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Volume 1 - 1998


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1-172

Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications

Author(s): Dr. Teni, Boulikas,

Abstract: Gene therapy is a newly emerging field of biomedical research aimed at introducing therapeutically important genes into somatic cells of patients; a new and revolutionary era in molecular medicine has begun. Diseases already shown to be amenable to therapy with gene transfer in clinical trials include cancer (melanoma, breast, lymphoma, head & neck, ovarian, colon, prostate, brain, chronic myelogenous leukemia, non-small cell lung, lung adenocarcinoma, colorectal, neuroblastoma, glioma, glioblastoma, astrocytoma, and others), AIDS, cystic fibrosis, adenosine deaminase deficiency, cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease,  1-antitrypsin deficiency, rheumatoid arthritis and a few others. Human diseases expected to be the object of clinical trials include hemophilia A and B, Parkinson’s disease, ocular diseases, xeroderma pigmentosum, high blood pressure, obesity and many others. The establishment of novel animal models for human disease, the discovery of new genes, and improvements in successful gene delivery open bright new prospects for molecular medicine. A wide variety of delivery vehicles for genes have been tested including murine retroviruses, recombinant adenoviral vectors, adeno-associated virus, HSV, EBV, HIV vectors, and baculovirus. Nonviral gene delivery methods use cationic or neutral liposomes, direct injection of plasmid DNA, and polymers. Various strategies to enhance efficiency of gene transfer have been tested such as fusogenic peptides in combination with liposomes, or polymers, to enhance the release of plasmid DNA from endosomes. Recombinant retroviruses stably integrate into the DNA and require host DNA synthesis; adenoviruses can infect nondividing cells but cause immune reactions leading to the elimination of therapeutically transduced cells. Adeno-associated virus (AAV) is not pathogenic, does not elicit immune responses but new strategies are required to obtain high AAV titers for preclinical and clinical studies. Wild-type AAVs integrate into chromosome 19 whereas recombinant AAVs are deprived of site-specific integration and may also persist episomally; HSV vectors can infect nonreplicating cells such as neuron cells, have a high payload capacity for foreign DNA but inflict cytotoxic effects. It seems that each delivery system will be developed independently of the others and that each will prove its strengths for specific applications. At present, retroviruses are most commonly used in human clinical trials followed by adenoviruses, cationic liposomes and AAV. Polymer-encapsulated syngeneic or allogeneic cells implanted into a tissue of a patient can be used to secrete therapeutic proteins; the method is in trials for 1 Boulikas: An overview on gene therapy amyotrophic lateral sclerosis using the ciliary neurotrophic factor gene, and can be extended to Factor VIII and IX for hemophilia, interleukin genes, dopamine-secreting cells to treat Parkinson's disease, nerve growth factor for Alzheimer's disease and other diseases. Ingenious techniques under development with great future prospects for human gene therapy, include the Cre-LoxP recombinase system to rid of undesirable viral DNA sequences used for gene transfer, use of tissue- specific promoters to express a gene in a particular cell type or use of ligands, such as peptides selected from random peptide libraries, recognizing surface molecules to direct the gene vehicle to a particular cell type, designing p53 “gene bombs” that explode into tumor cells, exploit the HIV-1 virus to engineer vectors for gene transfer, the combining of viruses with polymers or cationic lipids to improve gene transfer, the attachment of nuclear localization signal peptides to oligonucleotides to direct them to nuclei, and the invention of molecular switch systems allowing genes to be turned on or off at will. Although many human tumors are non- or weakly immunogenic, the immune system can be reinforced and instructed to eliminate cancer cells after transduction of patient’s cells ex vivo with the cytokine genes GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN- , and TNF- , followed by cell vaccination of the patient (e.g. intradermally) to potentiate T-lymphocyte-mediated antitumor effects (cancer immunotherapy). DNA vaccination with genes encoding tumor antigens and immunotherapy with synthetic tumor peptide vaccines are further developments in this exciting field. The genes used for cancer gene therapy in human clinical trials include a number of tumor suppressor genes (p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc, K-ras), and suicide genes (HSV-tk, in combination with ganciclovir, cytosine deaminase in combination with 5-fluorocytosine). Important in gene therapy are also the genes of bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, CFTR, LDL-R, TGF- , and leptin. Reports on human clinical trials using adenoviral and retroviral injections of the p53 gene have been very encouraging; future directions might go toward the use of genes involved in the control of tumor progression and metastasis. The molecular mechanisms of carcinogenesis have been largely elucidated and improvements in gene delivery methods are likely to lead to the final victory of the human race in the fight against cancer and other deadly diseases.

Keywords: gene therapy, gene transfer, clinical trials, cancer, immunotherapy, p53, adenovirus, retrovirus, adeno-associated virus, HIV-1, HSV-1, EBV, AIDS, tumor vaccines, IFN-γ, TNF-α, VEGF, retinoblastoma, purine nucleoside phosphorylase, HSV- tk, E1A, E1B, Cr



173-214

The challenge of liposomes in gene therapy

Author(s): Dr. Francis, Martin,

Abstract: Recently, liposomes have gained a special interest as gene delivery systems: over 30 human clinical trials for gene delivery using cationic liposomes have been approved; all these delivery methods use intratumoral, subcutaneous and other local delivery but not systemic delivery due to the toxicity of cationic lipids. Stealth liposomes (coated with polyethyleneglycol to camouflage the liposome and evade detection by the immune system) have a remarkable longevity in body fluids, have negligible toxicity with respect to their lipid components, reduce the toxicity of the encapsulated drug, and can deliver efficiently their doxorubicin payload (DOXIL) or cis-platin to tumor lesions. The mechanism of stealth liposome accumulation in tumors involves their extravasation through gaps in the endothelium of tumor vessels. DOXIL can sustain a much higher concentration of Doxorubicin in tumor tissue compared to free drug administration at comparable doses. Liposomes tagged with folate-PEG or with antibodies can target specific tissues. We propose that “stealth” liposomes, could find future applications to systemically deliver plasmid DNA with therapeutic genes (p53, HSV-tk, angiostatin) to primary tumors and their metastases leading to complete cancer eradication.

Keywords: liposomes, p53, doxorubicin



215-229

Gene transfer to the nervous system using HSV vectors

Author(s): Dr. Joseph C., Glorioso,,

Abstract: The natural history of Herpes simplex virus type 1 (HSV-1) infection in humans suggests its potential for development as a gene transfer vehicle suitable for nervous system applications. HSV-1 has a broad host range, does not require cell division for infection and gene expression and has evolved to persist in a life-long nonintegrated latent state without the expression of viral proteins or evidence of neurodegenerative disease in the immune competent host. The virus also has evolved a unique neuronal-specific promoter system that remains active during latency and fortuitously may be used to express therapeutic proteins without compromising the latent state. The establishment of latency also does not require the expression of viral lytic functions and thus removal of genes required for expression of the viral cascade of expressed products allows for safe vector design without the possibility of reactivation from latency. The HSV-1 genome is 152 Kb in length and of the 84 known genes (Roizman & Sears, 1996), approximately half are dispensable for virus replication in cell culture thereby providing considerable opportunity for introduction of foreign sequences. In this review, a brief overview of the biology of HSV relevant to vector design and progress in reducing virus cytotoxicity and its relevance to the level and duration of transgene expression is discussed. Methods for the expression of transgenes in the peripheral and central nervous system using the latency active promoters are described and strategies are suggested for potential applications to the treatment of neurodegenerative disease and cancer.

Keywords: HSV-1, mechanisms



231-239

The baculovirus vector system for gene delivery into hepatocytes

Author(s): Dr. Michael, Strauss,

Abstract: Gene therapy in the liver requires powerful vectors capable of mediating sufficient gene delivery and expression in affected hepatocytes. Viral vectors are amongst the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in vivo gene therapy strategies. We have recently shown for the first time that a genetically modified baculovirus Autographa californica can efficiently and specifically transfer genes into cultured liver cells from various origin. The efficiency of baculovirus-mediated gene transduction into hepatocytes was determined to approach 100% using appropriate virus titers. Apart from these features, potential advantages of baculovirus vectors are the nearly unlimited capacity for insertion of foreign DNA, a supposed restriction of viral promoters to the arthropod host and the ease of generating high vector titers. Uptake of the virus occurs via the endosomal pathway, most likely via a receptor that is currently under investigation. Baculovirus-mediated gene expression is transient in dividing cells, but prolonged expression can be achieved in non-dividing primary hepatocytes. Baculovirus-mediated gene transfer is feasible into ex vivo perfused human liver tissue. Systemic application of baculovirus vectors in vivo is hampered by the complement (C) system. Current attempts to facilitate baculovirus-mediated gene transfer in vivo include strategies for both, blocking or avoiding the C system and generation of new baculovirus vectors that are not affected by the C system. Alternatively, direct injection of baculovirus vectors was successful into normal mouse liver and into induced human hepatocarcinomas in nude mice. The potential of baculovirus vectors in vitro and the feasibility of vector application in vivo provide the basis for gene therapy strategies for metabolic diseases and tumors of the liver.

Keywords: liver, baculovirus, viral promoters



241-251

Delivery systems for the MDR1 gene

Author(s): Dr. Michael M., Gottesman,

Abstract: The acquired resistance of cancer cells to a wide variety of structurally unrelated anti-cancer drugs as well as the increased sensitivity of cells of the hematopoietic system to these same drugs have contributed to the limited success of long term cancer chemotherapy. The overexpression of the multidrug resistance (MDR1) gene was found to be associated with this acquired resistance and has been exploited to protect normal bone marrow cells from myelosuppression that may result in life threatening leukopenia and thrombocytopenia following high dose chemotherapy. Thus far, retroviral transfer of the MDR1 gene has been the main route of delivery into bone marrow cells. Although widely used as one of the most efficient vehicles for gene delivery, safety and other concerns associated with viruses cannot be ignored. We have explored other means of introducing the MDR1 gene into recipient cells. A variety of vectors can be introduced into cultured cells and bone marrow cells in vitro using lipofection. One new system under development uses lipofection to introduce an Epstein Barr Virus (EBV)-based episomal vector carrying the MDR1 cDNA. High efficiency transfection of cultured cells has been achieved with this system.

Keywords: MDR1, Drug Resistance, cancer



253-263

Tumor killing using the HSV-tk suicide gene

Author(s): Dr. Scott M., Freeman,

Abstract: The Herpes Simplex virus thymidine kinase gene (HSV-tk) has been widely used as a suicide gene in cancer gene therapy. Since the first demonstration of the ability of HSV-tk gene modified tumor cells to generate a bystander effect, a number of clinical trials have been initiated to treat human cancers. However, the mechanism of the HSV-tk mediated bystander tumor killing has remained controversial and is under intense investigation. The present report discusses the various mechanisms proposed by which the HSV-tk mediated bystander tumor killing occurs and highlights the importance of the host immune system in mediating the tumor killing. In addition, the present report also demonstrates that the initial tumor killing results in an inflammatory response leading to a cytokine cascade. This subsequently leads to an immune response resulting in an influx of macrophages and tumor infiltrating lymphocytes. Finally, strategies to augment the HSV-tk mediated bystander tumor killing by immunization are discussed and conclude with potential pitfalls of using HSV-tk/GCV system in a clinical setting.

Keywords: HSV-tk, cancer, apoptosis, suicide gene therapy



279-292

Hemophilia A: current treatment and future gene therapy

Author(s): Dr. Sheila, Connelly,

Abstract: The last two decades has seen significant progress in the treatment of hemophilia A. The development of highly purified and recombinant FVIII pharmaceutical products has dramatically increased the life expectancy and quality of life for many hemophiliacs. However, the high cost and short supply of these replacement products has resulted in their availability limited to less than 10% of the world’s hemophiliac population. Gene therapy for hemophilia A would provide prophylactic expression of FVIII and correction of the coagulation defect. A gene therapy protocol allowing simple, infrequent vector administration may extend hemophilia treatment to remote locations worldwide that currently lack access to FVIII replacement therapy. While progress has been made with each of the gene therapy vector systems described below, each still faces obstacles to its clinically utility. However, with the efforts that are currently directed toward overcoming these limitations, gene therapy for hemophilia A will ultimately become a reality.

Keywords: Hemophilia, FVIII gene, VSV G protein



293-300

Gene therapy for haemophilia

Author(s): Dr. Rob C, Hoeben,

Abstract: Gene therapy is an appealing prospect for the treatment of human diseases. In this chapter, I will describe the hopes that gene therapy has brought for hemophilia patients, as well as the hurdles that the researchers have encountered on the route that shall lead to the development of a clinically applicable protocol.

Keywords: haemophilia, factor VIII,



301-308

Kallikrein gene therapy in hypertension, cardiovascular and renal diseases

Author(s): Dr. Julie, Chao,

Abstract: Somatic gene delivery approaches have received wide attention in recent years as a new technique for studying gene expression and as a potential therapeutic tool in treating both inherited and infectious diseases. Hypertension, which is a polygenic disease influenced by environmental and dietary factors, shows abnormality of the tissue kallikrein-kinin system in its pathogenesis. To demonstrate potential therapeutic effects of gene delivery in treating hypertension, we introduced the human tissue kallikrein gene in the form of naked DNA or an adenoviral vector into hypertensive rats. A single injection of the kallikrein gene caused a sustained blood pressure reduction for several weeks in spontaneously hypertensive rats (SHR), two kidney-one clip (2K1C) Goldblatt hypertensive rats, and Dahl salt-sensitive (Dahl-SS) rats. The expression of human tissue kallikrein in rats receiving gene delivery was identified in tissues relevant to cardiovascular function including the kidney, heart, aorta, lung and liver. Adenovirus-mediated kallikrein gene delivery attenuated cardiac hypertrophy and renal damage in 2K1C and Dahl-SS rats fed on a high salt diet. Kallikrein gene delivery also caused significant increases in renal blood flow, glomerular filtration rate and urine flow as well as in water intake, urine excretion, urinary electrolyte output, kinin, nitrite/nitrate (NOx) and cGMP levels. These findings are consistent with the mechanisms of blood pressure reduction and enhanced renal function mediated via kinin through a NO-cGMP dependent signal transduction pathway following kallikrein gene delivery. The ability of kallikrein gene delivery to produce a wide spectrum of beneficial effects makes it an excellent candidate in treating hypertensive, cardiovascular and renal diseases.

Keywords: hypertension, kallikrein gene delivery



309-321

Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination

Author(s): Dr. Max L., Birnstiel,

Abstract: With the advent of gene therapy there has been a revival of immunotherapy of cancer. Preclinical studies with the so called tumor vaccines – syngeneic, irradiated tumor cells secreting cytokines – are at present entering clinical trials and hold much promise for efficacious treatment, maybe even cures, of cancer. However, autologous whole cell vaccines which are cytokine gene-modified are expensive and difficult to standardize. In addition, autologous tumor cell cultures, and hence tumor vaccines, cannot be prepared for all patients. For these reasons we have investigated alternative schemes for vaccination against cancer. We have developed vaccines on the basis of tumor antigen- derived peptides using a method, we called ”transloading”, to transfer peptides efficiently into cells. Such vaccines are chemically well defined and inexpensive to produce. We show in a preclinical mouse model system for mastocytoma, and to a somewhat lesser extent for melanoma, that peptide vaccines give excellent protection against tumor take, provided tumor antigen peptides are injected subcutaneously in conjunction with polycations like polyarginine. Immunohistological investigations on thin sections show that three days after injection, the vaccine bolus is heavily infiltrated by antigen presenting cells which take up peptides. We posit that such antigen presenting cells then migrate into draining lymph nodes where they activate naive T cells to become anti-tumor cytotoxic lymphocytes (CTL). The consequences of strong dependency of peptide sequence on the HLA-type of patients and possible remedies are discussed. One avenue to be tested in mouse models is the injection of mixtures of peptides covering a multiplicity of MHC-specific peptides. Another is the production of recombinant proteins as vaccines whose application is considerably less dependent on HLA-types of patients to be treated. In addition, such proteins would be expected to contain both class I and class II restricted peptides. Alternatively, artificial proteins incorporating many CTL epitopes of known tumor antigens, including members of the MAGE family, thus yielding a SUPERMAGE vaccine with utility for several human HLA- types could be designed. The prophylactic application of peptide/polyepitope vaccines can be envisaged.

Keywords: cancer, immunotherapy, HLA heterogeneity, peptide vaccines



323-332

Somatic transgenesis by immunoglobulin genes

Author(s): Dr. Maurizio, Zanetti,

Abstract: In this chapter we describe and discuss somatic transgenesis produced in adult immunocompetent mice using plasmid DNA containing immunoglobulin genes under control of tissue-specific regulatory elements. We review our experience to date and discuss the findings in relation to the known rules for intracellular usage of immunoglobulin genes in activated and differentiated B cells. Because immunoglobulin genes are controlled by B lymphocytes specific promoter and enhancer elements, somatic transgenesis is a new approach to selective targeting of B lymphocytes in vivo for transcription and long-term expression of exogenous immunoglobulin genes. Owing to the fact that transgenic immunoglobulins synthesized and secreted in vivo are immunogenic for the host and that immunoglobulin genes can be engineered to code for heterologous epitopes, ligands or receptors, somatic transgenesis offers unique features for the development of new strategies of DNA-based immunization and gene therapy.

Keywords: H chain transgene, immunoglobulin genes



333-344

Applications of gene therapy in transplantation

Author(s): Dr. Mary E. , White-Scharf,

Abstract: The success of organ transplantation is limited by the availability of donor organs and the requirement for long-term immunosuppression. Use of animal organs would circumvent the organ shortage, and the swine is the most likely candidate to serve as donor for xenotransplantation. Establishing specific tolerance to donor organs would eliminate the need for chronic immunosuppression. Specific tolerance to transplantation antigens across major histocompatibility complex (MHC) barriers has been demonstrated by establishing mixed bone marrow chimerism in the recipient. An alternative approach to establishing chimerism is the use of gene therapy to transduce autologous bone marrow cells with a vector containing one or more MHC antigens. Previous studies in miniature swine have shown that tolerance can be established across allogeneic barriers when a single MHC antigen is shared between donor and recipient. A gene therapy approach is particularly attractive in xenotransplantation since the availability of MHC-inbred miniature swine as donors allows the use of a single vector for transferring swine MHC genes into recipient progenitor cells. In this article, we review the basics for using a gene therapy approach to achieve specific immune tolerance and describe the models being used to evaluate its efficacy in allogeneic as well as in xenogeneic organ transplantation.

Keywords: MHC gene, transplantation



345-363

Myoblast transfer as a platform technology of gene therapy

Author(s): Dr. Peter, Law,

Abstract: Myoblasts divide profusely, and fuse during muscle regeneration, interiorizing MHC-I antigens and inserting myonuclei with the normal genome into muscles of genetically deficient recipients, where any replacement gene can be stably integrated and naturally expressed. Myoblasts are the natural source and vehicle for many gene therapies. Myoblast transfer therapy is completing US FDA Phase II clinical trials for Duchenne muscular dystrophy.

Keywords: Myoblast transfer, Clinical trials, Gene therapy, Duchenne muscular dystrophy, Viral vectors



365-379

Constitutive activation of fibroblast growth factor receptors in human developmental syndromes

Author(s): Dr. Daniel J, Donoghue,

Abstract: Fibroblast growth factor receptors (FGFRs) represent specific receptors for the fibroblast growth factors (FGFs), a family of at least 13 polypeptides. Ligand/receptor interactions between FGFs and their receptors are involved in many fundamental biological processes, particularly cell growth and differentiation during chondrogenesis and myogenesis. The four different human FGFR genes encode related glycoproteins with a common structure consisting of an N-terminal signal peptide, three immunoglobulin (Ig)-like domains, a single transmembrane domain, and an intracellular split tyrosine-kinase domain. FGFs, acting in concert with heparan sulfate proteoglycans, bind to FGFRs and result in their activation, involving homo- or hetero-dimerization of receptors, leading to trans-phosphorylation of the kinase domains. The activated receptors can then phosphorylate various intracellular proteins involved in signal transduction, although much remains to be learned concerning these signal transduction pathways downstream of activated FGFRs. Many mutations in different domains of FGFR1, FGFR2 and FGFR3 have recently been identified as causing various human craniosynostosis and dwarfism syndromes, and the molecular consequences of these mutations are beginning to be unraveled. Craniosynostosis syndromes, characterized by premature ossification and fusion of the cranial sutures of the skull, arise primarily from mutations in the extracellular domain FGFR2, although specific mutations in other FGFRs may also underlie related craniosynostosis syndromes. Skeletal dwarfism syndromes, characterized by disproportionate short stature and macrocephaly, arise predominantly from mutations in FGFR3 and include achondroplasia, the most common genetic form of dwarfism, as well as the thanatophoric dysplasias (type I and type II). Recent studies demonstrate that a common mechanism, constitutive activation of receptor signaling, underlies most of these disorders. The mutations responsible for the craniosynostosis and skeletal dwarfism syndromes map variously to either the extracellular domain, the transmembrane domain, or the tyrosine kinase domain of these receptors, suggesting multiple mechanisms of aberrant receptor activation. An overview of the developmental consequences arising from mutations in FGFR family members will be presented, including an examination of the molecular mechanisms underlying these defects.

Keywords: FGFR3; Thanatophoric Dysplasia; skeletal malformation; achondroplasia; receptor tyrosine kinase



381-398

Genes involved in the control of tumor progression and their possible use for gene therapy

Author(s): Dr. Georgii P, Georgiev,

Abstract: Three major groups of genes may be used for cancer gene therapy: (i) oncogenes and tumor suppressor genes; (ii) genes involved in the control of tumor progression and metastasis; and (iii) genes encoding proteins protecting the organism from tumor cells. Each group contains numerous genes, and the discovery of new important genes is an exciting prospect in cancer research. We are working on the search and characterization of the genes over- or under-expressed in metastatic comparing to non-metastatic tumors of the same origin. Two mouse systems are being used: (i) VMR-0 (non-metastatic mammary adenocarcinoma cells) - VMR-100-Liv and VMR-100-Ov cells (metastatic preferentially to the liver or ovaries, respectively); and (ii) CSML-0 - CSML-100 (mammary adenocarcinoma cells non-metastatic and metastatic to the lungs, respectively). Several different genes were found to be over-expressed in metastatic cells, but only few of them were shown to be necessary and sufficient for maintaining the metastatic phenotype using stably transfected cells and/or transgenic animals. Among them are the mts1 and c-met genes. The mts1 gene, encoding a calcium-binding protein of 101 amino acids of the S-100 family, was extensively characterized. Its expression induced a number of changes in cell functions connected with cytoskeleton features, attachment properties of the cell, mesenchyme formation and possibly tumor vascularization. As a multifunctional regulator, the mts1 gene is a promising target for gene therapy of cancer. Other genes identified are over-expressed only in few metastatic tumors and do not seem to be connected directly with the acquisition of the metastatic phenotype. However, during the transfection experiments some interesting features emerged for these genes, raising the possibility of their exploitation in cancer gene therapy. The most interesting is the tag7 gene encoding a new cytokine, 182 amino acids long, with a far distant relation to cytokines of the TNF-Lymphotoxin family. The tag7 gene is expressed in lymphoid cells, in a limited set of other normal cells, and in few cancer cells including myelomas. The Tag7 protein is secreted to the culture medium and possesses a strong cytotoxic activity inducing apoptosis. VMR-0 cells were stably transfected with a construct containing the tag7 gene under control of the CMV promoter. The original VMR-0 tumors killed mice in one month after subcutaneous transplantation; animals displayed large necrotic foci at this stage. However, the VMR-0/tag7 cells, synthesizing very low amounts of Tag7 protein, exhibited dramatically different growth properties: they grew much slower; even after 4 months, no mice were killed by tumors arising from the transplanted cells and no necrotic foci were formed. Histological analysis of VMR-0/tag7 tumors showed a strong inhibition in mitotic rates and an enhanced rate of apoptosis compared to VMR-0 tumors. The tumors induced by transplantation of a mixture of VMR-0 and VMR-0/tag7 cells also grew much slower than VMR-0 cells alone, suggesting an activation of the immune system against tumor (tumor vaccination effect), which may be mediated through induction of CTL cells. Experiments with nude mice gave similar results. In fact at later stages of development in nude mice, VMR-0/tag7 tumors were completely eradicated. It seems that the effect of tag7 expression is complex and includes 381 Georgiev et al: Genes involved in the control of tumor progression in gene therapy activation of an immune response as well as a direct cytotoxicity. The higher tag7 expression in culture cells is incompatible with cell survival. Experiments are in progress for further elucidating the role of Tag7 and its exploitation for the development of tumor vaccines.

Keywords: cancer, tag7 gene



399-406

The Apoptin® gene of chicken anemia virus in the induction of apoptosis in human tumorigenic cells and in gene therapy of cancer.

Author(s): Dr. Mathieu, Noteborn,,

Abstract: Apoptosis is an active physiological process for the elimination of superfluous or altered cells from a developing or adult organism. Aberrations in the apoptotic process cause various diseases, e.g. tumor formation. Chemotherapy for the treatment of cancer often exerts its cytotoxic effect via the induction of apoptosis and its success depends mainly on the presence of functional p53 and the absence of an overexpressed Bcl-2 proto-oncogene. Unfortunately, in more than 50% of the tumors functional p53 is lacking, and/or Bcl-2 is overexpressed, which often results in resistance to anti-cancer therapy. Apoptin,1, a protein derived from chicken anemia virus (CAV), can induce apoptosis in cultured (human) cells derived from various tumors, e.g., osteosarcoma, lymphoma, leukemia, hepatoma, melanoma, and from tumors of breast, colon, and lung. Tumor cells lacking p53 can undergo Apoptin-induced apoptosis, and over-expression of Bcl-2 even stimulates Apoptin- induced apoptosis. Downstream inhibitors of the p53-pathway, like Bcl-2, BAG-1 and CrmA, do not inhibit Apoptin-induced apoptosis, which indicates that Apoptin induces cell death via a different pathway or that Apoptin acts at a step far downstream the apoptotic cascade. Interestingly, Apoptin fails to induce apoptosis in human primary lymphoid, dermal, epidermal, endothelial and smooth muscle cells. Co-expression of a transforming agent with Apoptin in normal diploid cells results in apoptosis. The fact that Apoptin induces a p53-independent, and Bcl-2-stimulated type of apoptosis in human tumor cells, but not in normal diploid cells, renders Apoptin a potential anti-tumor agent. Gene-therapy strategies based on viral vectors expressing Apoptin are currently under development.

Keywords: cancer, Chicken Anemia Virus, apoptosis



407-418

Tissue-specific triple ribozyme vectors for prostate cancer gene therapy

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

Abstract: Ribozymes are naturally occurring catalytic RNAs which can be targeted to bind and cleave other RNA molecules with high specificity. Ribozyme-mediated inhibition of gene expression at the message level represents a powerful therapeutic tool. Implement of ribozymes in gene therapy has focused mainly on HIV and cancer. Use in HIV protocols has been widespread due to the wealth of targets offered by a well-characterized RNA retrovirus. Ribozyme utility in cancer has centered on oncogene inhibition in tumors with well defined genetics. As molecular mechanisms of prostate cancer are poorly understood, ribozyme targets must be universal with either delivery or expression firmly restricted to the area of interest. Overall, continued research on ribozyme design, delivery vehicles, tissue specificity, and the genetic makeup of disease will allow ribozymes to fulfill their considerable potential as therapeutic agents.

Keywords: ribozyme technology, cancer, HIV



419-434

A novel system for selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli

Author(s): Dr. Kazunari, Taira,

Abstract: If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In the DHFR expression vector, a sequence encoding either an active or an inactive ribozyme was connected either upstream (5'-connected active or inactive ribozyme) or downstream (3'-connected active or inactive ribozyme) of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). In the case of both 5'-connected and 3'-connected ribozymes, the active ribozyme did indeed cleave the primary transcript in vivo, whereas inactive ribozymes had no cleavage activity. We confirmed that cells that harbored the active ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored an inactive ribozyme-coding plasmid. Consequently, when cells were transformed with a mixture that consisted of active ribozyme-coding and inactive ribozyme-coding plasmids at a ratio of 1:1, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP. These results indicated that our positive selection system in vivo was functional and that, moreover, if the background "noise" could be removed completely in the future, it might usefully complement existing selection systems in vitro.

Keywords: DHFR, molecular cloning, E. coli



451-466

Isolation and characterization of an RNA that binds with high affinity to Tat protein of HIV-1 from a completely random pool of RNA.

Author(s): Dr. Penmetcha K. R., Kumar,

Abstract: The trans-activation (Tat) protein of human immunodeficiency virus type-1 (HIV-1) is vital for the replication of the virus. In a transcription assay in vitro in the presence of authentic TAR RNA, we found that authentic TAR RNA inhibits transcription from a template based on the CMV early promoter in a manner that is not related to the Tat/TAR interaction. Using variants of TAR RNA, we identified potential sequences of RNA that seems to be responsible for the inhibition of transcription. In addition, we isolated an RNA aptamer that can bind with high affinity to Tat protein specifically. The isolated aptamer appears to include two TAR-like RNA motifs for higher- affinity binding to Tat peptides. The aptamer's high affinity for Tat peptides, as well as the absence of any inhibitory effects on the transcription of unrelated genes, as opposed to those of TAR RNA, suggests that the novel RNA might be very useful as a Tat-specific decoy.

Keywords: trans-activation (Tat) protein, HIV, 11G-31 RNA



467-474

Sequence-specific control of gene expression by antigene and clamp oligonucleotides

Author(s): Dr. Claude, Hélène,

Abstract: Gene expression can be artificially controlled by synthetic oligonucleotides that bind either to the gene itself or to its messenger RNA. Binding of an antigene oligonucleotide to the major groove of DNA involves the formation of a triple helix. Covalent attachment of an intercalating agent to the third strand oligonucleotide strongly stabilizes the triple-helical complex. Oligonucleotide-intercalator conjugates inhibit transcription factor binding and transcription initiation. Introduction of N3'  P5' linkages into the oligonucleotide leads to stronger complexes that are able to arrest the transcription machinery at the elongation step. Cellular DNA is accessible to oligonucleotides within the chromatin structure of the cell nucleus as demonstrated for HIV proviral DNA in chronically infected cells. A triple helix can be formed on a single-stranded nucleic acid by an oligonucleotide made of two portions : one binds to the target sequence by forming Watson-Crick base pairs, the second one binds to this double-helical region to form a triple-helical complex. These clamp oligonucleotides are able to arrest replication of a single-stranded DNA or reverse transcription of a viral RNA. The antigene and clamp strategies can be adapted to a gene therapy protocol. A DNA expression vector is used to obtain a RNA transcript that can bind to DNA (antigene RNA) and block transcription.

Keywords: antigene, Clamp oligonucleotides, triple helix-formation,



475-482

Physical approaches to the study of chromatin fibers

Author(s): Prof. Jordanka, Zlatanova,

Abstract: Investigations of the structures of complex macromolecular assemblies (like chromatin fibers, microtubules, etc.) have traditionally utilized two approaches, which we term macroscopic and microscopic. The macroscopic methods include hydrodynamic and radiation-scattering techniques. While applicable to molecules in solution, they present results which are only averages over the molecules in the sample. If, as is usually the case, these structures are heterogeneous, interpretation can become hopelessly ambiguous. At the other extreme, the traditional high- resolution microscopic techniques (transmission electron microscopy and its numerous variants) while sensitive to even local variations in structure, impose often devastatingly harsh conditions on delicate biological structures. Recently, two kinds of microscopic methods have been developed which hold great promise for studies of macromolecular assemblies. The first is cryo-electron microscopy, which allows preservation of much of solution structures. Second, and potentially even more promising, are the various scanning probe microscopic methods, especially scanning force microscopy. In its present stage of development, this technique allows detailed structural studies under relatively mild conditions. Together, cryo-electron microscopy and scanning force microscopy have already provided new insights into the static structure of chromatin. Even more exciting are the prospects for imaging in liquid media, now under development. These hold promise for study of not only the statics, but dynamics as well, for functionally important structures like chromatin fibers.

Keywords: radiation-scattering techniques, chromatin fibers



483-494

Activation at a distance: involvement of nucleoprotein complexes that remodel chromatin

Author(s): Dr. Emery H, Bresnick,

Abstract: Large-scale sequencing of the human genome has confirmed that genes are often spread over several thousand base pairs on a chromosome. The cis-acting regulatory elements that control gene transcription can be located at a considerable distance from the respective gene. Significant advances in understanding the mechanism of eukaryotic gene transcription have revealed a great deal about how the RNA polymerase initiation complex assembles on the promoter region of genes. However, the fundamental problem of how distant genetic regulatory elements, such as enhancers and locus control regions, communicate with proximal elements to confer cell- and tissue-specific patterns of transcription remains largely unsolved. The principle focus of this chapter will be on the role of chromatin structure in transcriptional regulation by RNA polymerase II, with an emphasis on long-range activation.

Keywords: chromatin remodeling complexes, SWI/SNF, NURF, RSC, histone acetyltransferases, LCR



495-508

Dedicated sites of gene expression in the nuclei of mammalian cells.

Author(s): Dr. Dean A, Kackson,

Abstract: Establishing sites of transcription in the nuclei of higher eukaryotic cells is a very complex process. Before transcription can begin, a series of transcription factors must associate with their recognition motifs, within promoters and more remote activating sequences. Once bound, these factors and associated proteins are believed to form a complex that positions the RNA polymerase holoenzyme so that transcription can commence. As a consequence, active genes assume a specialized chromatin state across regions that define functional domains. Global nuclear architecture appears to stabilize these active domains by providing local environments dedicated to gene expression. As the spatial organization of these sites is unaffected by the removal of most chromatin they must be associated with a structural network. This nucleoskeleton, the associated transcription 'factories' and chromatin loops that arise as DNA binds proteins within factories are fundamental features of nuclear structure in higher eukaryotes. We argue that concentrating proteins needed to perform different steps of RNA synthesis within specialized nuclear compartments will be important in orchestrating events required for efficient gene expression.

Keywords: RNA, transport pathways,



509-528

Nuclear matrix: application to diagnosis of cancer and role in transcription and modulation of chromatin structure

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

Abstract: The nuclear matrix is involved in the processing of the genetic information and in the organization of chromatin. In recent years we have come to appreciate the organization of functional domains within the nucleus (e.g., transcript domains, RNA processing sites, sites of replication). The nuclear matrix is the foundation from which this organization is built, providing a scaffold upon which nuclear processes such as DNA replication and transcription occur. Chromatin is arranged into loop domains through the attachment of matrix associated regions (MARs) at the base of the loop to nuclear matrix proteins. Considering the role of the nuclear matrix in the organization and processing of the genetic information, it is not surprising to find that nuclear matrix proteins are informative in distinguishing cell types and disease states. For example, nuclear matrix proteins informative in the diagnosis of cancer, including breast cancer have been identified. Typically nuclear matrices are obtained following the salt extraction of nuclease-digested nuclei. However, recent studies show that cisplatin preferentially crosslinks MAR DNA to nuclear matrix proteins in situ, providing a complimentary method to identify informative nuclear matrix proteins. Transcribed, but not repressed chromatin, is associated with the nuclear matrix. Regions of a chromatin loop engaged in transcription are associated with the nuclear matrix through multiple dynamic interactions with nuclear matrix proteins. Nuclear matrix bound transcription factors, the transcription machinery and histone modifying enzymes are thought to mediate these dynamic attachments between the nuclear matrix and transcriptionally active chromatin. Core histones of transcriptionally active chromatin are dynamically acetylated, with histone acetyltransferases (HATs) and deacetylases (HDACs) catalyzing this reaction. Both of these enzymes, which are now known to be transcriptional coactivators and modulators, are associated with the nuclear matrix. We have proposed that these enzymes participate in the dynamic attachment of transcriptionally active chromatin with the nuclear matrix. Our recent studies show that cisplatin crosslinks nuclear matrix-bound transcription factors and transcription modulators to nuclear DNA in situ. This suggests that cisplatin will be most useful in the discovery of nuclear matrix MAR binding proteins involved in the organization of DNA and nuclear matrix bound transcription factors/modulators participating in the nuclear matrix association of transcriptionally active chromatin. (Supported by Medical Research Council of Canada)

Keywords: nuclear matrix, transcription factors, activators, transcriptionally active chromatin, Histone acetylation



529-542

Structural organization and biological roles of the nuclear lamina

Author(s): Dr. Yosef, Gruenbaum,,

Abstract: The nuclear lamina is a protein meshwork that lies on the nucleoplasmic side of the nuclear envelope and is associated with the peripheral chromatin. It is involved in several biological activities including: the mitotic disassembly and reassembly of the nuclear envelope, determination of the size and shape of the nucleus, higher order chromatin organization, cell differentiation, and apoptosis. Lamins are the major proteins of the nuclear lamina. They are type V intermediate filaments and, like all intermediate filaments, they form filamentous structures. Lamins can interact in vitro with specific DNA sequences, with chromosomal proteins and with several proteins of the inner nuclear membrane, including otefin, LBR, LAP1 and LAP2. In this paper we show that Drosophila lamin Dm0 and otefin proteins are required for the assembly of the Drosophila nuclear envelope. We also demonstrate that the lack of lamin Dm0 activity causes the dissociation of peripheral chromatin from the nuclear envelope, accumulation of annulate lamellae and lethality. In addition, we show that the carboxy (tail) domain of lamin Dm0 can interact in vitro with chromosomes and the central (rod) domain of lamin Dm0 is essential and sufficient for the in vitro assembly of lamin Dm0 into filamentous structures. These results are discussed in relationship to the biological roles of the nuclear lamina.

Keywords: membrane proteins, nuclear, membrane, lamina, βII PKC, LAP2β



543-549

Analysis of mutant p53 for MAR-DNA binding: determining the dominant-oncogenic function of mutant p53

Author(s): Dr. Wolfgang, Deppert,

Abstract: At least some mutant p53 proteins not simply have lost the wild-type p53 specific tumor suppressor function, but exhibit oncogenic functions on their own. Recently we showed that binding of mutant p53 to MAR/SAR elements is an activity specific for mutant p53 and clearly distinguishable from the previously reported DNA-binding activities of p53. Since MAR/SAR elements are considered to be important regulatory elements for a variety of nuclear processes, the interaction of mutant p53 with MAR/SAR elements might form the molecular basis for oncogenic potential of mutant p53. By employing different binding assays (the target-bound DNA binding assay, the South-western blotting technique and an adapted liquid phase binding assay), we studied MAR/SAR binding of various p53 proteins to different MAR/SAR elements. Murine mutant p53 bound different MAR/SAR elements with an approximately 1,000-fold higher affinity than murine wild-type p53. Analysis of MAR/SAR binding of human wild-type and mutant p53 proteins revealed also high affinity MAR/SAR binding of several human p53 mutant proteins (175 Arg His, 273 Arg Pro), but not of human wild-type p53, confirming that MAR binding is a general property of mutant p53. By antibody interference analysis using a panel of different p53- specific monoclonal antibodies and by deletion mutant analysis the MAR/SAR binding domain on mutant p53 was mapped, revealing a bipartite domain consisting of the mutated core region and the C-terminal 60 amino acids.

Keywords: p53, murine wt, MAR/SAR elements



551-580

Transcription-promoting genomic sites in mammalia: their elucidation and architectural principles

Author(s): Dr. Jurgen Bode,

Abstract: Scaffold/matrix attached regions (S/MARs) represent a relatively novel addition to the class of cis- acting DNA sequences in the eukaryotic genome. These elements are thought to operate via functional contacts to the protein backbone of the nucleus. S/MARs of several kilobases are found at the putative borders of several chromatin domains, and shorter elements with basically the same physicochemical properties occur in close association with certain enhancers or in introns. Accordingly, S/MARs can be situated either in nontranscribed regions or within transcription units. Biological roles that have been assigned to them include insulating and chromatin domain opening functions. These activities apparently are not separable, and both are compatible with the same kind of structure. In this contribution we present a series of recent results suggesting that S/MARs act as topological gauges with the potential to adapt their functions to environmental stresses. We also suggest that previously noted uncertainties regarding their activities may have arisen from inadequacies in the methods that were used for their characterization. We discuss the application of new, highly controlled site specific recombination methodologies that integrate single copies into controlled genomic positions to the study of transgene and S/MAR functions in cell cultures and in transgenic organisms.

Keywords:



581-590

Synthetic concatemers as artificial MAR: importance of a particular configuration of short AT-tracts for protein recognition

Author(s): Dr. Ken Tsutsui,

Abstract: The matrix attachment region (MAR), a class of sequences involved in organization of genomic DNA into looped superhelical domains, is also believed to be important in the regulation of nuclear functions such as DNA replication, transcription, and recombination. The association of MARs to the nuclear matrix is probably mediated by a variety of proteins which selectively bind to MAR. Because of the complex nature of MAR-protein interactions, it is still difficult to tell, from sequence information alone, whether or not a particular DNA region is a MAR. An abundant nuclear protein, termed SP120, is one of the major MAR binding proteins identified so far that selectively binds to AT-rich MARs of different origins in vitro. We have recently found that SP120 also binds to a GC- rich concatemer synthesized by random ligation of a short duplex oligonucleotide. Although the result seemed rather paradoxical at first, subsequent experiments with sequence-manipulated concatemers indicated that intactness of short homopolymeric AT-tracts harbored in the concatemer and a particular pattern of their distribution within it are prerequisites for the binding.

Keywords:



591-598

Replicon map of the human dystrophin gene: asymmetric replicons and putative replication barriers

Author(s): Dr. Sergey V. Razin,

Abstract: Using the replication direction assay and oligonucleotide probes designed on the basis of the known exon sequences of the human dystrophin gene we have made a replicon map of this giant gene. It has been found that dystrophin gene is organized into at least six replicons ranging in size from 170 to more than 500 kb. One of the replicon junctions (sites of replication termination) was mapped in intron 44, i.e. roughly in the same area where the major recombination hot spot is located. It is also worth mentioning that the central part of the dystrophin gene (exons 8 - 48) is organized into relatively short symmetrical replicons surrounded by two extended regions of apparently unidirectional replication (exons 1 - 8 and exons 49 - 64). These observations suggest for the first time that there should be certain signals for the termination of replication in euchromatic areas of the genome of higher eukaryotes. Furthermore, it may be concluded that the replication of the central part of dystrophin gene must be completed much faster than the replication of its ends. This may induce some topological stresses resulting in an increased rate of chromosomal rearrangements within this gene. The experimental approach used in our study may be helpful for fast analysis of the replication structure of other areas of the human genome provided that these areas are saturated with STS markers.

Keywords:



599-608

Initiation of DNA replication at the rat aldolase B locus

Author(s): Dr. Ken-ichi Tsutsumi,

Abstract: In higher eukaryotes, DNA replication initiates at multiple sites on each chromosome. Positioning and firing of the replication origins are not fixed, but different and selected origins may initiate at different times in a single cell cycle of particular cells through a range of complex mechanisms controlling, for example, cell differentiation. The origin region at the rat aldolase B locus (ori A1) has been found to encompass the promoter which governs liver-specific transcription. Ori A1 is, thus, thought to be a suitable target in investigating causal relationships among those under control of cell differentiation, i.e., firing or silencing of the origin, cell type-specific regulation of transcription, and positioning of nearby origins. In this article, we summarize our approach to elucidate such relationships. We describe sequence-dependent replication from ori A1, overlapping of essential regions required for replication and transcription, cell cycle-regulated binding of factors to the essential region, and then chromosomal state of the ori A1 region in the nucleus.

Keywords:



609-612

TARgeting the human genome to make gene isolation easy

Author(s): Dr. Michael A. Resnick,

Abstract: Considerable information is now available about the human genome and expressed sequences have been identified for most genes. Until recently there was no opportunity to specifically isolate genes or specific chromosomal regions from genomic DNA. We have utilized transformation- associated recombination (TAR) in yeast to isolate genes and specific regions from total human DNA. This has been demonstrated by the direct isolation of complete copies of rDNA, BRCA1, BRCA2 and HPRT genes with high fidelity as yeast artificial chromosomes (YACs). We propose that there are many utilities of TAR cloning including gene therapy and diagnostics.

Keywords:



613-628

Control of growth and proliferation by the retinoblastoma protein

Author(s): Dr. Robert J. White,

Abstract: The retinoblastoma susceptibility gene Rb is an important tumour suppressor. It will inhibit both growth and proliferation when introduced into many types of cell. Furthermore, it is frequently found mutated in a range of human cancers. It is therefore of considerable importance that we should understand fully how this gene operates. The RB gene product is a 110 kDa nuclear phosphoprotein that regulates the activity of a number of key transcription factors. In turn, its activity is controlled through phosphorylation by cyclin-dependent kinases in response to the availability of growth factors. It therefore provides a mechanism for coordinating gene expression with growth factor availability. One of the principle targets of RB is a transcription factor called E2F. E2F controls the expression of a panel of genes that promote proliferation. By down- regulating these genes through its inhibitory action on E2F, RB provides a restraining influence upon cell cycle progression. It has been less clear how RB is able to suppress the growth (increase in mass) of cells. However, recent studies have suggested that it may achieve this by repressing the production of rRNA and tRNA. Loss of control over the protein synthetic apparatus may constitute an important step in tumour development.

Keywords: Cancer. Retinoblastoma. Tumour suppression. Transcription. Translation



629-639

Transcriptional regulation of the H-ras1 proto- oncogene by DNA binding proteins: mechanisms and implications in human tumorigenesis

Author(s): Dr. D. A. Spandidos,

Abstract: Altered expression of ras genes is a common event in human tumors. Transcriptional regulation of the H-ras1 proto-oncogene occurs through nuclear factors that recognize elements in the promoter region of the gene, in the first and fourth intron and in the VTR unit and involves alternative splicing and specific methylation patterns, as well. Aberrant levels of the Ras p21 protein are detected in a variety of human tumors and are often correlated with clinical and prognostic parameters. Thus, understanding the regulation of the expression of ras genes provides a useful target for gene therapy treatments.

Keywords:



641-647

Periodicity of DNA bend sites in eukaryotic genomes

Author(s): Dr. Ryoiti Kiyama,

Abstract: We found that DNA bend sites are distributed regularly and periodically in the genomic DNA of eukaryotes. Their locations were conserved during molecular evolution in otherwise unstable intergenic regions of genomic DNA at intervals of approximately 700 bp, which corresponds to a length of four nucleosomes, suggesting their active role in chromatin organization. By further examination of these sites with respect to chromatin structure, we obtained evidence that these sites may act as signals for nucleosome phasing. Here, we summarize our results regarding periodic bent DNA in the human -globin, c-myc, and immunoglobulin heavy chain loci and discuss their biological functions.

Keywords:



649-660

DNA methyltransferase: a downstream effector of oncogenic programs; implications for therapy.

Author(s): Dr. Moshe Szyf,

Abstract: DNA MeTase is an attractive anticancer target. A molecular analysis of its regulation suggests that it is a downstream effector of many oncogenic pathways and that its down modulation can inhibit tumor growth. It is possible that DNA MeTase inhibitor will be effective in a broad spectrum of cancers because it lies downstream to nodal cellular checkpoints that could be activated by multiple ways. An important challenge is to design novel inhibitors of DNA MeTase that are highly specific. Such inhibitors are potentially important pharmacological agents with wide therapeutic applications.

Keywords:



661-679

Correlation between DNA methylation and poly(ADP-ribosyl)ation processes

Author(s): Dr. Paola Caiafa,

Abstract: The aim of this article is to show the close relationship between DNA methylation and poly(ADP- ribosyl)ation which are two important nuclear enzymatic mechanisms. An open question is to explain how some CpG dinucleotides, in particular those present into GpG islands, can maintain their unmethylated state in spite of the presence of active DNA methyltransferase in chromatin. This paper illustrates some data indicating that H1 histone is a possible trans-acting factor involved in protecting genomic DNA from full methylation and proposes that the somatic variant H1e, in its poly(ADP-ribosyl)ated isoform, is the protein capable of undertaking this role.

Keywords:



681-699

Poly (ADP-ribosyl)ation as one of the molecular events that accompany mammalian spermatogenesis.

Author(s): Dr. Piera Quesada,

Abstract: It is known that mammalian spermatogenesis is a synchronous process of cellular differentiation during which morphological changes occur, concomitantly with alterations in the complement of constituent proteins, that reflect differences in the mRNA populations coding for stage-specific proteins. Moreover, the most dramatic changes in chromatin structure observed in eukaryotes, take place during spermiogenesis, and the main nuclear processes occur in well-defined cell stages. Rat testis has been used as experimental model in a research project carried out at various levels, represented by rat germinal cells (primary and secondary spermatocytes, round spermatids), chromatin fractions (transcriptionally active chromatin, nuclear matrix, MARs) and purified nuclear proteins (histone and non-histone proteins). Specific experiments have been carried out in order to determine the poly(ADPR)polymerase content at different stages of germ-cell differentiation, the poly(ADP-ribose) amount, length and complexity inside the nucleus, and the poly(ADP-ribose) acceptors among tissue- and stage-specific nuclear proteins. The results indicate that regulation of the poly(ADPribosyl)ation system accompanies the earlier phases of the germinal cell differentiation. Indeed, poly(ADPribose)polymerase is particularly active in primary spermatocytes, being possibly implicated in the recombination events that characterize the pachytene phase of the meiotic division. Different classes of poly(ADPribose) modify different chromatin fractions (DNase I-sensitive, DNase I-resistant chromatin, and nuclear matrix) implicated in DNA replication, repair and transcription. Moreover, the H1 variant H1t, specifically expressed in pachytene spermatocytes, represents the main poly(ADPribose) acceptor, together with poly(ADPR)polymerase itself, in rat germ-cells. Its modification can amplify the role of histone H1 variants as modulators of chromatin structure.

Keywords:



701-706

embrane biogenesis: from mechanism to disease

Author(s): Dr. Volker Haucke,

Abstract: The biogenesis of membranes involves the continous flow of proteins and lipids which are selectively targeted to or retrieved from specific compartments within eukaryotic cells. While some diseases are caused by the impairment of particular protein transport pathways or mislocalization of a certain protein others may be related to altered signal transduction cascades resulting from defective endocytosis of plasma membrane receptors or other membrane trafficking defects. The implications of this hypothesis for our understanding of the proper functioning of a eukaryotic cell and for the treatment of human diseases are being discussed.

Keywords: membrane biogenesis-peroxisomal disorders-nerve terminal-endocytosis-autoimmune diseases-cancer



707-711

Cdc25 protein phosphatase: regulation and its role in cancer

Author(s): Dr. Jens W. Eckstein,

Abstract: The family of the Cdc25 dual-specific protein tyrosine/threonine phosphatases is critically involved in cell cycle control. The substrates of Cdc25 are cyclin-dependent kinases, which are regulated by the phosphorylation of threonine and tyrosine residues. Cdc25 regulation and activity reveals a complex network of counter-balancing mechanisms and puts it on the crossroads of fundamental cellular events like cell proliferation, cell cycle arrest and apoptosis. Our present knowledge of the biology and biochemistry of Cdc25 phosphatases makes them attractive targets for drug discovery efforts: (a) they phase critical, non-redundant cell cycle regulatory functions; (b) they are bona fide checkpoint genes; (c) they have tight substrate specificities and a well-defined mechanism of catalysis; (d) they are potential targets of at least two oncogenes (Raf1 and c-Myc) that are frequently altered in human cancers; (e) they co-operate with other oncogenes in cell transformation and thus are bona fide proto-oncogenes; and lastly (f) their expression is altered in tumors.

Keywords:



713-740

Nucleocytoplasmic trafficking: implications for the nuclear import of plasmid DNA during gene therapy

Author(s): Dr. Teni Boulikas,

Abstract: Trafficking of nuclear proteins from the site of their synthesis in the cytoplasm to the sites of function in the nucleus through pore complexes is mediated by nuclear localization signals (NLSs) on proteins to be imported into nuclei. Protein translocation from the cytoplasm to the nucleoplasm involves (i) the formation of a complex of karyopherin with NLS-protein, (ii) subsequent binding of karyopherin , (iii) binding of the complex to FXFG peptide repeats on nucleoporins, (iv) docking of Ran-GDP to nucleoporin and to karyopherin heterodimer by p10, (v) a number of association-dissociation reactions on nucleoporins which dock the import substrate toward the nucleoplasmic side with a concomitant GDP-GTP exchange reaction transforming Ran- GDP into Ran-GTP and catalyzed by karyopherin , and finally (vi) dissociation from karyopherin and release of the karyopherin /NLS-protein by Ran-GTP to the nucleoplasm. A number of processes have been found to be regulated by nuclear import including nuclear translocation of the transcription factors NF- B, rNFIL-6, ISGF3, SRF, c-Fos, GR as well as human cyclins A and B1, casein kinase II, cAMP-dependent protein kinase II, protein kinase C, ERK1 and ERK2. Failure of cells to import specific proteins into nuclei can lead to carcinogenesis. For example, BRCA1 is mainly localized in the cytoplasm in breast and ovarian cancer cells whereas in normal cells the protein is nuclear. mRNA is exported through the same route as a complex with nuclear proteins possessing nuclear export signals (NES). The majority of proteins with NES are RNA-binding proteins which bind to and escort RNAs to the cytoplasm. However, other proteins with NES function in the export of proteins; CRM1, which binds to the NES sequence on other proteins and interacts with the nuclear pore complex, is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells. Nuclear localization and export signals (NLS and NES) are found on a number of important molecules including p53, v-Rel, the transcription factor NF-ATc, the c-Abl nonreceptor tyrosine kinase, and the fragile X syndrome mental retardation gene product; the deregulation of their normal import/export trafficking has important implications for human disease. Both nuclear import and export processes can be manipulated by conjugation of proteins with NLS or NES peptides. During gene therapy the foreign DNA needs to enter nuclei for its transcription; a pathway is proposed involving the complexation of plasmids and oligonucleotides with nascent nuclear proteins possessing NLSs as a prerequisite for their nuclear import. Covalent linkage of NLS peptides to oligonucleotides and plasmids or formation of complexes of plasmids with proteins possessing multiple NLS peptides is proposed to increase their import rates and the efficiency of gene expression. Cancer cells are predicted to import more efficiently foreign DNA into nuclei compared with terminally differentiated cells because of their increased rates of proliferation and protein import.

Keywords: pore complex, nucleoporins, nuclear localization signals, karyopherin, nuclear export signals, mRNA export



741-748

The ATP-driven protein translocation-motor of mitochondria

Author(s): Dr. Martin Horst,

Abstract: The majority of mitochondrial proteins are encoded in the nucleus. In the cytosol they are synthesized as precursor proteins which are transported into mitochondria. Protein import into mitochondria requires a concerted action of a variety of different proteins. For the transport of precursor proteins across the mitochondrial inner membrane and their folding in the matrix the mitochondrial hsp70 (mhsp70) chaperone plays an essential role. Mhsp70 is found in at least two protein complexes within mitochondria: together with Tim44 and mGrpE, mhsp70 forms the import-complex and together with Mdj1 and mGrpE it forms a folding-complex. This review focuses on the function of the import-complex. It is believed that mhsp70 can act as a mechano- chemical enzyme that actively pulls precursor proteins across the inner membrane.

Keywords:




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