, and CD1a. DCs are susceptible to a variety of gene transfer protocols, which can be used to enhance biological function in vivo. Transduction of DCs with genes for defined tumor antigens results in sustained protein expression and presentation of multiple tumor peptides to host T cells. Alternatively, DCs may be transduced with genes for chemokines or immunostimulatory cytokines. Although the combination of ex vivo DC expansion and gene transfer is relatively new, preliminary studies suggest that injection of genetically modified autologous DCs may be capable of generating anti-tumor immune responses in patients with cancer. Preclinical animal studies showing potent antigen-specific tumor immunity after DC-based vaccination support this hypothesis and provide rationale to further evaluate this approach in patients. Preliminary human studies are now required to evaluate optimal DC dose, schedule of vaccination, route of delivery, and maturational state of cultured cells. Initiation of these phase I/II cell therapy-based studies will occur in collaboration with hospital-based transfusion facilities. Issues relating to cell harvesting, storage, culture methodology, and administration require the collaborative efforts of basic scientists, immunologists, clinical investigators, and transfusion medicine staff to ensure strict quality control of injected cellular products. This review is intended to provide a brief overview of clinical DC-based gene transfer.

Adenoviridae↗
PubMed1999

Lumbar transplant of neurons genetically modified to secrete galanin reverse pain-like behaviors after partial sciatic nerve injury.

The use of cell lines as biologic "minipumps" to chronically deliver antinociceptive molecules such as the peptide galanin near the pain processing centers of the spinal cord after nerve injury is a newly developing technology for the treatment of neuropathic pain. The neuronal rat cell line, RN33B, derived from E13 brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat preprogalanin (GAL) cDNA and the galanin-synthesizing and -secreting cell line, 33GAL.19, was isolated [1]. The 33GAL.19 cells transfected with the GAL gene expressed immunoreactivity (ir) for the GAL protein and synthesized low levels of GAL-ir at permissive temperature (33 degrees C), when the cells were proliferating, and increased GAL-ir during terminal differentiation at non-permissive temperature (39 degrees C) in vitro. A control cell line, 33V.1, RN33B cells transfected with the pCEP4 vector alone and similarly isolated by subcloning, contained no detectible GAL-ir at either temperature in vitro. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and mechanical hyperalgesia, were evaluated in the affected hindpaw after CCI and transplants. The 33GAL.19 and 33V.1 cells transplanted in the lumbar subarachnoid space near the spinal cord one week after CCI, survived at least seven weeks on the pial surface around the spinal cord and only the 33GAL.19 cells expressed GAL-ir in vivo after transplant. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced or eliminated during the two to seven week period after grafts of 33GAL.19 cells. The maximal effect on chronic pain behaviors with the GAL grafts occurred one to three weeks after transplantation. Transplants of 33V.1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of galanin supplied by transplanted cells near the lumbar spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of transplants of genetically modified neural cell lines that are able to deliver antinociceptive molecules, such as galanin, offers a safe and novel approach to pain management.

Animals↗
PubMed2005

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses.

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.

Antigen Presentation↗
PubMed2006

Genetically modified live attenuated parasites as vaccines for leishmaniasis.

Leishmaniasis causes significant morbidity and mortality worldwide and is an important public health problem. Even though it is endemic in developing countries in tropical regions of the world,in recent years economic globalization and increased travel has extended its reach to people in developed countries. Leishmania is usually spread by the bite of the female sandfly. In addition, naïve populations can be exposed to Leishmania infection through transfusion of blood and blood products from infected asymptomatic individuals. There are several clinical forms of leishmaniasis caused by different species of the parasite. In some cases, the only possible cure for this disease is drug treatment. However, prolonged use of such drugs has led to parasite drug resistance. At present there are no effective vaccines against Leishmania. Many vaccine strategies have been pursued, including the use of whole cell lysate, killed, avirulent or irradiated parasites. Additionally, DNA vaccines and purified or recombinant parasite antigens have also been tested. Most of these strategies have shown some degree of effectiveness in animal models but little or no protection in humans. There is now a general consensus among Leishmania vaccine researchers that parasite persistence may be important for effective protective response and could be achieved by live attenuated parasite immunization. In this article we reviewed the efforts in developing genetically defined live attenuated Leishmania parasites as vaccine candidates with the goal of achieving a low level of parasite persistence without being virulent in the host and inducing protective immunity.

Animals↗
PubMed2004

Differential rescue of poliovirus RNA replication functions by genetically modified RNA polymerase precursors.

We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

5' Untranslated Regions↗
PubMed2003

The genetic modifier Rpe65Leu(450): effect on light damage susceptibility in c-Fos-deficient mice.

PURPOSE: To test whether introduction of the Rpe65Leu(450) variant can overcome protection against light-induced photoreceptor apoptosis in mice without the activator protein (AP)-1 constituent c-Fos. METHODS: c-Fos-deficient mice (c-fos(-/-)) carrying the Leu(450) variant of RPE65 were compared with c-fos(-/-) mice with Rpe65Met(450). Expression of RPE65 was analyzed by Western blot analysis. Rhodopsin regeneration was determined by measuring rhodopsin after different times in darkness after bleaching. Susceptibility to light-induced damage was tested by exposure to white light and subsequent morphologic analysis. Activation of AP-1 and its complex composition was analyzed by electromobility shift assay (EMSA) and antibody interference. The contribution of AP-1 to apoptosis was tested by pharmacological inhibition of AP-1, using dexamethasone. RESULTS: Compared with RPE65Met(450), introduction of the RPE65Leu(450) variant led to increased levels of RPE65 protein, accelerated rhodopsin regeneration, loss of protection against light-induced damage, and AP-1 responsiveness to toxic light doses, despite the absence of c-Fos. c-Fos was mainly replaced by Fra-2. Application of dexamethasone restored resistance to light-induced damage. CONCLUSIONS: Increasing retinal photon catch capacity by introducing the Rpe65Leu(450) variant overcomes light damage resistance provided by c-fos deficiency. Thus, a variation of RPE65 at position 450 is a strong genetic modifier of susceptibility to light-induced damage in mice. Under conditions of high rhodopsin availability during exposure to light, Fra-2 and, to a minor degree, FosB substitute for c-Fos and enable light-induced AP-1 activity and thus photoreceptor apoptosis. Regardless of the AP-1 complex's composition, glucocorticoid receptor activation inhibits AP-1 and prevents apoptosis. Thus, not the absence of c-Fos per se, but rather impairment of AP-1 DNA binding is protective against light-induced damage. This impairment may result from the absence of c-Fos or glucocorticoid receptor-mediated transrepression.

Animals↗
PubMed1999

Effects of secretable SOD delivered by genetically modified cells on xanthine/xanthine oxidase and paraquat-induced cytotoxicity in vitro.

We designed a new eukaryotic expression vector for secretable superoxide dismutase (SOD), which expresses human SOD cDNA by fusing it to 1 connecting amino acid and the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene (IL-SOD(2) cDNA). The ILSOD(2) cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmid (pRc/CMV-ILSOD(2)). Rat lung epithelial-like cells (L2 cells) and rat skin fibroblasts (FR cells) were transfected with pRc/CMV-ILSOD(2) by lipofection. The extracellular SOD activities of IS(2)-L2 cells (L2 cells transfected with pRc/CMV-ILSOD(2)) and IS(2)-FR cells (FR cells transfected with pRc/CMV-ILSOD(2)) were 2-3 times higher than those of host cells. Initially, we investigated the protective effect of extracellular SOD secreted from these transformed cells (IS(2)-L2 and IS(2)-FR cells) on extracellular superoxide anion (xanthine/xanthine oxidase; X/XO treatment)-induced cytotoxicity in normal cells. The sensitivities of these transformed cells to X/XO-induced cytotoxicity was decreased significantly as compared with that of host cells. Although, the conditioned medium from IS(2)-L2 and IS(2)-FR cells protected against X/XO-induced cytotoxicity, the conditioned medium from host cells (L2 and FR cells) showed no significant effects on X/XO-induced cytotoxicity. Furthermore, the conditioned medium from transformed cells was more effective than that of host cells against lipid peroxidation by normal cells under conditions of oxidative stress. Second, we generated superoxide anions in the intracellular space by paraquat treatment. The transformed cells were more sensitive to paraquat-induced cytotoxicity than host cells. Following addition of catalase, the sensitivity of these genetically modified cells to paraquat became equivalent to that of host cells. These results indicated a protective effect of transfection with secretable SOD genes against extracellular superoxide anion-induced cytotoxicity although no such protective effect was observed against the intracellular cytotoxicity generated by paraquat treatment.

Animals↗