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J Nalbantoglu

Publications and source records attributed to J Nalbantoglu.

At least 37 records · Page 2Linked to original sources
PubMed1999

Intracerebral adenovirus-mediated p53 tumor suppressor gene therapy for experimental human glioma.

Malignant gliomas of astrocytic origin are good candidates for gene therapy because they have proven incurable with conventional treatments. Although mutation or inactivation of the p53 tumor suppressor gene occurs at early stages in gliomas and is associated with tumor progression, many tumors including high-grade glioblastoma multiforme carry a functionally intact p53 gene. To evaluate the effectiveness of p53-based therapy in glioma cells that contain endogenous wild-type p53, a clinically relevant model of malignant human glioma was established in athymic nu/nu mice. Intracerebral, rapidly growing tumors were produced by stereotactic injection of the human U87 MG glioma cell line that had been genetically modified for tracking purposes to express the Escherichia coli lacZ gene encoding beta-galactosidase. Overexpression of the p53 gene by adenovirus-mediated delivery into the tumor mass resulted in rapid cell death with the eradication of beta-galactosidase-expressing glioma cells through apoptosis. In long-term experiments, the survival of mice treated with the p53 adenoviral recombinant was significantly longer than that of mice that had received control adenoviral recombinant. During the observation period of 1 year, a complete cure was achieved in 27% of animals after a single injection of p53 adenoviral recombinant, and 38% of the animals were tumor free in the group receiving multiple injections of p53 adenoviral recombinant into a larger tumor mass. These experiments demonstrate that overexpression of p53 in gliomas, even in the presence of endogenous functional wildtype p53, leads to efficient elimination of tumor cells. These results point to the potential therapeutic usefulness of this approach for all astrocytic brain tumors.

Adenoviridae↗
PubMed1999

Molecular therapy for glioblastoma.

Glioblastoma (GB), the relatively frequent and most malignant form of primary brain tumor, is fatal within 1 to 2 years of onset of symptoms, despite conventional therapy. Molecular therapy promises to be an effective and possibly curative treatment. Several molecular strategies have been tested, either in animal models or clinical trials. These include: prodrug activating systems, introduction of tumor suppressor or cell-cycle-related genes, inhibition of growth factors and/or their receptors, inhibition of neovascularization, immunomodulatory maneuvers, oncolytic viruses and inhibition of matrix metalloproteinases. Of special interest for the development of optimal molecular therapy of GB, is the choice of the most efficient and least toxic gene vectors (adenovirus, retrovirus, herpes simplex virus), the route of administration of the therapeutic agent (intratumoral with or without debulking and intracarotid), avoidance of collateral damage to the perineoplastic neuropil and adequate preclinical studies. The ultimate molecular therapy will probably involve the application of multiple simultaneous (combinatorial) therapeutic modalities. The safety and efficiency of these in humans can only be judged by properly controlled therapeutic trials.

Adjuvants, Immunologic↗
PubMed1998

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Adenoviridae↗
PubMed1998

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells.

Recombinant adenovirus (AVR) promises to be an efficient vector in gene therapy for neuromuscular diseases, but in preclinical experiments the expression of therapeutic genes is shorter lived in immunocompetent animals than in immunocompromised hosts. Interferons (IFN), which are known to have a role both in early antiviral activity and in late cytotoxic immunoreaction against the virus or transduced cells, may influence the efficiency of gene transfer. In this study we investigated the role of IFNs in determining the efficiency of gene transfer by AVR. AVRs expressing beta-galactosidase (beta-gal) from either a cytomegalovirus (CMV) or a troponin-I promoter were used. Muscle cells were infected by AVR after exposure to various IFNs. The alphaIFN treatment significantly reduced (up to fivefold) the CMV promoter-driven gene expression in muscle cells in vitro and in immature muscles in vivo, while the least effective inhibitor was betaIFN. The decrease in gene expression by IFNs was more pronounced with the CMV-driven transgene than troponin-I promoter-driven one and was due to a decrease in transcript level. Intrinsic IFNs that are triggered by AVR administration can decrease the efficiency of gene transfer in muscle cells. Therefore the use of muscle specific promoters in AVR and/or IFN inhibitory agents will likely improve the prospects of effective gene therapy by AVR.

3T3 Cells↗
PubMed1998

Adenovirus-mediated dystrophin minigene transfer improves muscle strength in adult dystrophic (MDX) mice.

Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx) are both due to absence of the subsarcolemmal protein dystrophin. Recombinant adenovirus vectors (AdV) are considered a promising means for delivering a functional dystrophin gene to muscle. However, the usefulness of AdV for this purpose is limited by vector toxicity as well as immune-mediated elimination of infected fibers. In addition, studies to date of AdV-mediated dystrophin gene transfer have either failed to examine effects on muscle strength or been performed in immunologically immature neonatal animals with little baseline abnormality of force-generating capacity. In the present study, AdV-mediated dystrophin gene transfer was performed in adult mdx mice with pre-existent dystrophic pathology and muscle weakness. The main findings are as follows: (1) acute myofiber toxicity and gene transfer efficiency are both AdV dose-dependent, such that the therapeutic margin of safety is fairly narrow; (2) immunosuppressive therapy (FK506) prevents immune-mediated elimination of dystrophin-positive fibers but not the dose-dependent toxic effects; (3) at the optimal vector dosage and with effective immunosuppression, AdV-mediated dystrophin minigene transfer is capable of alleviating the loss of force-generating capacity as well as histopathological evidence of disease progression normally seen in adult mdx muscles over a 2-month period. These findings have important implications for the eventual application of AdV-mediated dystrophin gene transfer in DMD patients.

Adenoviridae↗
PubMed1998

Combinatorial blockade of calcineurin and CD28 signaling facilitates primary and secondary therapeutic gene transfer by adenovirus vectors in dystrophic (mdx) mouse muscles.

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.

Adenoviridae↗
PubMed1998

Differential role for protein kinase C-mediated signaling in the proliferation of medulloblastoma cell lines.

Recent studies have implicated protein kinase C (PKC)-mediated signaling in the proliferation of gliomas. In this study, we have investigated the role of PKC mediated signaling in the proliferation of medulloblastoma cell lines DAOY, D283-Med and D341-Med. By Western blot analyses, conventional PKC (cPKC) alpha was detectable in DAOY only, while atypical PKC (aPKC) zeta was present in all three cell lines. cPKC beta1, beta11, gamma novel PKC (nPKC) delta, and epsilon were not detectable in any of the cell lines. Antisense oligonucleotides to PKC alpha , Calphostin C (a specific PKC inhibitor) and prolonged treatment with phorbol 12-myristate 13-acetate (PMA) with down regulation of cPKCalpha caused a decrease in proliferation in DAOY and no effect on D283-Med. Furthermore, PMA treatment was also associated with upregulation of p21cip1 in DAOY. Since cPKCalpha is the only PMA responsive isoform in DAOY, this observation implicates the cPKCalpha isoform in the proliferation of DAOY but not in D283-Med. A comparison of DAOY and D283-Med showed a higher proliferation index in DAOY. In contrast, multiprobe riboquant ribonuclease protection assay revealed higher levels of p27kip1 and p21cip1 mRNA in D283-Med. These transcripts were barely detectable in untreated DAOY. These observations indicate possible significant molecular heterogeneity among medulloblastomas with implications for differing biology among medulloblastoma cell lines and tumors.

Cell Division↗
PubMed1997

Alzheimer's disease: current knowledge, management and research.

Alzheimer's disease is a common neurological condition, appearing as early as age 40 but increasing dramatically in incidence over age 85. Different genetic factors are at play, modified by events over a lifetime. Clinical diagnosis is possible through careful history taking with a reliable informant and a minimum number of laboratory tests. A relatively predictable natural history can be observed, with progression through stages of cognitive loss, functional impairment and behavioural disinhibition or apathy. New medications such as donepezil offer hope for improving or stabilizing symptoms. Such treatment can be administered by primary care physicians with experience in the diagnosis and management of Alzheimer's disease. Disease stabilization, or even prevention, may be possible in the future.

Adult↗
PubMed1997

Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin.

In situ DNA hybridization of an E4 adenoviral sequence amplified by in situ polymerase chain reaction (PCR) was used to mark adenovirus-containing myonuclei in muscles of immunocompetent and immunosuppressed mdx mice following intramuscular injection of adenoviral recombinants. The adenoviral recombinants contained a 6.3-kb dystrophin cDNA (minigene) driven by a cytomegalovirus (CMV) promoter/enhancer and thus, immunostaining for dystrophin of the same sections permitted correlation of adenoviral recombinant-containing myonuclei with dystrophin positivity of the same muscle fiber segments. As early as 2 hr post-injection of adenoviral recombinant, an appreciable number of adenoviral recombinant-positive (AVR+) myonuclei, and some partial dystrophin positive (pdys+) fibers were observed. Some fully dystrophin-positive (dys+) muscle fibers were present as early as 6 hr. The maximum number of fibers containing AVR+ myonuclei (observed by 72 hr) was maintained until 60 days in immunosuppressed, but not in immunocompetent, animals. In immunocompetent animals, the maximum number of dys+ fibers was observed at 10 days. The vast majority of these fibers contained AVR+ myonuclei; however, by 60 days, dys+ fibers disappeared with some AVR+ myonuclei persisting. Our studies suggest that widespread delayed inactivation of the dystrophin expression cassette is probably unlikely. Thus, optimization of immunosuppression could assure successful long-term dystrophin gene transfer for gene therapy.

Adenoviridae↗
PubMed1997

The immunosuppressant FK506 prolongs transgene expression in brain following adenovirus-mediated gene transfer.

First generation, replication-defective adenoviral vectors are highly effective for gene transfer into the central nervous system, but the host's immune response limits the utility of this vector for possible therapy of neurological disease or long-term gene transfer studies in experimental animals. We have demonstrated the effectiveness of FK506 (tacrolimus), a powerful immunosuppressant that readily crosses the blood-brain barrier, in maintaining adenovirus-mediated reporter gene transfer following stereotaxic injection of the recombinant (AdCMVlacZ) into mouse striatum. After 28 days, beta-galactosidase expression was reduced by 75% relative to day 10 in immunocompetent animals, accompanied by an inflammatory reaction in the region of transduced cells; however, in mice receiving daily s.c. injections of FK506, beta-galactosidase activity was maintained at the 10 days post-injection level.

Adenoviridae↗
PubMed1997

Impaired learning and LTP in mice expressing the carboxy terminus of the Alzheimer amyloid precursor protein.

Proteolytic processing of amyloid precursor protein (APP) through an endosomal/lysosomal pathway generates carboxy-terminal polypeptides that contain an intact beta-amyloid domain. Cleavage by as-yet unidentified proteases releases the beta-amyloid peptide in soluble form. In Alzheimer's disease, aggregated beta-amyloid is deposited in extracellular neuritic plaques. Although most of the molecular mechanisms involving beta-amyloid and APP in the aetiology of Alzheimer's disease are still unclear, changes in APP metabolism may be important in the pathogenesis of the disease. Here we show that transgenic mice expressing the amyloidogenic carboxy-terminal 104 amino acids of APP develop, with ageing, extracellular beta-amyloid immunoreactivity, increased gliosis and microglial reactivity, as well as cell loss in the CA1 region of the hippocampus. Adult transgenic mice demonstrate spatial-learning deficits in the Morris water maze and in maintenance of long-term potentiation (LTP). Our results indicate that alterations in the processing of APP may have considerable physiological effects on synaptic plasticity.

Aging↗
PubMed1997

Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.

Adenoviridae↗
PubMed1997

Enhanced expression of amyloid precursor protein in response to dibutyryl cyclic AMP is not mediated by the transcription factor AP-2.

The gene for amyloid precursor protein (APP) is expressed almost ubiquitously, with high levels of mRNA being detected in brain. The basal expression level of the APP gene can be modulated by physiological stimuli, and in this report we demonstrate that the second messenger cyclic AMP can regulate APP mRNA through transcriptional mechanisms. Northern blot analysis showed a 1.8-fold increase in steady-state levels of APP mRNA when the neuroblastoma x glioma hybrid cell line NG108-15 was treated with dibutyryl cyclic AMP. Although the upstream sequences of the APP gene do not contain a canonical cyclic AMP response element, transient transfection assays in NG108-15 cells using different portions of the APP promoter showed an increase in reporter gene activity mediated by sequences located between -303 to -204 and -488 to -2991. Cotransfection assays carried out in HepG2 cells with AP-2, a cyclic AMP-regulated transcription factor, failed to activate the APP promoter through the AP-2 consensus sequence (GCCNNNCGG) located at position -205. Electrophoretic mobility shift analysis revealed that the AP-2 binding activity present in HeLa nuclear extracts fails to recognize the APP AP-2 consensus sequence. We conclude that increases in cyclic AMP levels can lead to an up-regulation of APP gene transcription through at least two different regions of the APP promoter. This increase does not involve the AP-2 consensus sequence present in the APP promoter located at position -205, and, moreover, this putative site is not recognized by the transcription factor AP-2.

Amyloid beta-Protein Precursor↗
PubMed1997

Transcriptional regulation of amyloid precursor protein during dibutyryl cyclic AMP-induced differentiation of NG108-15 cells.

Early expression of amyloid precursor protein (APP) during development of the nervous system suggests that this protein may play an important role first in axogenesis and later in synaptogenesis. To study regulation of APP mRNA expression in neuronal cells, NG108-15 neuroblastoma x glioma cells were induced to differentiate in the presence of dibutyryl cyclic AMP. Steady-state levels of APP mRNA and APP isoforms increased gradually, concomitantly with the appearance of differentiated phenotype. Northern blot analysis showed a three-fold increase in APP expression at day 6 of dibutyryl cyclic AMP treatment. Nuclear run-on assays and transient transfections performed using APP promoter/reporter constructs confirmed a twofold increase in the rate of APP gene transcription. The stability of the mRNA was unchanged, with differentiated and nondifferentiated cells having the same half-life of about 21 h. These results strongly suggest that APP mRNA induction in the differentiated NG108-15 cells is due to an increase in the rate of transcription of the gene.

Amyloid beta-Protein Precursor↗
PubMed1997

Adenovirus-mediated wild-type p53 gene transfer and overexpression induces apoptosis of human glioma cells independent of endogenous p53 status.

Mutation or inactivation of the p53 tumor suppression gene is an early alteration in the transformation of glial cells to gliomas. To study the effect of exogenous wild-type p53 on glioma cell growth, human glioma lines U251 MG, U87 MG and A172 were infected with an adenovirus vector expressing either wild-type p53 or bacterial lacZ. Rapid cell death occurred only in the p53-transduced cell lines and was characterized by nuclear condensation, formation of nucleosomal DNA ladders, and positive in situ end-labeling of DNA, suggesting that apoptosis had been induced. The U87 MG cell line that contains wild-type p53 as evidenced by wild-type p53-dependent transcription activity also underwent apoptosis within 2 to 3 days after infection. These results suggest that the presence of endogenous wild-type p53 does not preclude apoptosis by overexpression of exogenous p53.

Adenoviridae↗
PubMed1997

Gene therapy research for Duchenne and Becker muscular dystrophies.

Gene therapy is a promising option for the definitive treatment of Duchenne and Becker muscular dystrophies. Presently, gene therapy for Duchenne and Becker muscular dystrophies is still in the preclinical stage with dystrophin-deficient animals (the mdx mouse and a golden retriever dog strain) serving as convenient models. The thrust of research during the past 18 months has focused on two approaches: adenovirus-mediated dystrophin gene transfer and upregulation of a natural dystrophin analogue, utrophin. In the area of adenovirus-mediated gene transfer, substantial progress has been made in characterizing and mitigating the deleterious immune responses to the vector and transgene proteins. Furthermore, new adenovirus vectors have been created with reduced immunogenicity and increased insert gene capacity, which enhance the longevity of the transgene expression. Additional efforts are underway to develop safe and efficient routes of administration of the adenovirus vector carrying the dystrophin expression cassette. The prospects of utrophin upregulation as an attractive strategy for treatment of Duchenne and Becker muscular dystrophies was greatly enhanced by the demonstration of a substantial mitigation of the dystrophic phenotype of the transgenic mdx mouse overexpressing utrophin.

Animals↗