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Volume 7 - 2002


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1-13

Dynamic histone acetylation and its involvement in transcription

Author(s): Dr. James R. Davie,

Abstract: Histones are subject to a variety of posttranslational modifications, the most studied being acetylation of the N terminal lysine residues. Acetylation is a dynamic event mediated by the actions of histone acetyltransferases and deacetylases. The exact function of this event in transcription has remained an enigma for several reasons. The enzymes that catalyze this dynamic event act on histones, but are also capable of affecting the properties of non- histone proteins including transcription factors. Also, some histone acetyltransferases can acetylate the histones along a specific gene, while simultaneously acetylating the histones over an entire region of the genome. More confusing are the observations that the acetylation pattern of histones along one transcriptionally active gene may differ significantly from that along another. Perhaps some of these discrepancies can be explained by the dynamic interplay between histone acetyltransferases and deacetylases, and the proximity of a gene to these enzymes. Thus, to fully appreciate the role of acetylation in transcription, we must further understand the dynamic nature of this event.

Keywords: histone acetylation, histone acetyltransferases and deacetylases, transcription



15-23

Tumor therapy using radiolabelled antisense oligomers- aspects for antiangiogenetic strategy and positron emission tomography

Author(s): Dr. Kalevi JA Kairemo,

Abstract: Angiogenesis provides a putative target for radiochemotherapy as endothelial cells on vascular wall are sensitive for radiation and by destructing of one endothelial cell may lead to death hundred of tumor cells. Endothelial cells in the angiogenic vessels within solid tumors express several proteins that are absent or faintly expressing in established blood vessels, including αv integrins (Hammes, 1996) and receptors for certain angiogenic growth factors (Hanahan, 1997) (Risau, 1997). Recently, vascular endothelial cell growth factor (VEGF)-induced invasiveness has been inhibited specifically by ETS-1 antisense oligonucleotide. ETS-1 gene expression can be induced, while there are several other systems with constant expression. In this paper, we extent use of oligos from conventional biokinetic studies to therapeutic use by comparing radioactive oligos to peptide counterparts. Radiolabelled oligos have a potential of having both direct antisense inhibition and radiation effects. Previously we have shown theoretically that oligonucleotide therapy may be effective with internally labelled (P-32, P-33 and S-35) oligodeoxynucleotide phosphorothioates. This has also been demonstrated in vitro using P-33 (Kairemo et al, 1999). We investigate also the possibility of using 15-mer oligodeoxynucleotide phosphorothioates (oligos) or oligomers in which the phosphate-ribose backbone has been replaced with polyamide backbone (peptide nucleic acids). The absorbed organ doses of these radiolabelled compounds were estimated from biodistribution data. Subcellular biodistribution was used in evaluation of the best targeting inside the cell with one oligomer. Our results indicate that oligos can give significantly up to 130-fold higher absorbed organ doses in oligos than in peptides. Mainly this is due to slower biokinetics of oligos (35-fold slower half-lives). For imaging, positron emitters such as F-18 and Br-76, offer an advantage for radiopharmacokinetic studies (Wu at al., 2000). We have therefore calculated the subcellular dosimetry for these isotopes in different cell dimensions (nuclear diameter 6-16μm, cellular diameter 12-20μm).

Keywords: antisense therapy, oligonucleotides, phosphorus radioisotopes, sulphur radioisotopes, AIDS, cancer, dosimetry, positron emission tomography



25-35

Strategy of sensitizing tumor cells with adenovirus- p53 transfection

Author(s): Dr. Jekunen Antti,

Abstract: Loss or malfunction of the p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy. While it may be the most frequently mutated gene in human tumor samples, the function of p53 is critical for maintaining the integrity of the cellular genome in its responses to treatment with cytotoxic agents. Intact p53 protein in nuclei of normal cells acts as a transcriptional activator for a group of genes involved in cell cycle arrest, DNA repair and apoptosis. The transfection of adenovirus p53 (adeno-p53) alone has been shown in ovarian cancer cell culture models to inhibit cell growth and to promote apoptosis regardless of the endogenous p53 status of the cells. Both mutant p53 in the tumor cells and the loss of p53 function were associated with resistance to chemotherapeutic agents. There are various reports of at least additive interactions between adeno-p53 and several chemotherapeutic agents in a number of cancers, e.g. bladder cancer, NSCLC, prostate cancer, breast cancer, and ovarian cancer both in vitro and in vivo. The mechanisms of these interactions are unknown, but they may depend on the chemotherapeutic agents used, the targets and critical tissues, and the intracellular signal transduction pathways affected.Results obtained with a speculative treatment regimen consisting of oligonucleotide therapy and p53 transfection suggest that p53 expression in tumor cells may improve their sensitivity to routine chemotherapy, e.g. docetaxel and irinotecan, which are efficacious drugs possessing different modes of action: prevention of depolymerization of tubulin and specific DNA topoisomerase I inhibition, respectively. It is known, however, that even these new agents cannot achieve responses in all tumors, and that in some tumors the efficacy, once established, diminishes along with the treatment. In these cases of resistant tumors or recurrences and relapses, combined treatment with adeno-p53 and chemotherapeutic agents may be an attractive strategy for inhibiting the progression of local cancers. In fact, the ground is ready for a rapid practical development of adeno-p53, which itself causes only minimal side-effects after administration, e.g. injection site rashes and fever, and an immunostimulation that seems to be quite mild and transient in nature. Future cancer therapy strategies may consist of effective chemotherapy coupled to molecular medicine specifically targeting tumor cells. So far, we do not have proper means in molecular medicine for achieving high enough tumor access with any of the current systemic virus vectors having the proper level of selectivity between tumor and normal cells. We have already some clinical experience, however, with intratumoral approaches that ensure the highest possible concentrations inside NSCLC, ovarian cancer and head and neck cancer tumors. It seems that there is clear evidence of good tolerability at non-maximal doses, but unfortunately, only modest activity when the construct is used alone. We review here the published data on the use of adenovirus p53 for sensitizing tumors to chemotherapeutic agents and outline perspectives for the future.

Keywords:



37-42

Antigenicity and immunogenicity of HIV envelope gene expressed in baculovirus expression system

Author(s): Dr. Pradeep Seth,

Abstract: Human immunodeficiency virus type I (HIV-1) envelope gene was expressed in Spodoptera frugiperda (Sf21) cells. DNA constructs encoding env-tat-rev genes were cloned into the baculovirus expression vector pBacPAK9. Recombinant baculovirus was prepared by cotransfection with linearized wild type virus DNA. Western blotting of cell extracts containing recombinant HIV-1 proteins demonstrated expression of HIV-1 gp160 and its complete cleavage products gp120 and gp41. A time course experiment suggested that the maximum expression was observed at 48-hrs post infection. In order to measure the biological activity recombinant HIV envelope proteins were used for lymphocyte proliferation assay. The results demonstrated that recombinant gp160 and its cleavage products were antigenically and functionally authentic.

Keywords:



43-59

Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags

Author(s): Dr. José de la Fuente,

Abstract: Expression library immunization (ELI) combined with analysis of expressed sequence tags (ESTs) were used to identify genes transcribed in a cell line (IDE8) that was originally derived from embryos of Ixodes scapularis. A cDNA expression library was constructed from the IDE8 cells and cDNA clones were screened by ELI. Mice injected with cDNA clones were then infested with I. scapularis larvae. cDNA clones affecting larval feeding or development were subjected to single pass 5’ sequence analysis and the non-redundant sequences were putatively identified by sequence identity using the protein Basic Local Alignment Search Tool (BLAST) algorithm. Sequences of the clones were grouped according to the predicted function of the encoded proteins. 351 cDNAs that affected larval feeding and/or development were identified, of which 316 cDNA clones contained non-redundant sequences and 101 produced a significant identity to sequences reported previously. Gene ontologies could be assigned to 87 clones. Vaccination of mice with plasmid DNA followed by tick infestation resulted in identification of cDNA clones that inhibited tick infestation or promoted tick feeding. cDNAs that inhibited tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins, and proteins with unknown function. cDNA clones that promoted tick feeding were identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function. Herein, we describe the sequence analysis of I. scapularis ESTs selected by ELI that affected larval tick feeding and/or development. These proteins may be useful for incorporation into vaccine preparations designed to interrupt the life cycle of I. scapularis and/or interfere with transmission of pathogens.

Keywords: Key words: tick, vaccine, tick cell culture, cDNA library immunization, EST, expression library immunization



61-68

Delayed intratracheal injection of manganese superoxide dismutase (MnSOD)-plasmid/liposomes provides suboptimal protection against irradiation- induced pulmonary injury compared to treatment before irradiation

Author(s): Dr. Joel S. Greenberger,

Abstract: Ionizing irradiation results in cellular production of reactive oxygen species (ROS), which cause DNA strand breaks, lipid peroxidation or other cellular damage leading to cell death. Antioxidant enzymes neutralize these ROS and provide cellular protection against sources of oxidative stress including ionizing irradiation. Intratracheal injection of the transgene for antioxidant protein MnSOD in plasmid/liposome (PL) complex 24 hours before irradiation has been shown to protect the murine lung from irradiation-induced organizing alveolitis/fibrosis. To determine whether intratracheal injection of MnSOD-PL at later times of macrophage infiltration and inflammation following irradiation had a detectable protective effect against irradiation fibrosis, control non- injected or MnSOD-PL complex injected C57BL/6J mice were irradiated to 20 Gy. Subgroups received a delayed injection of MnSOD-PL at day 1, 80, 90 or 100 after irradiation and all were followed for the development of organizing alveolitis/fibrosis. While mice injected with MnSOD-PL prior to irradiation demonstrated the best level of protection, we observed that mice injected with MnSOD-PL at 80 or 100 days after irradiation also showed significant protection of the lung compared to irradiated, control mice. Thus, delayed administration of MnSOD-PL has detectable radioprotective effects on C57BL/6J mouse lung but pre-irradiation injection remains the optimal treatment paradigm.

Keywords: MnSOD, reactive oxygen species, pulmonary fibrosis



69-73

Regulation of vascular endothelial growth factor by hypoxia

Author(s): Dr. Ilana Goldberg-Cohen,

Abstract: The past few decades have singled out the growth of new blood vessels, termed angiogenesis, as a key process in the course of normal development as well as in pathological disease processes. VEGF, an endothelial cell specific mitogen, is now accepted as a key mediator of angiogenic events and as such may be a powerful tool in manipulating the growth of new blood vessels. VEGF expression is regulated to a great extent by hypoxia. The lack of oxygen to supply a tissue triggers several molecular mechanisms that increase VEGF mRNA transcription, stability and translation, and thus upregulate the expression of VEGF protein. This review focuses on the increase in VEGF mRNA stability through its recognition by the RNA binding protein HuR. Binding of HuR to its cognate site on the 3 ́UTR of VEGF mRNA results in a several fold increase in VEGF mRNA stability, possibly due to the masking of a nearby binding site for ribonucleases. Mastering the regulatory mechanisms of VEGF expression is of great importance for the future manipulation of VEGF and angiogenesis in the disease setting.

Keywords: VEGF (vascular endothelial growth factor), hypoxia, HuR



75-89

Gene therapy antiproliferative strategies against cardiovascular disease

Author(s): Dr. Vicente Andrés,

Abstract: Excessive cellular proliferation is thought to contribute to the pathogenesis of several forms of cardiovascular disease (e. g., atherosclerosis, restenosis after angioplasty, and vessel bypass graft failure). Therefore, candidate targets for the treatment of these disorders include cell cycle regulatory factors, such as cyclin-dependent kinases (CDKs), cyclins, CDK inhibitory proteins (CKIs), tumor suppressors, growth factors and their receptors, and transcription factors. Importantly, animal models of atherosclerosis have demonstrated an inverse correlation between neointimal cell proliferation and atheroma size, suggesting that excessive cell growth prevails at the onset of atherogenesis. Cell growth may also predominate at the onset of human atherosclerosis. Thus, given that affected humans often exhibit advanced atherosclerotic plaques when first diagnosed, the potential benefit of antiproliferative strategies for the treatment of atherosclerosis in clinic is doubtful. The antiproliferative approaches used so far in the setting of vascular obstructive disease have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is spatially localized and develops over a short period of time (typically 2-12 months). Vascular interventions, both endovascular and open surgical, allow minimally invasive, easily monitored gene delivery. Thus, gene therapy strategies are emerging as an attractive approach for the treatment of vascular proliferative disease. In this review, we will discuss the use of gene therapy strategies against cellular proliferation in animal models and clinical trials of cardiovascular disease.

Keywords: atherosclerosis, restenosis, bypass graft failure, cell cycle, gene therapy



91-97

Regulation of the Sp/KLF-family of transcription factors: focus on post-transcriptional modification and protein-protein interaction in the context of chromatin

Author(s): Dr. Toru Suzuki,

Abstract: The Sp1- and Krüppel-like zinc finger transcription factor family is a rapidly expanding and highlighted group of factors given important biological roles. Understanding specific regulation is important to dissect individual functions. In this collective review, the regulation of this family of transcription factors with a particular focus on post-transcriptional modification and protein-protein interaction in the context of chromatin will be discussed. Studies by ourselves and others show that the zinc finger DNA-binding domain region of these factors mediates important regulatory interactions and modifications which may explain at least in part their specific regulation. Their possible implications in gene therapy are discussed.

Keywords: transcription factors, gene regulation, chromatin, Sp1, acetyltransferase, nucleosome remodeling



99-102

Detection of MET oncogene amplification in hepatocellular carcinomas by comparative genomic hybridization on microarrays

Author(s): Dr. Nagy A. Habib,

Abstract: The oncogene MET localized on human chromosome 7q21-31 encodes a transmembrane protein with tyrosine kinase activity and is believed to be implicated in progression of colorectal cancer. The aims of the study were to determine whether overexpression and amplification of the MET oncogene confers a selective growth advantage to hepatocellular carcinomas. Comparative genomic hybridization on microarrays was used in the analysis of DNA from 32 liver tumors (6 hepatocellular carcinoma; 16 colorectal liver metastases; 3 cholangiocarcinomas; 2 adenomas; 2 fibrolamellar; 3 unclassified) to screen for sequence copy number changes. The results revealed a MET gene amplification in hepatocellular carcinoma, cholangiocarcinoma, and colorectal liver metastases tumors. Moreover, one of the patients with hepatocellular carcinoma showed MET amplifications in both tumor and non- tumor samples, with the tumor having approximately 12.8 copies of the MET target locus per cell. These findings suggest that amplifications in the MET gene may play an important role in hepatocarcinogenesis.

Keywords: MET oncogene, amplification, hepatocellular carcinoma, microarrays, comparative genomic hybridization



103-111

HMG-CoA-reductase inhibition-dependent and - independent effects of statins on leukocyte adhesion

Author(s): Dr. Dr. T. Chavakis,

Abstract: Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, a key enzyme for cholesterol biosynthesis and isoprenoid intermediates. Increasing evidence suggests that statins might affect inflammatory processes including leukocyte recruitment, yet, the underlying mechanisms are not defined. In this study two different pathways for inhibition of leukocyte adhesion by statins are described. (i) Coincubation with lovastatin inhibited adhesion of LFA-1 (CD11a/CD18, αLβ2)-transfected K562 cells to ICAM-1 and of p150.95 (CD11c/CD18, αXβ2)-transfected K562 cells to both ICAM-1 and fibrinogen (FBG), whereas adhesion of Mac-1 (CD11b/CD18, αMβ2)-transfected K562 cells was not affected. Moreover, only LFA-1-mediated adhesion to ICAM- 1 but not Mac-1-mediated adhesion to FBG or urokinase-receptor (uPAR)-mediated adhesion to vitronectin (VN) of myelo-monocytic U937 cells was blocked by coincubation with lovastatin. The antiadhesive effect of lovastatin was independent of HMG-CoA-reductase inhibition, as it was not reversible in the presence of mevalonate, farnesyl- pyrophosphate or geranyl-pyrophosphate. In purified systems, lovastatin only blocked the ICAM-1/LFA-1 interaction but not the ICAM-1/Mac-1, FBG/Mac-1 or the VN/uPAR interactions. (ii) In contrast, preincubation of U937 cells for up to 18 h with lovastatin completely abrogated LFA-1-, Mac-1- and uPAR-dependent cell adhesion to the respective ligands. This anti-adhesive function of lovastatin was dependent on HMG-CoA reductase inhibition, since mevalonate or the isoprenoid intermediates restored adhesion, while no downregulation of integrin- or uPAR-expression was observed. Thus, two distinct pathways, involving a direct interaction with LFA-1 and p150.95 and an indirect inhibition of cell adhesion through disruption of cholesterol and/or isoprenoid metabolite biosynthesis are induced by statins. These functions can explain at least in part the inhibition of leukocyte adhesion and the associated antiinflammatory role of statins

Keywords: leukocyte, adhesion, β2-integrins, urokinase-receptor, statins, lovastatin, HMG-CoA reductase



113-133

Current progress in adenovirus mediated gene therapy for patients with prostate carcinoma

Author(s): Dr. Salih Sanlioglu,

Abstract: Prostate cancer is the most frequently diagnosed male cancer in the world. Like all cancers, prostate cancer is a disease of uncontrolled cell growth. In some cases tumors are slow growing and remain local, but in others they may spread rapidly to the lymph nodes, other organs and especially bone. Although surgery and radiation can cure early stages of organ confined prostate carcinoma (stages I and II), there is no curative therapy at this time for locally advanced or metastatic disease (stages III and IV). The likelihood of postsurgical local recurrence increases with capsular penetration as detected in 30 % of the patients at the time of radical prostatectomy. Moreover, 10-15 % of patients have metastatic cancer at the time of diagnosis. Considering the fact that 60 % local recurrence is observed in patients receiving radiation therapy with or without adjuvant hormonal ablation therapy, it is generally believed that androgen ablation therapy simply delays the progression of prostate carcinoma to a more advanced stage. In addition, the overall ten-year survival rate of patients with locally recurrent prostate cancer is only around 35 %; thus; the ultimate progression into androgen independent prostate carcinoma appears to be inevitable. Gene therapy arose as a novel treatment modality with the potential to decrease the morbidity associated with conventional therapies. Therefore, gene therapy is expected to lower the incidence of tumor recurrence and finally improve the outcome of patients with recurrent and androgen independent prostate carcinoma. Viral vectors are most commonly used for the purpose of gene therapy. Currently, there are a total of 40 clinical trials being conducted using viral vectors for the treatment of prostate carcinoma. 22 out of 40 clinical protocols (55 %) approved for the treatment of prostate cancer utilize adenovirus vectors. Most of these adenovirus mediated therapeutic approaches employ either selectively replicating adenoviruses or suicide gene therapy approaches. In this review, we mainly concentrated on the progress in adenovirus mediated gene therapy approaches for prostate cancer. Analysis of the death ligand mediated gene therapy approach was also discussed in detail, while our novel findings were incorporated as an example for up-to-date approaches used for adenovirus mediated gene therapy against prostate carcinoma.

Keywords: Prostate cancer, adenovirus, gene therapy, immunomodulation, apoptosis, inducible promoters



135-151

Gene therapy for vascular diseases

Author(s): Dr. Claudio Napoli,

Abstract: Currently, successful pharmacological treatments are unavailable for many vascular diseases. Many patients undergo surgical interventions and then present with recurrence of symptoms. Recently, gene therapy using both non-viral and viral delivery has emerged as a novel tool to treat patients with vascular diseases. Here we discuss the requirement to develop suitable gene delivery vectors for vascular diseases. Our expanding knowledge of the pathogenesis of vascular diseases has allowed the identification of several gene therapy strategies and many candidate genes. Gene therapy using both gene knockout and gene overexpression has been considered. In pre- clinical studies, antisense and decoy oligonucleotides have been successfully employed to knockout the expression of stimulatory genes such as cell cycle promoters and growth factors. Furthermore, overexpression of inhibitory genes such as cell cycle inhibitors and nitric oxide and overexpression of genes to promote therapeutic angiogenesis have been shown potential in animal models. The progress of pre-clinical studies to treat vein graft failure, restenosis, myocardial and peripheral ischemia and hypertension and the development of clinical trials will be discussed. Despite the quite promising findings with clinical trials, particularly with therapeutic angiogenesis, improved gene transfer vectors and methods for safe long-term gene transfer are still required to bring gene therapy to clinical practice.

Keywords: Atherosclerosis, gene therapy, adenoviruses, vascular diseases.



153-165

Angiogenic gene therapy for improving islet graft vascularization

Author(s): Dr. Hengjiang Dong,

Abstract: Clinical islet transplantation is considered a curative treatment for type 1 diabetes, but long-term survival and function of implanted islets is greatly compromised by a number of adverse events. In addition to immune rejection and recurrent autoimmunity, the survival and function of islets is determined by the rate and degree of islet revascularization, an essential process termed angiogenesis that is required for the development of new vessels within islet grafts to derive blood from the host vasculature. Rapid and adequate revascularization is crucial for islet survival and function. Delay in islet revascularization can deprive islets of oxygen and nutrients, resulting in islet cell death and early graft failure. There is evidence that despite the infusion of sufficiently large amounts of islets (~11,000 islets/kg body weight) per diabetic recipient, less than 30% of islet mass becomes stably engrafted post transplantation. In this article, we will review the molecular basis of islet revascularization and highlight the importance of developing novel therapeutic strategies to stimulate angiogenesis within islet grafts and enhance islet graft vascularization post transplantation. Such strategies, when applied in conjunction with islet transplantation, are expected to improve the viability of transplanted islets and provide long-term survival of functional islet mass post transplantation, thereby increasing the overall success rate of islet transplantation.

Keywords: Type 1 diabetes, islet transplantation, islet revascularization, VEGF, gene transfer.



167-177

G-CSF Receptor-mediated STAT3 activation and granulocyte differentiation in 32D cells

Author(s): Dr. Akihiro Kume,

Abstract: Granulocyte colony-stimulating factor (G-CSF) receptor (GcR) mediates growth and differentiation signals in the granulocyte/monocyte lineage of hematopoietic cells. To investigate the differentiation signal via GcR, a conditional receptor activation system was constructed. Wild-type and mutant GcRs were controlled by fusion to a molecular switch derived from the hormone binding domain of the estrogen receptor (ER). GcR-associated signaling molecules were analyzed in 32D progenitor cells that possess a potential of granulocyte differentiation. While the wild-type GcR-ER fusion molecule induced a granulocyte differentiation in 32D cells, a substitution of phenylalanine for tyrosine 703 (Y703F) in GcR resulted in a differentiation block. The activation of the JAK1 and JAK2 kinases was indistinguishable between the cells expressing the wild-type fusion and the Y703F mutant, and phosphorylation of the STAT5 transcription factor was comparable, too. On the other hand, tyrosine phosphorylation of STAT3 was significantly decreased following activation of the Y703F mutant compared to the wild-type GcR fusion. The results suggested that tyrosine 703 was responsible, at least in part, for transmitting a differentiation signal via STAT3 in 32D. The fusion system with the estrogen binding domain provides a valuable tool to analyze mutant effector proteins in the natural cellular milieu while bypassing the endogenous counterparts.

Keywords: STAT3, G-CSF receptor, granulocyte differentiation, estrogen binding domain, selective amplifier gene



173-179

Calcium induces apoptosis and necrosis in hematopoetic malignant cells: Evidence for caspase- 8 dependent and FADD-autonomous pathway

Author(s): Dr. Marek Los,

Abstract: One of the killing mechanisms employed by Natural Killer (NK) cells and Lymphokine-Activated Killer (LAK) cells is the perforation of the cellular membrane that causes the increase of cytoplasmic calcium concentration and disturbs further the homeostasis of other ions. Cytoplasmic calcium influx, exceeding the tolerated physiologic threshold in cell signaling events, can induce either apoptosis or necrosis depending on its final concentration. Despite several years of intensive research and identification of some molecular targets of action like e.g. calpains, calcineurin or calreticulin, the exact mechanism of calcium-induced cell death is not known in detail. We show here that death pathways triggered by calcium rely on a novel, caspase-8-dependent and Bcl-2-inhibitable pathway that is FADD-adaptor molecule -independent. This is shown in a leukemic cell model. The experimental system employs either cells that lack the expression of casapase-8 or cells genetically modified to overexpress, Bcl-2, or a FADD- dominant negative mutant (FADD-DN).

Keywords: A23187, apoptosis, Bcl-2, caspase-8, FADD, necrosis



181-209

The current status and future direction of fetal gene therapy

Author(s): Dr. A.L. David,

Abstract: Application of gene therapy in utero has been considered as a strategy for treatment or even prevention of early onset genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy. Prenatal gene transfer may target rapidly expanding stem cell populations that are inaccessible after birth, permit induction of immune tolerance against vector and transgene and allow permanent gene transfer by use of integrating vector systems. Application of this therapy in the fetus must be safe, reliable and cost-effective. Recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. Prenatal studies in animal models are being pursued in parallel with adult studies of gene therapy, but they remain presently at the experimental stage.

Keywords: fetal gene therapy; adenovirus; retrovirus; lentivirus; adeno-associated virus; Sendai virus; liposome



211-219

The role of EBV and genomic sequences in gene expression from extrachromosomal gene therapy vectors in mouse liver

Author(s): Dr. Michele P. Calos,

Abstract: A plasmid vector containing Epstein-Barr virus (EBV) sequences and the full genomic SERPINA1 locus encoding the gene for α1-antitrypsin is capable of providing long-term, high-level expression when transfected into mouse liver. It was unclear which viral and genomic sequences were required for efficient expression of this transgene in vivo. We tested here the requirement for EBV sequences for retention and expression of plasmid DNA in normal and replicating liver in vivo. The results showed that EBV sequences provided increased retention and expression of an extrachromosomal vector containing the full SERPINA1 transgene, in addition to the expression provided by the full gene alone. We also minimized the SERPINA1 sequence and determined which portions were necessary for persistent, high expression levels. Finally, we demonstrated that the SERPINA1 sequence can act to enhance expression of a heterologous gene cloned within it. Expression from a factor IX minigene was increased ~50-fold when it was expressed from within the SERPINA1 sequence, compared to a vector containing the factor IX minigene alone. The results presented here demonstrate that a significant amount of genomic sequence may be required for persistent, high levels of expression in vivo and that the persistence of plasmid DNA in dividing tissues and expression levels are enhanced by inclusion of EBV sequences on the vector.

Keywords: Epstein-Barr virus (EBV); extrachromosomal gene therapy, SERPINA1 sequence, α1-antitrypsin (AAT)



221-228

Site-specific kidney-targeted plasmid DNA transfer using nonviral techniques

Author(s): Dr. Hiroki Maruyama,

Abstract: Kidney-targeted gene transfer has the potential to be one of the most important tools for broadening our understanding of renal disease processes and for revolutionizing the treatment of renal diseases. We reviewed the literature on kidney-targeted plasmid DNA transfer using nonviral techniques: naked plasmid DNA, cationic lipid/DNA complex, hemagglutinating virus of Japan (HVJ)-liposome complex, anionic artificial viral envelope-type HVJ-liposome complex, cationic polymer/DNA complex, electroporation, ultrasound-microbubble, and hydrodynamics-based transfection. Gene-transfer methods using nonviral techniques are administered via renal arterial, renal venous, pelvic, ureteric, or direct routes into the glomerulus, tubules, or interstitial fibroblasts. Gene transfer can be achieved with varying degrees of transfection efficiency and duration of gene expression. Thus, we can select the most effective gene transfer technique to deliver the appropriate therapeutic gene to the particular sites involved in various renal diseases

Keywords: renal artery, renal vein, ureter, pelvis, glomerulus, tubules, interstitium, lipoplex, polyplex, hydrodynamic-based transfection, electroporation, ultrasound-microbubble



229-238

Hepatocyte-targeted delivery of Sleeping Beauty mediates efficient gene transfer in vivo

Author(s): Dr. Clifford J. Steer,

Abstract: Currently, most gene therapy studies utilize viral vectors that can potentially produce immunological and toxic side effects. To circumvent these limitations, we evaluated the efficiency of nonviral hepatocyte-targeted in vivo delivery of plasmids that mediate stable genomic integration of transgenes via the Sleeping Beauty (SB) transposon system. We constructed plasmids that express a reporter green fluorescent protein (GFP) transposon and the SB transposase, required for transgene insertion into genomic DNA, from either a single plasmid (cis) or two different plasmids (trans). The constructs were compacted to an average diameter of < 50 nm with lactosylated polyethyleneimine, a polycation, for targeting to the hepatocyte asialoglycoprotein receptor. Intravenous administration of the cis plasmid resulted in greater efficiency of transgene integration in mouse liver compared to transposase expression from a separate plasmid. Furthermore, by western blot analysis and fluorescence microscopy, delivery of the cis plasmid to rat livers resulted in transgene expression that persisted for months even after regeneration from partial hepatectomy. Southern blot analysis of the regenerated livers indicated that SB mediated genomic integration of the GFP transgene at random sites, and this correlated with disappearance of SB transposase. In conclusion, receptor-mediated targeted delivery of a transposon system capable of transgene integration and stable expression provides an attractive alternative to viral vectors for gene therapy to the liver.

Keywords: asialoglycoprotein receptor, gene therapy, genomic integration, polyethyleneimine, Sleeping Beauty transposon



239-243

PRL-3 as a target for cancer therapy

Author(s): Dr. Zeng Qi,

Abstract: A group of protein tyrosine phosphatases (PTPs), the PRL family, has been implicated in the contribution and progression of cancer metastasis. The PRL family consists of three members: PRL-1, -2 and -3. This small (20kD) class of prenylated protein tyrosine phosphatase contains a PTP signature motif (VHCXAGXXR) at their active sites and a catalytic domain, similar to dual specificity phosphatases. The three closely related PRLs share 76-87% identities in their amino acid sequences, with a unique C-terminal prenylation motif and with significant sequence homology to Cdc14p, a mitotic regulator, and PTEN/MMAC1, the tumor suppressor (Zeng et al, 1998a). PRL-1 was identified as an immediate-early gene which was induced in mitogen-stimulated cells and regenerating liver. PRL-3, along with PRL-2, was identified subsequently by using PRL-1 sequence to search mouse EST database. Recently, PRLs have been implicated in the process of oncogenic transformation and cancer metastasis. We suggest that PRLs might be important modulators in the process of cancer metastasis and are therefore potential targets for therapeutic intervention of cancer.

Keywords: PRL-1, -2 and -3, prenylated phosphatase, cancer metastatasis, phosphatase inhibitor, therapeutic target and cancer therapy



245-254

Protective effect of heat shock proteins: potential for gene therapy

Author(s): Dr. David S. Latchman,

Abstract: The heat shock proteins (hsps) are expressed in normal cells but their expression is enhanced by a number of different stresses including heat and ischaemia. They play important roles in chaperoning the folding of other proteins and in protein degradation. In the heart and the brain, a number of studies have shown that prior induction of the hsps by a mild stress has a protective effect against a more severe stress. Moreover, over-expression of an individual hsp in cardiac or neuronal cells in culture and in the intact heart or brain of either transgenic animals or using virus vectors, also produces a protective effect, directly demonstrating the ability of the hsps to produce protection. These findings indicate the potential importance of developing procedures for elevating hsp expression in a safe and efficient manner in human individuals using either pharmacological or gene therapy procedures.

Keywords: heat shock proteins (hsps); Drosophila protein, gene therapy



255-272

Lung cancer gene therapy

Author(s): Dr. RA Xu,

Abstract: Lung cancer is the most lethal cancer worldwide. Although progress has been made in prevention, early detection, and treatment, mortality from this disease is still increasing. Current treatments in clinical trials have yielded only very limited results, and it is therefore necessary to develop new therapeutic strategies. Gene therapy is a novel field of medicine that may signal a more promising future for patients with lung cancer. Several studies on lung cancer therapy have held out the promise of treatment methods, including the alteration of intracellular molecular defects, the introduction of suicide genes, the inhibition of angiogenesis, and the augmentation of specific antitumor immunity. Various methods have been used to achieve specific gene transduction and effective gene expression. Clinical trials indicate that a combination of different treatment modalities is needed to obtain better results in lung cancer therapy. This review will summarize and discuss some recent advances and the potential future applications of gene therapy approaches in lung cancer.

Keywords: lung cancer, gene therapy, tumor suppressor genes, growth factor pathway targets, suicide gene therapy, angiogenesis, immunotherapy



273-289

Advances in cationic lipid-mediated gene delivery

Author(s): Dr. Pierre Lehn,

Abstract: Over previous years, problems associated with virus-mediated gene delivery have stimulated the synthesis and biological evaluation of non-viral vectors as a possible alternative for gene therapy applications. Of the various non- viral vectors, cationic lipids have come forward as effective gene delivery agents, although it is clear that their transfection efficiency must be increased in order for them to become of real therapeutic value. This can be achieved by overcoming both the intracellular and extracellular barriers they encounter while conveying the transgene towards the nucleus of the target cells. The purpose of this review is to highlight the advances made to date in facing these challenges by paying particular attention to the design of the cationic lipid itself and the complexes (termed lipoplexes) formed on interacting with DNA. Because the structures of all three parts of a cationic lipid – the cationic headgroup, the hydrophobic moiety and the connecting linker – are important determinants of transfection efficiency, each will be considered here in turn, with special attention focused on recent studies including our own work. In addition, the stability of the lipoplex in the extracellular medium and the features of its intracellular trafficking towards the cell nucleus will be assessed from both chemical and biological viewpoints. In conclusion, the future will probably see the development of sophisticated modular self-assembling gene delivery systems incorporating various functional elements to face the various biological barriers encountered. Such vectors can be envisaged as ‘virus-like’ systems which share the levels of gene delivery efficiency of their viral counterparts, but coupled with the safety of purpose-made organic molecules.

Keywords: gene therapy, gene delivery, transfection, cationic lipid, synthetic vector, lipoplex



291-298

Unusual chemical hypersensitivity of the d(GA)n• d(TC)n repeat in vivo dependent on functional lactose repressor

Author(s): Dr. Sergei M. Mirkin,

Abstract: A microsatellite, d(GA)n•d(TC)n, was inserted upstream of an inducible promoter in an Escherichia coli plasmid and its structure was probed by chemical footprinting in vivo. Hyper-reactivity to the single-strand DNA specific chemical, chloroacetaldehyde, was observed within the repeat, pointing to a structural transition within it. Surprisingly, hyper-reactivity of the d(GA)n•d(TC)n repeat diminished upon increased negative supercoiling caused by transcription. Furthermore, the fine modification pattern of the repeat was inconsistent with H-DNA or other known conformations that it adopts in vitro. Finally, functional lactose repressor appeared to be required for chemical hyper-reactivity of the repeat. We believe, therefore, that unanticipated binding of the lactose repressor to the d(GA)n•d(TC)n repeat, which is non-homologous to its regular binding sites, leads to elevated chemical sensitivity of the repeat in vivo.

Keywords: d(GA)n•d(TC)n, microsatellites, primers, operator deletions, repressor deletion




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