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Volume 9 - 2005


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1-6

Single-cell RT-PCR analysis of cerebrospinal fluid B cell clonality and immunoglobulin heavy and light chain variable region gene mutation in diffuse large B-cell lymphoma

Author(s): Dr. Xiaoli Yu,

Abstract: Fluorescence-activated cell sorting and reverse transcription-polymerase chain reaction were used to examine cerebrospinal fluid and blood before and during treatment of a patient with diffuse large B cell lymphoma. A monoclonal population of IgM-expressing B cells that used a rearranged VH4 heavy chain variable region and Vκ3 light chain variable region was found. The persistent monoclonal B cell response in cerebrospinal fluid correlated with neurologic deterioration during treatment.

Keywords: B cell clonality, lymphoma



7-14

A novel IL-6/IL-12 family cytokine IL-27 and its antitumor activity

Author(s): Dr. Takayuki Yoshimoto,

Abstract: Interleukin (IL)-12 is a heterodimeric pro-inflammatory cytokine, which plays a critical role in a link between innate and adaptive immunities and in the development of type 1 cell-mediated immunity. Novel heterodimeric cytokines IL-23 and IL-27 with structural and functional similarities to IL-12 have been identified. These novel cytokines establish the IL-6/IL-12 family, whereas they mediate distinct cellular functions and roles, resulting in the coordinate regulation of Th1 development and type 1 cell-mediated immunity. Recent studies have revealed that both IL-23 and IL-27 possess a potent antitumor activity. Although IL-12 has a powerful antitumor activity against various tumors, IL-12 therapy has been limited by its systemic toxicities. Therefore, IL-23 and IL-27 may be alternative attractive candidates as therapeutic agents against tumors. This review summarizes recent advance on the roles of IL-27 in immune regulation and its antitumor activity.

Keywords: IL-27, IL-12, IL-23, antitumor activity, CD8+ T cells



15-22

Circular dumbbell AP-1 and E2F decoy oligodeoxynucleotide based antiproliferative gene therapy

Author(s): Dr. In-Kyu Lee,

Abstract: Excessive proliferation of cells is a characteristic finding in a wide variety of diseases including post-angioplasty restenosis, diabetic nephropathy, and malignant disease. It is well known that the transcription factors AP-1 and E2F play a critical role in cell proliferation and cell cycle regulation. Therefore, sequence-specific inhibition of AP-1 and E2F by decoy oligodeoxynucleotides (ODNs) is an attractive method to treat the above mentioned diseases. However, one of the main limitations of the conventional decoy ODNs is that they are easily degraded by intracellular nucleases. To rectify this problem, we have developed novel circular dumbbell (CD) decoy ODNs for AP-1 and E2F. The CD decoy ODNs for AP-1 and E2F significantly decreased the expression of genes that are transactivated by these factors and blocked cell proliferation in vascular smooth muscle cells, mesangial cells, and U2OS and C33A cancer cell lines in vitro. Moreover, we demonstrated the effectiveness of CD-AP-1 and CD-E2F decoy ODN based gene therapy in animal models of restenosis and diabetic nephropathy. Therefore, our data suggest that CD-AP-1 and CD-E2F decoy ODN based antiprolilferative gene therapy could provide a new therapeutic strategy for the treatment of restenosis following angioplasty, diabetic nephropathy and cancer.

Keywords: CD-AP-1 and E2F, decoy oligodeoxynucleotide, gene therapy



23-32

Retardation of atherosclerosis in immunocompetent apolipoprotein (apo) E-deficient mice following liver-directed administration of a [E1−, E3−, polymerase−] adenovirus vector containing the elongation factor-1α promoter driving expression of human apoE cDNA

Author(s): Dr. George Dickson,

Abstract: Although gene transfer of human apolipoprotein E (apoE), a 34-kDa circulating glycoprotein, to the liver of apoE- deficient (apoE-/-) mice using recombinant adenoviral vectors (rAd) is antiatherogenic, its full therapeutic potential has yet to be realized. First generation vectors led to immune clearance of transduced hepatocytes, while an improved vector with adenovirus regions E1, E3 and DNA polymerase deleted also had transient effects due to cellular shutdown of the cytomegalovirus (CMV) promoter. Here, we have studied an alternative promoter from the cellular elongation factor 1α (EF-1α) gene, injecting 6-8 week old apoE-/- mice intravenously with 2x1010 virus particles (vp) of the [E1−, E3−, polymerase−] rAd vector Ad-EF1·-apoE. Plasma apoE levels were low (18-55 ng/ml) and failed to reduce plasma cholesterol or normalize the adverse lipoprotein profile. By contrast, the hyperlipidaemic phenotype of apoE-/- mice treated with Ad-CMV-apoE (2x1010 vp) was transiently normalized. Nevertheless, at termination (265 days) the aortic lesion areas in animals given Ad-EF1·-apoE were significantly reduced by 15% (P<0.05) compared to untreated animals, a decrease approaching that in Ad-CMV-apoE-treated mice (23%; P<0.02). Importantly, the attenuation of apoE transgene expression noted with the CMV promoter was absent with the EF-1α promoter, which gave relatively sustained, albeit low, levels of plasma apoE throughout the study period.

Keywords: Atherosclerosis; apolipoprotein E; gene therapy; adenovirus



33-40

A rational approach to the systemic treatment of cancer involving medium-term depletion of arginine

Author(s): Dr. DN Wheatley,

Abstract: Arginine catabolizing enzymes have been applied to cancerous material for over 60 years. The scattered reports in the literature during this period have been reviewed on several previous occasions. This article will be concerned with reports mainly over the last 6 to 7 years on the ability of arginine catabolizing enzymes not only to inhibit proliferation, but to kill tumour cells. The selectivity of action is based on the inability of many tumour cells to circumvent arginine deprivation by utilizing (recycling) various precursors available through the urea cycle. While this offers an immediate window of opportunity for treating melanomas and hepatocellular carcinomas in particular, in vitro treatment can be customized so that even those tumour cell lines with intact urea cycles can be targeted, making the protocol more generally applicable. Since in vitro studies have provided convincing evidence of the efficacy of arginine degrading enzymes, and animal tumour models responded similarly, this treatment has moved on into clinical and veterinary trials. Initial findings are encouraging, which could be effective with many tumour types, from leukemias to melanomas. This is made even more attractive because arginine deprivation protocols can “stage” tumour cells for combination therapy where cells have not been killed outright by deprivation. This is also selective because deprived normal cells will have become quiescent but soon recover on restitution of the missing nutrient, whereas tumour cells in cycle can be hit by low doses of cycle-dependent cytotoxic drugs.

Keywords: cancer, cell culture, models, arginase, arginine deiminase, arginine decarboxylase, transhepatic arterial embolism, catabolism, citrulline, therapy



41-46

Vascular endothelial growth factor as an effector of mast cell-induced tumor angiogenesis

Author(s): Dr. Domenico Ribatti,

Abstract: The current wisdom is that tumors are endowed with an angiogenic capability and that their growth, invasion and metastasis are angiogenesis-dependent. Tumor cells are surrounded by an infiltrate of inflammatory cells, namely lymphocytes, neutrophils, macrophages and mast cells (MC), which communicate via a complex network of intercellular signaling pathways, mediated by surface adhesion molecules, cytokines and their receptors. This review article summarizes: i) the MC mediators involved in angiogenesis; ii) the experimental evidence concerning the role played by MC in tumor angiogenesis; iii) the role played by vascular endothelial growth factor contained in MC secretory granules in tumor angiogenesis.

Keywords: angiogenesis; mast cells; tumor progression; vascular endothelial growth factor



47-50

Usage of U7 snRNA in gene therapy of hemoglobin E disorder, an in silico study

Author(s): Dr. Viroj Wiwanitkit,

Abstract: Hemoglobin (Hb) E disorder is an important hemoglobinopathy with the highest endemicity in Southeast Asia. People with hemoglobin E disease have a mild hemolytic anemia and mild splenomegaly. The microcytosis is attributed to the beta thalassemic nature of the beta E (βE) globin gene, whereas the in vitro instability of HbE does not contribute to the phenotype. Here, the author performs a bioinformatic analysis to study the effect of nucleic acid sequence change due to SnRNA repair in the hemoglobin E disorder on the secondary structure of beta globin chain. Answering this question, a computer-based study for amino acid sequence comparison and protein structure modeling is performed. The database Pubmed was used for data mining of the nucleic acid sequence for human beta globin chain. Then the mutation beta 26, GAG-AAG, was experimentally performed to derive primary sequence in Hb E disorder. Then modified U7 snRNA (U7.623) was experimentally direct inserted, as previously described, into the sequence. According to this study, the secondary structure of human beta globin chains of normal and SnRNA repaired Hb E are calculated and presented. According to this study, there is no significant difference in the secondary structures between both chains. Here, the author can reassure that modified U7 snRNA might be a good future tool for gene therapy in Hb E disorder.

Keywords: βglobin, hemoglobin E, modified U7 snRNA



51-60

Raf/MEK/ERK signaling: Implications in the infection and pathogenesis of viruses associated with AIDS

Author(s): Dr. Shaw M. Akula,

Abstract: Acquired immunodeficiency syndrome (AIDS) is characterized by failure of the immune system that is overwhelmed by an organized and well orchestrated invasion by various opportunistic pathogens. Cancers associated with AIDS are predominantly caused by viruses. Oncoproteins are directly involved in the initiation of neoplastic transformation. Recently, the presence of activating mutations in Raf has been described in a variety of cancers. Raf expression enhances Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of cells. KSHV is an etiology for Kaposi’s sarcoma (KS); a condition commonly associated with AIDS. In addition, Raf associated signaling also regulates expression of various growth factors (GFs)/inflammatory cytokines (ICs). Since, AIDS is a condition that is regulated by aberrant GF/IC expression, we analyzed a possible role for Raf expression in the infection and pathogenesis of AIDS associated viruses in this review. This review also attempts to rationalize on why Raf associated signaling could well be a novel target to treat disease conditions due to viruses associated with AIDS.

Keywords: KSHV, HHV-8, AIDS, Raf



61-76

Cytokine gene transfer in the therapy of autoimmune diseases

Author(s): Dr. Detlef Neumann and Diana Boraschi,

Abstract: Delivery of DNA coding for disease-modifying proteins has been experimented as a therapeutic option for over 15 years. Since the first clinical trial in 1989, about 900 trials have been approved worldwide in order to demonstrate safety, feasibility, and therapeutic benefits, cancer being the most common disease indication. Systems for efficient, safe, and targeted delivery of DNA encoding therapeutic proteins are actively studied and include viral vectors (mostly retro- and adenoviruses), non-viral vectors, and delivery of naked plasmid DNA. In autoimmune diseases, classical therapies are aiming at inhibiting either lymphocyte activation (immunosuppressive agents), or the downstream inflammatory effector mechanisms responsible for organ and tissue destruction (anti-inflammatory drugs). Newer therapies are being developed targeting inflammatory and immunostimulating cytokines, with the use of the so-called “cytokine traps” (recombinant and chimeric proteins and antibodies which capture and inhibit pathological cytokines) or of recombinant regulatory cytokines able to inhibit inflammatory cytokine production. In this context, gene therapy and DNA vaccination are very promising approaches, which could avoid problems connected with life-long treatments with protein drugs and achieve optimal efficacy. In this review, two parallel approaches will be examined, delivery of genes encoding cytokine inhibitors (regulatory cytokines, cytokine inhibitors including cytokine receptors), and DNA vaccination with genes coding for the pathogenic cytokines, to trigger an endogenous anti-cytokine response with therapeutic effects.

Keywords: gene transfer, DNA vaccination, cytokines, chemokines, autoimmunity



77-88

IGF-IR blockade strategies in human cancers

Author(s): Dr. Choon-Taek Lee,

Abstract: Growth factor receptor signals, like those from insulin-like growth factor (IGF)-I receptor (IGF-IR), are required for carcinogenesis and tumor progression in many human malignancies. The concept of targeting specific tumorigenic receptors has been validated by the successful clinical application of multiple new drugs, such as trastuzumab and gefitinib. Genetic blockade of IGF-IR has been accomplished by antisense, dominant negative inhibition, siRNA, and triplex formation mediated by plasmid vector transfection, oligonucleotide, or viral transduction, whereas non-genetic blockade of IGF-IR has been accomplished using soluble IGF-IR, monoclonal antibodies, and IGF-IR tyrosine kinase inhibitor. IGF-IR blockade induces apoptosis of cancer cells by blocking antiapoptotic signaling pathways resulting in the regression of established tumors. However, the lack of a suitable means for inducing an effective IGF-IR blockade remains an obstacle to the clinical application of IGF-IR blockade strategies. Furthermore, the structural similarity between IGF-IR and insulin receptor increases the importance that any inhibitor used in the clinic be both highly specific as well as effective. Here we review the current status of IGF-IR blockade strategies for cancer treatment and suggest possible future development directions.

Keywords: IGF-IR, cancer, antisense, dominant negative inhibition, small molecule



89-106

Catalytic nucleic acid enzymes for the study and development of therapies in the central nervous system

Author(s): Dr. Carol A. Kruse,

Abstract: Nucleic acid enzymes have been used with great success for studying natural processes in the central nervous system (CNS). We first provide information on the structural and enzymatic differences of various ribozymes and DNAzymes. We then discuss how they have been used to explore new therapeutic approaches for treating diseases of the CNS. They have been tested in various systems modeling retinitis pigmentosum, proliferative vitreoretinopathy, Alzheimer's disease, and malignant brain tumors. For these models, effective targets for nucleic acid enzymes have been readily identified and the rules for selecting cleavage sites have been well established. The bulk of studies, including those from our laboratory, have emphasized their use for gliomas. With the availability of multiple excellent animal models to test glioma treatments, good progress has been made in the initial testing of nucleic acid enzymes for brain tumor therapy. However, opportunities still exist to significantly improve the delivery and efficacy of ribozymes to achieve effective treatment. The future holds significant potential for the molecular targeting and therapy of eye diseases, neurodegenerative disorders, and brain tumors with these unique treatment agents.

Keywords: ribozyme, catalytic RNA, gliomas, brain tumors, proliferative vitreoretinopathy, Alzheimers disease



107-112

Targeting of cancer gene therapy with antibodies or their genes against tumor-associated antigens

Author(s): Dr. Masahide Kuroki,

Abstract: Gene therapy is expected to play a major role in future cancer treatment. Actually various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. This overview summarizes the current use of anti-tumor-associated antigen (TAA) antibodies in cancer gene therapy. Current data suggest that antibodies or their genes against TAAs can be used for targeting viral vectors for cancer gene therapy.

Keywords: Cancer gene therapy, viral vector, tumor-targeting, tumor-associated antigen



121-134

Graft engineering for autologous stem cell transplantation

Author(s): Dr. Luis F. Porrata,

Abstract: To date, the main therapeutic goal of the stem cell autograft has been the collection of sufficient stem cells in order to achieve hematologic engraftment following high-dose chemotherapy in the setting of autologous stem cell transplantation (ASCT). Efforts to improve the clinical outcomes of ASCT have recently focused on the impact of autograft contaminating tumor cells and methods related to their removal (purging). In the last five years, emerging evidence suggesting the impact of autograft lymphocyte content on post-ASCT immune reconstitution with dramatic improvement on clinical outcomes suggests a critical role for autograft immune cell content. Herein we review the clinical evidence suggesting that autograft engineering directed at immune cell enrichment may directly impact clinical outcomes in patients undergoing ASCT for malignant disorders.

Keywords: absolute lymphocyte count, apheresis machine, autograft absolute lymphocyte count, autologous stem cell transplantation, graft engineering, number of apheresis collections, purging, stem cell collection, stem cell dose, stem cell mobilization



135-142

Visualization of transfer of a fluorescently-labeled membrane raft protein to T cells using lentivirus

Author(s): Dr. William Rodgers,

Abstract: Lentivirus vector systems have been developed for the safe delivery of foreign genes to target tissues. However, the use of these systems for delivering specific proteins to target cells has been largely unexplored. To test this concept, the lentivirus expression plasmid pLenti was utilized to overexpress in producer cells a YFP-fusion protein that is specifically targeted to glycolipid-enriched membrane rafts, which is the site of virus assembly. Our data show that virus generated in producer cells that expressed the YFP fusion protein were able to effectively label target cells by a 2-3 hr incubation with the virus. Labeling of the target cells was specific to the lentivirus, as it was blocked by pre- incubating the virus with antibody to the surface protein, and it was not affected by pre-treating the target cells with cyclohexamide. T cells that were labeled using the lentivirus underwent a robust stimulation following crosslinking the T cell receptor, thus showing that T cells labeled using lentivirus remained responsive to extracellular cues. Altogether, these results show that overexpression of foreign proteins in lentivirus producer cells can yield protein-loaded viruses, which can then function to deliver the protein to target cells. Thus, our findings suggest an avenue for targeting specific proteins to cells where foreign gene expression is not feasible.

Keywords: Rafts, lentivirus, T cells



143-152

Gene editing of the wild-type APOE3 gene to the dysfunctional variants APOE2 or APOE4 using synthetic RNA-DNA oligonucleotides (chimeraplasts)

Author(s): Dr. James S. Owen,

Abstract: Plasma apolipoprotein E (apoE) is secreted by liver (>90%) and macrophages, and protects against atherosclerosis by contributing to cholesterol homeostasis and by locally restricting lesion development. Three common isoforms arise from single nucleotide polymorphisms (SNPs): apoE2, the rarest variant, differs from wild-type apoE3 by an R158C substitution and causes recessive Type III hyperlipidaemia, whereas apoE4 (C112R) produces a dominant hyperlipidaemia. Molecular explanations for these relationships are poorly defined, but most likely reflect differences in receptor binding and/or intracellular trafficking. Potentially, single-base mutations can be corrected in genes by using synthetic RNA-DNA oligonucleotides (chimeraplasts), which harness cellular mismatch repair mechanisms. Here, we evaluate gene editing of the APOE3 gene in HepG2 hepatoblastoma cells and THP-1 monocyte-macrophages, with the aim of producing new human clonal cell lines that secrete apoE2 or apoE4 for subsequent biological investigations. Initially, we used polyethyleneimine (PEI) to transfer chimeraplasts and showed that brief centrifugation improved conversion efficiency. A dose-dependent conversion was seen by PCR- RFLP analysis in HepG2 cells and confirmed by direct sequencing. Additional increases in conversion efficiency were also noted when receptor-dependent uptake was exploited, using galactotetraose-PEI and mannose-PEI for HepG2 and THP-1 cells, respectively. Unexpectedly, however, the conversions appeared unstable as analysing 50 clones isolated by limiting dilution from chimeraplast-treated THP-1 and HepG2 cells consistently revealed no genotypic changes. Whether this instability is due to cytotoxic or apoptotic effects of the transfection complex, or to a recent suggestion that cells have effective defence mechanisms that counteract targeted genome sequence alterations, remains to be established.

Keywords: Apolipoprotein E; atherosclerosis; chimeraplasty; transfection



153-168

Enhancement of cancer gene therapy with modified viral vectors and fusion genes

Author(s): Dr. Akseli Hemminki,

Abstract: The major obstacle in cancer gene therapy continues to be insufficient transduction of tumor cells and consequently poor therapeutic effect. However, several approaches have been developed to improve gene transfer rates. First, alternative viral vectors can be explored to find optimal gene transfer vehicles for each purpose. Secondly, viral vectors can be re-targeted to cancer cells, which can simultaneously enhance gene transfer to tumors and diminish undesired side effects in healthy tissue. In addition, it is possible to exploit viral replication per se to destroy cancer cells. To avoid side effects and increase the safety of these oncolytic agents, replication can be limited to tumor cells by partially deleting areas of the viral genome or by using tissue specific promoters to drive viral genes responsible for replication. Instead or in addition to modifying the gene transfer vector, one possibility is to modify the therapeutic gene so that the resulting therapeutic protein can spread to surrounding cells and thus compensate for low gene transfer efficiency and enhance therapeutic outcome.

Keywords: cancer gene therapy, viral vectors, adenovirus, targeting, oncolytic viruses, protein transduction domains



169-180

Mouse and pig nonviral liver gene therapy: success and trials

Author(s): Dr. Salvador F. Aliño,

Abstract: Success has been achieved with nonviral gene delivery to mouse liver, resulting in long-term therapeutic plasma levels of human α-1 antitrypsin (hAAT) protein, employing the hydrodynamic procedure. Now we contribute to explore the mechanism involved in the successful procedure, with the aim of circumventing the existing serious limitations for application to clinical practice. The results from mouse hydrodynamic gene transfer experiments support the following: a) the procedure mediates good dose-response efficacy, limited liver toxicity and long-term gene expression; b) the mechanism of gene delivery appears to be unspecific and involves transient inversion of intrahepatic flow, sinusoidal blood stasis and massive fluid endocytic vesicles in hepatocytes. In addition, since increased intrahepatic pressure induced by blood flow inversion could trigger hepatocyte DNA uptake, we have studied the efficacy of pig liver gene delivery by catheter-mediated retrodynamic injection of hAAT gene through the hepatic vein. The results show that in the same way as in mouse experiments: a) maximal plasma levels of human protein are observed on day 10-15 after injection, but the amount of protein is three orders of magnitude lower; b) maximal liver injury is observed two hours after retrodynamic perfusion, though it was very limited - suggesting that catheter perfusion is a mild procedure and/or that perfusion is limited to a very small region of the liver; c) electron microscopy shows the massive presence of large vesicles lining the membrane of hepatocytes alongside endothelial cells, though in contrast to the observations in mice, the vascular endothelium is very continuous and/or scarcely fenestrated.

Keywords: Gene transfer, gene delivery, liver, α-1 antitrypsin, genomic DNA, nonviral vector, fluid endocytosis, naked DNA, pig, catheter



181-182

Elongation of α globin chains, does excessive amino acids and helices relate to clinical manifestation?

Author(s): Dr. Viroj Wiwanitkit,

Abstract: Haemoglobin variants in which a disorder results in chain elongation are unusual. For α globin chain, the elongation of haemoglobin is believed to relate to the clinical manifestations such as those in hemoglobin H diseases. Although the primary structures of Hb disorders with elongated α globin chains are well-known the secondary structure of them is not well documented. The study on the tertiary structures of the elongated part in those Hb can help explain more in the pathogenesis of the disorders is needed. Here, the author performs a bioinformatic analysis for nanohematology to study the secondary structures of three well described Hb disorders, Hb Pak Num Po, Hb Pakse and Hb Constant Spring with elongated α globin chains. According to the analysis, the trend of increase aberration relating with increased excessive amino acids and excessive helical residues are noted. However, generalization to other Hb disorders, than these 3 disorders, needs further experimental studies..

Keywords: Hb disorder, α, globin, elongation, helix, amino acid



193-202

Using methyl methanesulfonate (MMS) to stimulate targeted gene repair activity in mammalian cells

Author(s): Dr. Timothy R. Schwartz,

Abstract: We describe a simple, efficient and inexpensive means of stimulating targeted gene repair in mammalian cell lines. Methyl methanesulfonate (MMS) is an alkylation agent capable of inducing DNA lesions and subsequent single strand breaks (ss breaks) and double strand breaks (ds breaks). This damage activates the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways, a response that can enhance the frequency of synthetic oligonucleotide-directed gene repair. We show in mammalian cells that the conversion frequency of a mutant base pair to wild-type in an integrated enhanced green fluorescent protein gene is increased by pretreatment with MMS. This stimulation is dose-dependent and correlates to the level of ds breaks induced by MMS treatment. These ds breaks result in Rad51p nuclear-relocalization and foci formation, a specific indication that HR pathway has been activated. In presence of MMS the cell cycle is slowed with an accumulation in S phase where coincidently, the highest level of gene repair takes place. Our data suggest that MMS-induced DNA damage elicits a cellular response that stimulates gene repair in mammalian cells and provides a direct method for elevating levels of gene correction.

Keywords: gene repair, MMS, single-stranded oligonucleotides, DNA damage, cell cycle



203-216

Cancer therapy by means of irreversible tumor blood flow stasis: Starvation tactics against solid tumors

Author(s): Dr. Katsuyoshi Hori,

Abstract: Despite extensive research efforts, effective therapies for advanced cancers have not yet been established, and development of successful treatment strategies remains the most important task in the field of oncology. Three significant problems in conventional chemotherapy using cytotoxic drugs require attention: (i) choosing the most effective drug for individual patients, (ii) delivering a sufficient dose of drug to tumor, and (iii) minimizing severe side effects of anticancer drugs. Because the cancer cells are themselves the direct target of the drugs, these three problems cannot be avoided. We recently showed that AC7700 (currently AVE8062) a derivative of combretastatin A-4, achieved irreversible stasis of tumor blood flow (TBF), thereby causing necrosis of tumor tissue by halting the supply of nutrients. Such effects were unrelated to cancer type, in that they were found for various solid tumors. In this review, we summarize our research on AC7700 and tumor vessels and discuss how AC7700 causes stasis of TBF and why the blood flow does not resume. This technique of attacking tumor by means of blocking TBF largely avoids the three problems typically encountered in conventional cancer chemotherapy that were mentioned above. We propose that such starvation tactics constitute a new therapeutic approach to solid tumors, including refractory cancers which are resistant to conventional cytotoxic drugs and recurrent cancers that have acquired drug resistance.

Keywords: Tumor blood flow, Tumor vessel, Combretastatin A-4, AC7700, Microcirculation, Necrosis



217-228

Papillomavirus biology and therapeutic approaches

Author(s): Dr. Patricio I. Meneses,

Abstract: Human papillomaviruses (HPV) are ubiquitous in the population and typical infection of the dermis can result in development of cutaneous warts that usually clear on their own. However, infection of the genital tract can result in persistent infections lasting 12-30 months or in the development of cervical carcinoma decades after initial infection. 500,000 cases of cervical carcinoma are detected annually worldwide. This leads to an approximately 288,000 deaths yearly, with most of these cases occurring in developing nations. This review will describe the underlying mechanisms during HPV mediated pathogenesis, the role of the specific viral antigens and explore ongoing efforts towards development of therapeutic interventions for treatment of HPV associated cancers.

Keywords: Papillomavirus, basal cells, E2 protein, E6 protein, E7 protein, L1 capsid antigen, L2 antigen, papillomavirus associated malignancies



229-246

Towards understanding the epigenetics of transcription by chromatin structure and the nuclear matrix

Author(s): Dr. Stephen A. Krawetz,

Abstract: The eukaryotic nucleus houses a significant amount of information that is carefully ordered to ensure that genes can be transcribed as needed throughout development and differentiation. The genome is partitioned into regions containing functional transcription units, providing the means for the cell to selectively activate some, while keeping other regions of the genome silent. Over the last quarter of a century the structure of chromatin and how it is influenced by epigenetics has come into the forefront of modern biology. However, it has thus far failed to identify the mechanism by which individual genes or domains are selected for expression. Through covalent and structural modification of the DNA and chromatin proteins, epigenetics maintains both active and silent chromatin states. This is the “other” genetic code, often superseding that dictated by the nucleotide sequence. The nuclear matrix is rich in many of the factors that govern nuclear processes. It includes a host of unknown factors that may provide our first insight into the structural mechanism responsible for the genetic selectivity of a differentiating cell. This review will consider the nuclear matrix as an integral component of the epigenetic mechanism.

Keywords: potentiation; epigenetics; DNA methylation; histone modification; RNA-mediated silencing, nuclear matrix; gene regulation; epigenetic therapy



247-256

Evolutionary dynamics of drug resistance in cancer

Author(s): Dr. Dominik Wodarz,

Abstract: Detailed molecular research has advanced treatment options against cancers significantly. Targeted drugs are being developed which attack specific abnormalities in the cancer cells. An especially promising example of this is the treatment of chronic myeloid leukemia (CML) with Imatinib mesylate. While therapy is often successful in early CML stages, therapy fails during the advanced blast crisis stage. The reason for this failure is drug resistance. In order to design strategies to overcome the problem of drug resistance, it is important to understand the principles according to which drug resistant cancer cells evolve. This requires mathematical models. Here, such a mathematical approach is reviewed. The mathematical framework is applied to CML, and some preliminary predictions and insights regarding the prevention of resistance are discussed.

Keywords: drug resistance, mathematical model, drug resistant mutants, cellular turnover



257-264

In vivo delivery technique of nucleic acid compounds using atelocollagen: Its use in cancer therapeutics targeted at the heparin-binding growth factor midkine

Author(s): Dr. Yoshifumi Takei,

Abstract: The largest obstacle in developing a therapeutic based on nucleic acid compounds, including antisense DNA, ribozyme and small interfering RNA, lies in the necessity of achieving effective transfection into the target organ and tissue. In this review, we introduce a technique to deliver nucleic acid compounds to tissue in vivo using atelocollagen. Atelocollagen, a pepsin-digested product of type I collagen derived from the dermis of cattle, is liquidified at low temperature and gels at 37ÆC. These unique properties of atelocollagen provide a nuclease- resistant in vivo environment and tissue maintenance of nucleic acid-based injected therapeutics, thus ensuring maximal treatment efficacy. In this review we introduce antisense DNA, morpholino antisense oligomer and siRNA targeting midkine a heparin-binding growth factor that is overexpressed in various tumors of humans and discuss their application in cancer therapy. All three nucleic acid compounds were transfected into xenografted tumor cells in vivo at high efficiency when they were mixed with atelocollagen. We proved that siRNA mixed with atelocollagen stayed in the tumor for at least eight days and was maintained intact. This review will be practically useful for developing a nucleic acid-based therapeutic against various diseases, including cancers.

Keywords: midkine, antisense oligodeoxynucleotide, small interfering RNA, cancer therapy, in vivo delivery system, gene transfer and atelocollagen



265-268

Rho/Rho-kinase as potential therapeutic targets for CNS injury

Author(s): Dr. Toshihide Yamashita,

Abstract: Axons of the adult central nervous system are capable of only a limited amount of regrowth after injury, and that an unfavorable environment plays major roles in the lack of regeneration. Some of the axon growth inhibitory effects are associated with myelin. Three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. The p75 receptor, in complex with the Nogo receptor, transduces the signal from all of the myelin-derived inhibitors found to date. The p75 receptor, in response to the myelin-derived proteins, induces activation of Rho, which is one of the key regulators of cytoskeletal organization. Inhibition of Rho or Rho-kinase, downstream effector of Rho, promotes axon regeneration in vivo. These findings establish Rho and Rho-kinase as key players in inhibiting the regeneration of the central nervous system, and launched a new wave of studies that aim to promote regeneration of injured axons by modulating this inhibitory pathway.

Keywords: central nervous system, axon, Rho, regeneration



269-280

Oncolytic virotherapy of cancer with vesicular stomatitis virus

Author(s): Dr. John Hiscott,

Abstract: Recent basic and pre-clinical studies have demonstrated that several innocuous, non-disease causing, replication competent viruses can selectively replicate in and kill a large panel of human tumor cells, clear bone marrow of leukemic cells and effectively arrest metastatic spread of tumors, while apparently sparing normal cells. These studies have revived interest in the clinical applicability of these oncolytic (onco=cancer; lytic=killing) viruses. Although it is as yet unknown why oncolytic viruses preferentially target and kill tumor cells, it appears that during the evolution of malignancies, genetic abnormalities accumulate that, while providing the cancer cells with growth and survival advantages, compromise the normal antiviral program of transformed cells. Defects in the Interferon (IFN) antiviral signaling network within transformed cells have been implicated in preferential oncolysis; according to this model, IFN-related defects allow VSV and other oncolytic viruses to replicate to high titers, uninterrupted by the host antiviral response, resulting in high virus production and virus induced lysis. The goal of our research program is to understand the molecular basis of virus-induced oncolysis and to identify important changes in the genetic response to virus infection in tumor cells. Basic, pre-clinical and clinical studies will attempt to harness what we believe is a potent new form of cancer therapy with promise in many areas of cancer treatment.

Keywords: VSV, oncolysis, apoptosis, virotherapy



281-290

Adenoviral-mediated overexpression of p16, p53 or TGFβ1 induces apoptosis of BPH cells

Author(s): Dr. Yi Lu,

Abstract: Benign prostate hyperplasia (BPH), is the most commonly occurring benign disease in men older than 50 years of age. This report has provided a novel gene therapy strategy for BPH by adenoviral-mediated gene transfer of negative growth regulators p53, p16 and transforming growth factor β1 (TGF β1). The effects of these adenoviral vectors on an established human BPH cell line, BPH-1, were examined. The apoptotic status of cells after p53, p16 and TGFβ1 expression was evaluated by DNA fragmentation, TUNEL (terminal deoxynucleotiyl transferase- mediated dUTP nick end labeling) staining, and flow cytometry. All these three gene products suppressed cell growth and induced apoptosis in BPH-1 cells. These results suggested that adenoviral-mediated gene transfer of p53, p16 and TGFβ1 may have a potential therapeutic application in treatment of BPH.

Keywords: BPH, gene therapy, p16, p53, TGFβ1



291-300

Adenoviral (Ad) vectors are widely used in gene therapies, recombinant viral vaccines, and basic science studies. The vectors can deliver therapeutic genes into cells to recover the lost function of some genes, to enhance the ability of host immune systems, or to increase the sensitivity of cancer cells to chemotherapeutic drugs. Several adenoviral vector systems have been developed: first-generation vectors are those with deletion of the E1a and E1b genes, second-generation vectors with deletions of the E1 and another viral gene, and gutless vectors removed all coding sequences for viral proteins. The first- and second-generation vectors are relatively easy to construct and produce. The gutless Ad vectors are substantially superior to any other adenoviral systems for achieving high-level and long- term expression in vivo. The recent developed oncolytic adenoviruses can selectively replicate in cancer cells but not in normal cells, allowing the spread of the viruses throughout the tumors. This review focuses on the advantages and limitations of adenovirus vectors and discusses potential strategies for further improvement of vectors for gene therapy.

Author(s): Dr. H. Sam Zhou,

Abstract: Adenoviral (Ad) vectors are widely used in gene therapies, recombinant viral vaccines, and basic science studies. The vectors can deliver therapeutic genes into cells to recover the lost function of some genes, to enhance the ability of host immune systems, or to increase the sensitivity of cancer cells to chemotherapeutic drugs. Several adenoviral vector systems have been developed: first-generation vectors are those with deletion of the E1a and E1b genes, second-generation vectors with deletions of the E1 and another viral gene, and gutless vectors removed all coding sequences for viral proteins. The first- and second-generation vectors are relatively easy to construct and produce. The gutless Ad vectors are substantially superior to any other adenoviral systems for achieving high-level and long- term expression in vivo. The recent developed oncolytic adenoviruses can selectively replicate in cancer cells but not in normal cells, allowing the spread of the viruses throughout the tumors. This review focuses on the advantages and limitations of adenovirus vectors and discusses potential strategies for further improvement of vectors for gene therapy.

Keywords: adenovirus, vectors, gutless vector, replication, gene therapy



301-316

Designing smart nano-apatite composites: the emerging era of non-viral gene delivery

Author(s): Dr. Toshihiro Akaike,

Abstract: Transfer of desirable genetic sequences into mammalian cells is an essential tool for analysis of gene structure, functions and regulation, and pivotal for gene therapy and DNA vaccination strategies. Considering some severe limitations of viral systems including immunogenicity, carcinogenicity and so on, synthetic non-viral systems are highly desirable in the above applications. However, existing non-viral techniques are extremely inefficient compared to the viral ones. We have recently developed a new class of non-viral technology based on biodegradable apatite materials having simplicity and flexibility of preparation, size regulation and surface functionalization for carrying genetic materials to selective or a wide variety of cell types in an effective manner. Moreover, while the materials have high affinity for DNA due to their cationic surface charge and confers high stability of condensed DNA towards serum, they could release the associated DNA rapidly during endosomal acidification, resulting in notable level of transgene expression. We will particularly focus here on the recent development of the highly efficient synthetic device for gene delivery and expression based on controllable growth of nano-apatite particles. Mg2+ or CO32- incorporation into the apatite particles caused significant inhibition of particle-growth, resulting in retention of nano-sized particles which contributed remarkably to the cellular uptake of DNA and its subsequent expression, leading to 5 to 100-fold higher transgene expression than the existing ones. Moreover, for cell-specific and more efficient transgene delivery, we could successfully assemble a desirable cell-recognizable protein and a highly hydrophilic protein onto the DNA/crystal surfaces, thereby conferring dual surface properties; one facilitating cell-specific delivery and the other blocking non-specific interactions. Thus, considering the efficiency, cell-targetability, biodegradability and simplicity, this newly developed gene delivery technology is highly promising over other existing ones for both basic research laboratories and clinical settings.

Keywords: non-viral vector, gene delivery, transfection, magnesium, carbonate, apatite, endocytosis, pH sensitivity, protein expression, cell targeting, asialofetuin, transferrin



317-324

Recombinant Sindbis virus expressing functional GFP in the nonstructural protein nsP3

Author(s): Dr. Guangpu Li,

Abstract: Sindbis virus vectors usually express foreign genes cloned in the structural region of the viral genome. In this report, we tested the possibility of expressing genes in the nonstructural region. We made a recombinant virus with a GFP gene inserted in the nsP3 sequence. The resulting Toto1101/GFP virus was infectious and grew to the same high titer as the parental virus. The nsP3-GFP fusion protein, like nsP3 itself, was phosphorylated. With confocal fluorescence microscopy, we found that nsP3-GFP, as a component of viral RNA replication complex, was on the plasma membrane early in infection (2 h post-infection), but quickly moved to punctate intracellular structures and remained there throughout the course of infection. The observed fluorescence indicates that GFP is functionally expressed in this nonstructural region and other marker or therapeutic genes should be able to be expressed in the same fashion, thus improving the utility of these viral vectors.

Keywords: Sindbis, Semliki, alphavirus, Togavirus, RNA virus, GFP



325-326

Identification of putative genes for hereditary persistence of fetal hemoglobin (HPFH)

Author(s): Dr. Viroj Wiwanitkit,

Abstract: Hereditary persistence of fetal hemoglobin (HPFH) is an important hemoglobin disorder. It is noted that that the 3'-juxtaposed region (3'JR) position exerted a positional effect associated with activation of fetal gene in HPFH. In this study, computer prediction and gene homology programs was used to reveal gene homologues to this area. The identified gene can be a considerable point in gene therapy. The results of in silico analysis of the breakpoint revealed 3 most highly homologuegenes (alignment score between 50 and 80) including a) Homo sapiens G-γ globin and A-γ globin genes, b) Homo sapiens β globin region (HBB@) on chromosome 11 and c) Homo sapiens chromosome 11, clone CTD-2643I7.

Keywords: Hereditary persistence of fetal hemoglobin, putative, gene



327-328

HIV-1 gp120 and human platelet glycoprotein GPIIIa: does structural homology exist?

Author(s): Dr. Viroj Wiwanitkit,

Abstract: Chronic thrombocytopenia is a common hematologic disorder in patients infected with the human immunodeficiency virus (HIV). Although often asymptomatic, the thrombocytopenia may be associated with a variety of bleeding abnormalities. Antigenic homology between HIV-1 gp120 and human platelet glycoprotein GPIIIa was reported. Here, the author performed a study to assess the structural homology between HIV-1 gp120 and human platelet glycoprotein GPIIIa. To answer this question, a computer-based study for amino acid sequence comparison and protein structure modeling is performed. According to this study, gp-120 is totally difference from human platelet glycoprotein GPIIIa. It seems that the structural homology between HIV-1 gp120 and human platelet glycoprotein GPIIIa might not exist. This implies that gp-120 might not be an important underlying factor for ITP in HIV-infected patients.

Keywords: HIV, thrombocytopenia, gp1202



329-338

Summary Electroporation (EP) has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids for gene therapeutic applications. Skeletal muscle is a preferable target tissue for a number of reasons including long-term secretion of therapeutic proteins for systemic distribution and promotion of strong humoral and cellular immune responses post-vaccination. All of these DNA-mediated applications are significantly improved by in vivo EP. We have demonstrated previously that constant-current EP is effective for intramuscular plasmid delivery in mammals and does not cause permanent damage to cells. Numerous other factors impact plasmid uptake and expression after intramuscular injection followed by EP, such as plasmid size, formulation, concentration, injection volume, intensity of the electric field, target muscle, and species and age of the treated subject. These improvements in the conditions of EP can increase the efficacy of plasmid transfer and lower the total amount of plasmid and DNA vaccines required to generate targeted levels of biologically active proteins or antibodies.

Author(s): Dr. Ruxandra Draghia-Akli,

Abstract: Electroporation (EP) has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids for gene therapeutic applications. Skeletal muscle is a preferable target tissue for a number of reasons including long-term secretion of therapeutic proteins for systemic distribution and promotion of strong humoral and cellular immune responses post-vaccination. All of these DNA-mediated applications are significantly improved by in vivo EP. We have demonstrated previously that constant-current EP is effective for intramuscular plasmid delivery in mammals and does not cause permanent damage to cells. Numerous other factors impact plasmid uptake and expression after intramuscular injection followed by EP, such as plasmid size, formulation, concentration, injection volume, intensity of the electric field, target muscle, and species and age of the treated subject. These improvements in the conditions of EP can increase the efficacy of plasmid transfer and lower the total amount of plasmid and DNA vaccines required to generate targeted levels of biologically active proteins or antibodies.

Keywords: electroporation, plasmid, gene transfer, skeletal muscle



339-342

The kinase activity of c-Abl is known to be tightly regulated. Mechanisms of c-Abl kinase regulation can be classified as intramolecular or intermolecular interactions. Crystallization of the amino (N)-terminal region of c-Abl has revealed that intramolecular folding of the N terminus onto the kinase domain represses intrinsic kinase activity. With regard to intermolecular interactions, recent studies suggested that trans-acting molecules bind to a proline-rich (PR) regions in the carboxyl (C)-terminal portion of c-Abl and activate the kinase. In this review, we focus on c-Abl kinase activation and the mechanisms of substrate phosphorylation mediated by adaptor molecules which bind to the C-terminal PR regions of c-Abl kinase.

Author(s): Dr. Tomoyuki Shishido,

Abstract: The kinase activity of c-Abl is known to be tightly regulated. Mechanisms of c-Abl kinase regulation can be classified as intramolecular or intermolecular interactions. Crystallization of the amino (N)-terminal region of c-Abl has revealed that intramolecular folding of the N terminus onto the kinase domain represses intrinsic kinase activity. With regard to intermolecular interactions, recent studies suggested that trans-acting molecules bind to a proline-rich (PR) regions in the carboxyl (C)-terminal portion of c-Abl and activate the kinase. In this review, we focus on c-Abl kinase activation and the mechanisms of substrate phosphorylation mediated by adaptor molecules which bind to the C-terminal PR regions of c-Abl kinase.

Keywords: Abl, adaptor protein, trans-acting regulation, substrate phosphorylation



343-358

Dendritic cell-based immunotherapy: A promising approach for treatment of cancer

Author(s): Dr. Bhaskar Saha,

Abstract: The accumulating evidence in favor of tumor immunosurveillance indicates that immunotherapies may prove effective for the treatment of cancer. Many current approaches against cancer immunotherapy are often limited in their potential to induce effective anti-tumor immune responses. However, recent approach with dendritic cell based therapy proves to be an effective method for induction of anti-tumor immune response. In this review we discuss the effectiveness and complications associated with DC based immunotherapy and new strategies being perused for effective anti cancer response.

Keywords: Dendritic cell-based immunotherapy, dendritic cells, immunotherapy, antigen, Peptides and proteins, DNA and RNA, Viral vectors, cell fusion



359-370

P210 BCR-ABL tyrosine kinase prevents apoptotic cell death through multiple pathways converging at mitochondrial membranes

Author(s): Dr. Manuela Mancini,

Abstract: Extended survival of clonal hematopoietic progenitors associated with the bcr-abl expression has a key role in the pathogenesis of Chronic Myeloid Leukemia (CML) and may concur to promote the disease progression towards the fully transformed, drug-resistant phenotype of blast crisis. Here we demonstrated that p210 bcr-abl protein tyrosine kinase (TK) activity protects bcr-abl-transducing 32D cell clones from apoptotic death through multiple pathways preserving mitochondrial membrane integrity. P210 TK prevents Trail/DR4, Bim and Bax induction and caspase 9 and Bid activation and upregulates the expression of anti-apoptotic BclxL in the cytoplasm. Moreover, it precludes the integration and aggregation of pro-apoptotic signals Bid, Bax and Bak at mitochondrial membranes letting, in turn, pore opening and apoptogenic molecule leakage from intermembrane spaces. Our results underscore the central role of Akt serine/threonine kinase, activated by p210 TK downstream of phosphatidyl- inositol 3 kinase (PI3K), in leukemic progenitor failure to undergo apoptotic death and support that pharmacological targeting of its enzymatic activity may implement the effects of TK inhibitor Imatinib mesylate (IM).

Keywords: Chronic Myeloid Leukemia, p210 bcr-abl tyrosine kinase, apoptosis, mitochondrial membranes, Akt



371-376

A Human Papillomavirus (HPV) - based pseudoviral gene delivery system for the non-viral, Episomally Replicating Vector pEPI-1

Author(s): Dr. Armin Baiker,

Abstract: Non-viral episomal vector systems might allow a safe and reproducible genetic modification of eukaryotic cells and organisms. However, they share with all non-viral systems the problem of low efficient delivery to the target cells. To overcome this limitation we have utilized a Human Papillomavirus type 16 (HPV16)-based pseudoviral gene delivery system for the non-viral episomally replicating plasmid vector pEPI-1. Pseudoviral particles harbouring pEPI-1 (pEPI-PV) were prepared by co-transfection of the pEPI-1 plasmid DNA and a plasmid expressing the capsid proteins L1 and L2 of HPV16. Here we demonstrate that the resulting pEPI-PV were able to transduce target CHO cells with high efficiency under maintenance of the episomal character of pEPI-1. The combination of HPV-based pseudoviral gene delivery and non-viral, episomally replicating plasmids will provide a powerful new tool for biotechnical and possibly in vivo biomedical applications.

Keywords: pseudovirus; non-viral gene delivery; pEPI-1; episome; HPV 16



377-392

The complexity of p73 isoforms in human neoplasia

Author(s): Dr. Maria Giulia Rizzo,

Abstract: p73, the homologue of p53, is a nuclear protein whose ectopic expression, in p53+/+ and p53 -/- cells, recapitulates the most well-characterized p53 effects, such as growth arrest, apoptosis and differentiation. Altered expression of the p73 gene has been reported in neuroblastoma, lung cancer, prostate cancer and renal cell carcinoma as well as in breast cancer, ovarian tumor, melanoma and hematopoietic neoplasia. p73 has a complex genomic organization that largely results from an alternative internal promoter in intron 3 generating NH2-terminally deleted dominant- negative proteins (∆N-p73) and differential splicing of the COOH-terminal exons (α, β, γ, δ, ε, ζ, Ë, Ë1 isoforms), of which the two major forms are p73α and p73β. These different splicing variants at the COOH-terminus were shown to have variable homo- and heterotypic interactions between themselves and with p53 whereas the ∆N-p73, that lacks the transactivation domain, exerts a dominant negative function towards p53 and p73 activity. Therefore, it is likely that the various products of this gene participate, in different ways, in a complex network that regulates cell growth, death, and differentiation giving rise to a family of proteins that adds a new level of complexity to the understanding p73 signalling in cancer cells. Indeed, several studies demonstrated that expression of p73 is markedly enhanced during differentiation of myeloid leukemic cells. We and others have shown that leukemic blasts from acute myeloid leukemia (AML) patients exhibit an increased expression of shorter p73 isoforms (γ, δ, ε). In addition, we described a distinct expression pattern of the ∆N-p73 isoform in the peculiar subset acute promyelocytic leukemia (APL) as compared to other AMLs. Here, we provide an overview of the potential role of p73 isoforms in acute myeloid leukemias (AMLs). We speculate that a complex p73 isoform profile with alterate expression pattern of a particular p73 variant (∆N-p73 in APL) might represent non-mutational mechanisms of leukemogenesis whose study can shed light on the pathogenesis of AMLs.

Keywords: p73, isoforms, human neoplasia, leukemias



393-400

Mutations of tumour suppressor gene P53 (TP53) in tumour tissue and cellular urine sediments in urinary bladder cancer

Author(s): Dr. Thorsten H. Ecke,

Abstract: TP53 mutations are frequently correlated with tumour development in bladder cancer. One function of TP53 is the suppression of apoptosis. We compared TP53 mutation in tumour tissue and urine sediment of urinary bladder cancer patients. We examined 32 patients with urinary bladder cancer. Screening for TP53 mutations in tumour tissue and urine sediment: Amplification of the TP53 gene by polymerase chain reaction (PCR) for the exons 5, 6, 7 and 8; Temperature gradient gel electrophoresis (TGGE) was used to analyse the TP53 mutations. We detected mutations of TP53 in invasive bladder cancer in 5 of 5 cases (100%); in superficial bladder cancer we found 20 of 27 cases (74%) with mutation of TP53. In 14 of 25 patients (56%) we could find TP53 mutation mobility shifts in the same exons analyzed in both tumour tissue and urine sediment. In some cases we could localize the same mutation of TP53 in both materials. TP53 mutations are detectable in early bladder cancer stages by PCR-TGGE and sequencing, which might influence therapeutic strategies. The identification of gene mutations in extracorporal samples, such as urine sediment, is an important area of research having implications for tumour diagnosis and monitoring. This study show that TP53 mutation are detectable in tumour tissue and in cellular urine sediment; beside that we demonstrate that TP53 mutation frequently occur in higher stages and grades of bladder tumours. The included review of the literature and the results of this study make clear that scientific work in the field of tumour markers in urinary bladder cancer is an important theme in urology nowadays and in future.

Keywords: Bladder cancer, p53, tumour suppressor gene, mutation, urine sediment



401-416

T cell activity in glioma chemoresponsiveness and genetics

Author(s): Dr. Christopher J. Wheeler,

Abstract: The dismal prognoses suffered by malignant primary brain tumor (glioma) patients remain unchanged over the past two decades despite significant improvements in the treatment of distinct tumors. Immunotherapy, and vaccine therapy in particular, represents a promising experimental approach to treat malignant gliomas, but major challenges still remain to render vaccination clinically effective. Combining vaccination with distinct therapies may be beneficial in this regard. For example, clinical chemotherapeutic responsiveness of glioblastoma multiforme (GBM) appears enhanced after therapeutic vaccination, and correlates with age-associated levels of nascent CD8+ T cells in patients. This review places these findings in the backdrop of previous research into glioma immunity, and introduce preliminary data validating a causative influence of T cells on glioma chemosensitivity. Further preliminary data suggesting an increase in a chromosomal marker of chemosensitivity in glioma tissue after therapeutic vaccination, and its bearing on the mechanism of T cell-induced glioma chemosensitivity is also discussed. Further insight into the dynamics of immune-tumor interactions promises to extend the reach of vaccine therapy by delineating the potential for immune synergy with conventional or targeted treatments.

Keywords: glioma chemoresponsiveness, Genetics, malignant gliomas, Cancer vaccines and dendritic cells, antigen-specific T cells, Molecular and cellular interactions, anti-glioma immunity, endogenous immune suppression, T cell-induced glioma chemosensitivity



417-422

Synthetic riboregulators – an alternative means to control gene expression

Author(s): Dr. Beatrix Suess,

Abstract: During the last years, the important role RNA plays for regulating gene expression in all organisms has become obvious. Consequently, several recent approaches aim to utilize the outstanding chemical properties of RNA in developing artificial RNA regulators for conditional gene expression systems. Rational design, in vitro selection and in vivo screening systems have been combined to create a versatile set of RNA based molecular switches. These tools rely on diverse mechanisms and exhibit activity in several organisms. In this review, we summarize recent developments in the application of synthetic riboswitches for gene regulation in vivo.

Keywords: engineered riboswitches, aptamer, ribozyme, antisense, conditional gene expression



423-430

Craniosynostosis: current treatment and future therapy

Author(s): Dr. Michael T. Longaker,

Abstract: Craniosynostosis, the premature fusion of cranial sutures, is one of the most common congenital craniofacial conditions. It can lead to severe neurological and cognitive deficits in children. Surgery, with its inherent risks, is currently the only therapeutic modality. In the investigation of growth factors involved in cranial suture biology, including the fibroblast growth factors (FGF), transforming growth factor-β (TGF-β), bone morphogenetic proteins (BMP), and BMP antagonists, potential targets for biologically based therapy are identified. With new developments in nucleic-acid based therapeutics, it is conceivable that these growth factors can be targeted with gene therapy to serve as an adjunct to minimally invasive surgery.

Keywords: Craniosynostosis, Gene Therapy, Fibroblast Growth Factors, Transforming Growth Factor-β, Bone Morphogenetic Proteins



431-444

Mammalian integrin-targeted gene delivery: A common approach for advanced viral and non-viral vectors

Author(s): Dr. Toshihiro Akaike,

Abstract: Development of efficient viral as well as non-viral vectors has received considerable attention for their successful applications in gene therapy and DNA vaccination programs in addition to their routine uses in proteomics and genomics. A major impediment to proper implementation of the DNA transfer vectors is the lack of a suitable cell membrane crossing device on their surface limiting transgene expression level in a variety of cells or tissues. Among the approaches designed so far for enhancing DNA delivery through receptor-mediated endocytosis, integrin- dependent devices in the viral and non-viral routes have shown tremendous potential for the above applications. Both genetically and synthetically oriented integrin recognizable domains, such as RGD motif (or peptide) in the DNA delivery devices remarkably and selectively increase gene expression level. Through this review we will focus on the recent successes in designing such devices with proper implementation both in the viral and non-viral aspects and particularly the latest finding of accelerated gene delivery based on extracellular matrix (ECM) protein- embedded calcium phosphate (CaP) nano-crystals.

Keywords: integrin, RGD, collagen, fibronectin, gene delivery, transgene expression, calcium phosphate nano-particles, adenovirus, retrovirus, non-viral vectors



445-456

Caffeine affects the level of gene repair in mammalian cells; implications for a role of DNA replication in the correction of single base mutations

Author(s): Dr. Eric B. Kmiec,

Abstract: Single stranded DNA oligonucleotides have been used to direct the correction of point mutations in mammalian cells. The introduction of a single-stranded oligonucleotide sets in motion a cascade of events that eventually leads to a stalling of DNA replication forks, which enables the oligo-mediated correction event to occur more readily. This is due to the fact that cells in S phase are more amenable to gene correction and a role for DNA replication in the gene repair reaction has been established. Here, we develop a mechanistic view of how cell cycle arrest might influence the gene repair reaction using the radiosensitizing agent, caffeine, as a tool. First, we demonstrate that ATM activation is enhanced and sustained by caffeine then we identify multiple roles for caffeine in the modulation of the gene repair reaction. Finally, we show that caffeine-treated cells retain the oligonucleotides for longer periods of time, perhaps enabling the oligo to assimilate into the target site more readily. Our results may define ATM activation upon the entry of the oligonucleotide as a likely block to the proliferation of corrected cells.

Keywords: repair, caffeine, oligonucleotides, cell cycle, replication, DLD-1




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