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Volume 4 - 1999


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1-10

The peculiar organization of telomeres in Drosophila melanogaster

Author(s): Dr. , Sergio Pimpinelli,

Abstract: Telomeres are specialized DNA-protein complexes at the ends of eukaryotic chromosomes. They protect the chromosome extremities by preventing potential chromosome damages such as loss of terminal sequences during chromosome replication and by preventing chromosome fusions and degradation. The telomeres of most organisms contain short terminal GC-rich repeats due to the activity of telomerase, a ribonucleoprotein DNA polymerase. Telomeres have gained an exceptional widespread interest in human biology because of their involvement in aging and carcinogenic processes. Despite the fact that the telomere concept was elaborated in Drosophila melanogaster, this organism lacks telomerase and its telomeres are dramatically different from those in other organisms. Telomere loss in Drosophila is prevented by two specific non-LTR transposons, called HeT-A and TART, that appear to be dispensable for chromosome stability. Recent studies have permitted to isolate proteins involved in telomere stability in Drosophila. In particular it has been shown that the heterochromatin protein 1 (HP1) plays an essential role in telomere capping. HP1 is of a special interest because it is a highly conserved protein; HP1 homologues have been identified in many different organisms. Most important, three HP1-like proteins have been found in humans. Future studies will tell us if some of the human HP1 proteins have conserved a functional telomeric localization as in Drosophila.

Keywords: Telomeres, Drosophila melanogaster, telomerase, HeT-A, TART, heterochromatin protein, 1 (HP1), telomere capping



11-22

Somatic cell nuclear transfer as a tool for investigating ageing processes in mammals

Author(s): Dr. , Paul G. Shiels,

Abstract: The development of nuclear transfer technology has opened up a new frontier in the investigation of the processes which contribute to ageing in mammals. This review seeks to assess the individual hypotheses that have been proposed to account for the development of the ageing phenotype and to ask how they correlate with observations made on cloned mammals. In sheep derived by nuclear transfer there appears to be prematurely shortened telomeres, indicative of increased age. The animals, however, are physiologically normal, consistent with a redox model of ageing where mitochondrial damage is the key contributory factor. The application of nuclear transfer technology to the study of ageing phenomena and its use in experimentally redressing aspects of the ageing phenotype is discussed.

Keywords: Mammalian cloning, nuclear transfer, ageing, aging, oxidative damage, telomere, rDNA, mitochondria. nucleolus



23-31

Semliki Forest virus vectors for in vitro and in vivo applications

Author(s): Dr. , Kenneth Lundstrom,

Abstract: Rapid virus generation, broad host range, efficient RNA replication in the cytoplasm, and high expression levels are features that have made the use of Semliki Forest virus (SFV) vectors attractive. High-level expression of G- protein coupled receptors has allowed specific binding and functional studies in a variety of mammalian cell lines. Furthermore, high infection efficiency (75-90%) has greatly facilitated gene expression and localization studies in primary neurons in culture. The establishment of SFV infections of large-scale suspension cultures has resulted in the production of hundreds of milligrams of recombinant receptors now available for structural studies. SFV vectors have been shown to preferentially infect neurons in hippocampal slice cultures, which will facilitate studies on gene expression, transport, and protein localization in neuronal tissue. Injection of replication-deficient SFV vectors into rat brain resulted in local, high-level transient expression in vivo. Recent vector improvements have included the generation of SFV vectors with low-to-moderate transgene expression resulting in more physiological expression levels that are similar to those seen in native tissue. Novel SFV vectors, which have recently been developed, permit prolonged survival of infected host cells.

Keywords: Semliki Forest virus, gene therapy, vaccine, LacZ, G-protein coupled receptors



33-43

Ovine adenovirus vectors promote efficient gene delivery in vivo

Author(s): Dr. Christian Hofmann,

Abstract: The use of vectors derived from human adenoviruses in gene therapy is limited by pre-existing humoral immunity against these vectors in many individuals. We have recently reported the use of a vector derived from ovine adenovirus (OAV) isolate 287 that can transduce cells in vivo (Hofmann et al., 1999). In this report we present data regarding the physical stability of the OAV particles and demonstrate their ability to infect mice pre- immunized with a human adenoviral (hAd) vector. The tissue distribution of the vector delivered by several routes of administration is also examined and shown to be different from that observed for vectors derived from human adenoviruses. The construction and rescue of a recombinant virus that expresses the green fluorescent protein gene will further facilitate studies on the tropism of OAV in vivo.

Keywords: ovine adenovirus vectors, pre-existing antibodies, human α1-antitrypsin, viral tropism



45-58

Efficient expression of ribozyme and reduction of stromelysin mRNA in cultured cells and tissue from rabbit knee via Adeno-associated Virus (AAV)

Author(s): Dr. Dennis Macejak,

Abstract: The potential use of Adeno-associated virus (AAV) as an efficient gene delivery vector is increasingly being recognized in human gene therapy. We have investigated the utility of recombinant AAV (rAAV) vectors for the delivery and expression of hammerhead ribozymes targeted against a cellular mRNA encoding a matrix metalloproteinase, stromelysin. Stromelysin expression has been linked to the pathogenesis of human osteoarthritis. We have constructed several rAAV backbone plasmids containing single or multiple hammerhead ribozymes expression cassettes under the control of either the tRNA, U1, or U6 promoter, and have used these plasmids to generate rAAV. These rAAV vectors also contain a selectable marker, the truncated nerve growth factor receptor (NGFR) driven by the cytomegalovirus immediate early gene promoter. rAAV expressing stromelysin-specific ribozyme transduced ex vivo cultured rabbit synovial fibroblasts (RSFs) with a greater than 95% efficiency. Stable ribozyme expression can readily be detected throughout the life span of RSFs in culture. Furthermore, ribozyme mediated knockdown of stromelysin mRNA was detected in RSFs infected by a rAAV containing the tRNA-based transcription unit.

Keywords: hammerhead ribozymes, Adeno-associated virus, gene delivery, osteoarthritis, stromelysin, nerve growth factor receptor



59-74

Glial cell line-derived neurotrophic factor (GDNF) gene therapy in an aged rat model of Parkinson's disease

Author(s): Dr. Martha C. Bohn,

Abstract: The chronic delivery of neuroprotective factors to specific regions of the CNS via gene therapy offers a new strategy for the treatment of neurodegenerative disorders. The neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) is a potent dopaminergic (DA) trophic factor that ameliorates the behavioral and histological consequences of lesioning DA neurons in rodent and primate models of Parkinson’s disease. GDNF gene therapy may therefore have potential use in the treatment of Parkinson's disease. We have observed previously that an adenoviral (Ad) vector harboring a GDNF minigene protects DA neurons from degeneration in the young rat brain. However, as Parkinson’s disease occurs primarily in aged populations, we have also studied the effects of GDNF gene delivery in an aged rat model of Parkinson’s disease. In the aged (20 month) Fischer 344 rat, an Ad vector was used to deliver GDNF either to the DA cell bodies in the SN or to the DA terminals in the striatum. One week following gene delivery, the neurotoxin 6-hydroxydopamine (6-OHDA) was injected unilaterally into the striatum to cause progressive degeneration of DA neurons. Injection of GDNF vector into either the striatum or SN provided significant cell protection against 6-OHDA. However, only striatal injections of Ad GDNF protected against the development of behavioral and neurochemical changes that occur in the DA-depleted brain. The results of this study are reviewed here and the behavioral and cellular effects of GDNF gene delivery into striatal versus mesencephalic sites are discussed.

Keywords: Key Words: Glial cell line-derived neurotrophic factor, GDNF, gene therapy, ex-vivo, Parkinson's disease, neurotrophic factors, dopaminergic neurons, striatum, 6-OHDA, neurodegenerative disorders, brain-derived neurotrophic factor



75-82

Structural insights into NF-κB/IκB signaling

Author(s): Dr. Gourisankar Ghosh,

Abstract: The vital cellular activities of growth, differentiation, reaction to stimuli, and apoptosis are controlled by the coordinated expression of a vast number of genes. Regulation of gene expression occurs primarily at the level of transcription. The process begins as one of a multitude of mitogen- induced signaling events triggers the intricate and exquisitely regulated signal transduction pathways. These lead ultimately to the recruitment of transcription factors on specific promoter/enhancer sites. One example of how complex cell signaling can lead to the temporal activation of transcription in a specific set of genes is illustrated by the transcription factor NF-κB. Since its discovery almost a decade and a half ago, NF-κB has fascinated researchers because of the complexity of the NF-κB signaling pathway. Several recently determined crystal structures of a number of NF-κB complexes have given a new dimension of understanding of the biochemistry behind NF-κB. We will review these structures in light of their functions.

Keywords: NFκB/IκB signaling, apoptosis, mitogen-induced signaling, transcription factors,



83-98

Mammalian c-Jun N-terminal kinase pathway and STE20-related kinases

Author(s): Dr. Tse-Hua Tan,

Abstract: The c-Jun N-terminal kinases (JNKs) belong to a subgroup of mitogen-activated protein kinases (MAPKs) that are activated by environmental stress, proinflammatory cytokines, and mitogenic stimuli in mammalian cells. Studies on the JNK pathway in mammalian cells demonstrate that JNK regulates the transcriptional activities of many transcription factors, and that JNK is required for the regulation of cell proliferation and apoptosis. Studies on jnk-deficient mice reveal that JNK is involved in the response to immunological stimuli and in embryonic morphogenesis. JNK, as other MAPKs, is regulated by a kinase cascade. JNK activation is mediated by dual phosphorylation on the motif, Thr-Pro- Tyr. To date, this phosphorylation is known to be mediated by the MAPK kinases (MAP2Ks), MKK4 and MKK7. MKK4 and MKK7 are activated by MEKK1 and other MAPK kinase kinases (MAP3Ks). The MAPK kinase kinase kinases (MAP4Ks) including HPK1, GCK, and homologous kinases, which have a kinase domain related to yeast STE20, can activate the JNK signaling cascade. These mammalian STE20-related MAP4Ks may be involved in integrating various stimuli to the JNK cascade. The signaling specificity of mammalian JNK pathway may be controlled by scaffold proteins that interact with kinases at different levels in the pathway.

Keywords: c-Jun, N-terminal Kinase, STE20-related Kinases, JNK signaling, MEKK1, MAPK



99-107

Nucleocytoplasmic trafficking and glucocorticoid receptor function

Author(s): Dr. Joanne G.A. Savory and Yvonne A. Lefebvre,

Abstract: The glucocorticoid receptor (GR) is a ligand activated transcription factor that redistributes between nucleus and cytoplasm in response to the addition and withdrawal of steroidal ligands. Localization of the receptor in the cell is dynamic and changes in GR localization reflect the shifting of equilibria between several competing cellular pathways. Since the naïve receptor is transformed from a transcriptionally inert cytoplasmic factor to a potent sequence-specific, DNA-bound transcriptional regulator, delimiting the controls on receptor localization is seminal to understanding how receptor activity may be manipulated or controlled within the cell. A number of recent reports have begun to reveal that the controls on GR trafficking are more sophisticated than previously expected and point to an important role for trafficking controls in the regulation of the steroid response.

Keywords: glucocorticoid receptor, nuclear import, steroid agonists, chaperone, importin, nuclear export signal, nuclear localization signal, steroid hormone receptors, transcription facto



109-118

Ribozyme-dependent inactivation of lacZ mRNA in E. coli: a feasibility study to set up a rapid in vivo system for screening HIV-1 RNA-specific ribozymes

Author(s): Dr. Sadhna Joshi,

Abstract: Ribozymes are potentially useful tools with widespread applications in gene therapy of several diseases. In order to assess the in vivo cleavage efficiency of human immunodeficiency virus (HIV)-1 RNA-specific ribozymes, a bacterial indicator cell system could be developed in which the degree of inhibition of β-galactosidase activity would correlate with ribozyme activity. The suitability of this indicator cell system was assessed using a ribozyme targeted against the env coding region within the HIV-1 RNA. To this end, a pGEM4Z-based plasmid was engineered wherein oligodeoxynucleotides containing a hammerhead ribozyme and its target site were cloned in frame within the lacZ coding region that encodes for the α fragment of β-galactosidase. Extra nucleotides were included in the insert to ensure that the lacZ open reading frame was not interrupted due to a frameshift or nonsense mutation. In E. coli indicator cells harbouring this plasmid, ribozyme-mediated cleavage of the target site provided in cis and the subsequent loss of β-galactosidase activity should correlate with ribozyme activity. However, frameshift mutations were observed upon sequence analysis of plasmid DNA isolated from the selected light blue to white colonies. Because these mutations affected the production of the β-galactosidase α fragment, a direct correlation between β-galactosidase and ribozyme activities could not be established in vivo. Thus, in clones which demonstrated visibly lower β-galactosidase activities than the control, the effect of the frameshift mutations on lacZ mRNA translation can not be discounted. In clones expressing ribozymes but displaying dark blue colour, it is possible that lacZ mRNAs were cleaved but that the β-galactosidase substrates used were sensitive enough to allow detection of proteins translated from residual lacZ mRNA transcripts. The use of alternative β-galactosidase substrates with less sensitivity may enable the use of the proposed indicator cell system.

Keywords: Ribozyme, lacZ, mRNA, HIV-1 RNA, bacterial indicator cell system, β-galactosidase, env coding region, pGEM4Z-based plasmid, hammerhead ribozyme



119-132

Direct redox modulation of p53 protein: potential sources of redox control and potential outcomes

Author(s): Dr. Hsiao-Huei Wu, Mark Sherman, Yate-Ching Yuan,

Abstract: Appropriate response to environmental stressors is essential for life. Many stressors, such as UV light, ionizing radiation, reactive oxygen intermediates (ROI), heat shock and hypoxia alter the redox potential of the cell. Recently, it has been shown that some of these stressors promote direct oxidation of specific protein cysteine residues resulting in either up-regulation or down-regulation of protein activity in the cytosol. In higher eukaryotes, the p53 tumor suppressor gene is a central component of stress response and its activation results in either cell cycle arrest or apoptosis. In cultured cells, p53 appears to become activated by some stressors (hydrogen peroxide, heat) predicted to directly increase cellular redox potential. However, in vitro studies indicate that p53 protein oxidation inhibits its ability to bind its consensus sequence DNA. If p53 is unable to bind consensus sequence DNA, p53 is predicted to be incapable of activating the p21WAF1/CIP1 gene, responsible for mediating G1 cell cycle arrest. Two proteins previously shown to reduce oxidized cytoplasmic proteins, Redox factor-1 and thioredoxin reductase, have been shown to play important roles in maintaining p53 activity, suggesting that they may be responsible for keeping p53 in the reduced state inside the cell. Analysis of the p53 crystal structure revealed several well-conserved cysteine residues exposed on the protein surface that may be susceptible to oxidation. Based on this analysis we predict that cysteine residues 124, 176, 182, 242 and 277 are primary candidates for redox regulation. In this communication, we review the data demonstrating p53 regulation by direct alteration of p53 cysteine residue oxidation, propose a testable mechanism by which p53 oxidation may occur, and discuss the possible implications of p53 oxidation on cell growth control and DNA repair.

Keywords: oxidation, cysteine, pyrrolidine dithiocarbamate, stressor, sulfhydryl



133-141

HIV-1 DNA integration: advancing anti-HIV-1 gene therapy approaches by blocking and modulating the process

Author(s): Dr. Roger J. Pomerantz,

Abstract: The efficient replication of retroviruses requires integration of the double-stranded DNA copy of viral RNA into chromosomal DNA of the infected host cell. Integration for all retroviruses, including human immunodeficiency virus type 1 (HIV-1), represents the stable incorporation of viral DNA into that of the host and depends upon the function of the viral encoded enzyme, integrase (IN). Among various classes of animal viruses, integration of viral DNA is a unique and defining step in the life cycle of retroviruses and results in the permanent establishment of the viral genome in the infected host cell. The practical consequences of this activity is the presence of a viral DNA template capable of directing the production of progeny virus for the life span of the infected cell. This template persists even despite the imposition of therapeutic regimens available currently that involve the combinatorial use of inhibitors against HIV-1 reverse transcriptase (RT) and protease (PR) that are effective in reducing circulating virus in most HIV-1 seropositive patients. Efficient blockade of integration, or the steps preceding integration, would be far preferable than attempts to suppress the production of viral products from cells that already harbor an HIV-1 provirus. The actual mechanistic details for integration of all retroviruses is remarkably similar and such an understanding is vital in a new era of rational drug design that not only relies upon an intimate knowledge of the structure and function of individual viral products, but alternatively utilizes those details to manipulate these products with the goal of halting viral replication completely or ablating the presence of infected host cells in the seropositive patient. Several small molecule inhibitors of HIV-1 integrase are in development with the eventual hope of including such compounds in combination therapy with PR and RT inhibitors. However, there are anti-IN gene therapy strategies being pursued that are highlighted in this review and which can be employed to inhibit function of the protein with a similar goal of blocking HIV-1 infection within host cells.

Keywords: Gene Therapy, Retroviruses, Integration



143-158

In eukaryotic cells, DNA replication, RNA processing and ribosome assembly all occur in the nucleus, while protein synthesis occurs in the cytoplasm. These activities are physically separated by the nuclear envelope. The inner nuclear membrane and the nuclear lamina are involved in organizing nuclear structure and regulating nuclear events including: nuclear organization, DNA replication, nuclear assembly and disassembly, apoptosis, and correct spacing of nuclear pores. In order to perform these activities the inner nuclear membrane and the nuclear lamina contain a unique set of proteins, including lamin type A and B, otefin, Young Arrest, LAP1, TMPO/LAP2, emerin, LBR and MAN. The proteins of the inner membrane form a complex network of interactions between themselves and with chromatin. Mutations in several of these proteins also result in known diseases. In this paper we review these interactions and discuss their possible roles in normal cell activity and in apoptosis. In addition, we demonstrate that specific regions in lamins can interact with the core histones H2A and H2B. We also show that Thymopoietin (TMPO)/ Lamina Associated Polypeptide 2 (LAP2) gene is alternatively spliced to form a large family of proteins with different size and different distribution within the nucleus. These results are discussed in relationship to the biological roles of the nuclear lamina.

Author(s): Dr. Amos J. Simon,

Abstract: In eukaryotic cells, DNA replication, RNA processing and ribosome assembly all occur in the nucleus, while protein synthesis occurs in the cytoplasm. These activities are physically separated by the nuclear envelope. The inner nuclear membrane and the nuclear lamina are involved in organizing nuclear structure and regulating nuclear events including: nuclear organization, DNA replication, nuclear assembly and disassembly, apoptosis, and correct spacing of nuclear pores. In order to perform these activities the inner nuclear membrane and the nuclear lamina contain a unique set of proteins, including lamin type A and B, otefin, Young Arrest, LAP1, TMPO/LAP2, emerin, LBR and MAN. The proteins of the inner membrane form a complex network of interactions between themselves and with chromatin. Mutations in several of these proteins also result in known diseases. In this paper we review these interactions and discuss their possible roles in normal cell activity and in apoptosis. In addition, we demonstrate that specific regions in lamins can interact with the core histones H2A and H2B. We also show that Thymopoietin (TMPO)/ Lamina Associated Polypeptide 2 (LAP2) gene is alternatively spliced to form a large family of proteins with different size and different distribution within the nucleus. These results are discussed in relationship to the biological roles of the nuclear lamina.

Keywords: Apoptosis, nuclear assembly, histone, thymopoietin



159-170

Models of cationic liposome mediated transfection

Author(s): Dr. Robert Malone,

Abstract: Synthetic gene delivery vectors are being developed for in vivo gene transfer applications, and these systems may circumvent the risks inherent in the use of recombinant virus vectors. The majority of synthetic delivery systems are based on the use of cationic amphiphiles to coat and condense polynucleotides, and to facilitate the uptake and release of the polynucleotide payload into somatic cells. Cationic amphiphile-based vector technology has benefited from over a decade of mechanistic and structure-function analyses, but many aspects of the pharmacology, toxicology, and cell biology of these systems remain unresolved. Important outstanding issues include the structure and characteristics of active and inactive particles, in vivo distribution and clearance of particles, and the cellular events involved in binding, cytoplasmic delivery, and nuclear uptake. These processes are interdependent, and therefore difficult to isolate and examine experimentally. The wide range of compounds, methodologies, and polynucleotides used to study these phenomena further complicate development of general principles. This article focuses on the molecular and cellular processes involved in cationic amphiphile-mediated transfection. Both primary data and current literature will be used to illuminate the complexity that impacts on the development and application of this class of synthetic gene delivery vectors.

Keywords: cationic lipid, liposome, gene delivery, DOTAP, transfection



171-176

Essentials of radionanotargeting using oligodeoxynucleotides

Author(s): Dr. Kalevi J. A. Kairemo,

Abstract: Antisense oligomers may be used for carrying radiation source into a specific location inside a tumour cell. Effects of radioactive labeled oligos may be exerted both via direct antisense inhibition and radiation. This radionanotargeting approach may provide several benefits to conventional treatment modalities, and radiation is minimized in adjacent tissue. In addition, a combination of radiation and antisense activity of oligodeoxynucleotide may result in synergistic interaction, as there are two different treatment modalities hitting a single mechanism of action. We have previously shown that oligonucleotide therapy is effective with internally labeled oligodeoxynucleotide phosphorothioates P-32, P-33 and S-35. Here, we review our results and discuss the role of radionanotargeting. We refer to our previous results of a large selection of radionuclides; we have calculated in vivo subcellular tissue distribution for oligodeoxynucleotide phosphorothioates using decay characteristics of ten β- and Auger-emitting radionuclides. The absorbed nuclear doses of these radiolabelled oligonucleotides were estimated in different cellular dimensions using the subcellular biodistribution data. These results indicate that Auger-emitter isotopes do not give higher absorbed cell nuclear doses than the isotopes suitable for internal labeling of oligo phosphorothioates. The best isotopes for subcellular targeting were P-33 and S-35 giving smallest variation of nuclear dose in the cell dimensions we studied (nuclear diameter 6-16 μm, cellular diameter 12-20 μm). Therefore, we conclude that radionanotargeting by oligonucleotides may provide synergistic interaction and should be carried on with short range β-emitters suitable for internal labelling of oligonucleotides unless relative biological effectiveness of Auger-emitters could be remarkably improved. Further preclinical evaluation of radionanotargeting based on radio-oligos should be continued.

Keywords: Antisense, oligonucleotide therapy, radionanotargeting, radionuclide, phosphorothioates, Auger electron emitter, cancer



177-182

ets-1 mRNA as target for antisense radio- oligonucleotide therapy in melanoma cells

Author(s): Dr. Kalevi J. A. Kairemo,

Abstract: Angiogenesis provides a novel target for anticancer therapy, in particular radiochemo-therapy as endothelial cells in the vascular wall are sensitive to radiation. Antisense phosphorothioate oligodeoxynucleotides (AS-PODNs) may serve as vehicles for carrying cytotoxic or radioactive agents into a particular intracellular location. Radiolabelled AS-PODNs have the potential of having both antisense and radiation effects. Recently, vascular endothelial growth factor (VEGF)-induced invasiveness was shown to be specifically inhibited by AS-PODN directed against ets-1. Previous studies have shown that radio-oligonucleotide therapy may be effective with AS-PODNs internally labelled with 32P, 33P or 35S. Theoretically, 35S gave the smallest variation in nuclear dose in the different cell dimensions studied (Kairemo et al., Cancer Gene Ther 1998; 5: 408-12). This means that cell nuclear targets should be treated with the short range β-emitters 35S or 33P for optimal radio-oligonucleotide therapy. Here we explore this possibility using 33P labeled 17-mer AS-PODNs directed against ets-1 in human melanoma cells in vitro. Inhibition of cell growth was observed in the following order: labeled AS-PODN > nonlabeled AS-PODN > labeled sense PODN > nonlabeled sense PODN > transfection agent. Even with a single 33P at the 5 ́-end of the AS- PODN melanoma cell uptake of label was approximately 0.5 mBq/cell. The nuclear doses in this experiment varied from 1.9 to 3.7 cGy. Thus, in vitro and in vivo use of radio-oligonucleotide therapy utilizing 33P radionanotargeting, e.g. in angiogenesis through ets-1, are highly recommended.

Keywords: Antisense, phosphorothioate, oligonucleotide therapy, radiation, phosphorothioates, angiogenesis, endothelial cell, ets-1



183-191

Electronic microarray for DNA analysis

Author(s): Dr. Lana Feng,

Abstract: Nanogen has developed a microelectronic technology that allows fast and accurate transport and hybridization of DNA on a semiconductor microchip. Based on this technology, we have developed electronic assays for genetic analysis of single nucleotide polymorphisms in several different applications. We have also developed electronic hybridization based short tandem repeats analysis for genetic identification and forensics. Our data show that these assays work in multiple formats under different electronic conditions. A beta instrument is capable of performing these tests on 100-site arrays in a semi-automated fashion. We are also working towards developing an integrated system, which could potentially become a portable device.

Keywords: microarray, electronic hybridization, short tandem repeat, single nucleotide polymorphism, forensics



193-202

Rapid generation of recombinant herpes simplex virus vectors expressing the bacterial lacZ gene under the control of neuronal promoters

Author(s): Dr. Alasdair R. MacLean,

Abstract: We describe the development of herpes simplex virus type 1 (HSV-1) RL1 negative mutants as vectors expressing the lacZ reporter gene under the control of neuronal specific promoters and their expression in neuronal cell cultures. Two neuronal promoters were used in this study 1) the rat neurone specific enolase (NSE) promoter, which is expressed in all neurones and 2) the mouse GABAA receptor delta (δ) subunit, which is expressed within specific regions of the brain (cerebellum, hippocampus and thalmus). The lacZ gene was also placed under the control of two viral promoters: 1) the HCMV IE promoter, a 'universally' strong promoter, and 2) the HSV-1 gD promoter, a strong delayed early lytic promoter. Initial expression experiments with recombinant viruses were carried out on neuronal and non-neuronal cell lines and primary mouse hippocampal cells. The promoter/reporter cassettes were introduced into one of two sites in the HSV-1 genome: firstly a non essential gene in the unique long region; and secondly at the LAT locus in the long terminal repeats just downstream from the LAT P2. Two high frequency methods of generating recombinant HSV are described. The first step for both involves insertion, into a non-essential genomic region, of a unique restriction enzyme site (Pac I), which is used to digest the HSV DNA into 2 arms. In the first method the insert, flanked by Pac I sites is isolated and in vitro ligated into the digested HSV vector to generate recombinant virus at a frequency of 10-90%. In the second method the DNA to be inserted is flanked by HSV DNA and co-transfected with the digested viral DNA. In vivo recombination across the digested ends of the HSV DNA and through the sequences to be inserted generates recombinants at a frequency of up to 100%.

Keywords: HSV-1, lacZ, neuronal promoter, GABAA receptor, recombination



203-208

Recent progress in gene therapy for eye diseases

Author(s): Dr. Andrius Kazlauskas,

Abstract: Gene therapy for eye diseases is still developing and requires further effort to bring it to clinical trials. Both a better understanding of the pathological aspect of the diseases and establishment of better viral systems is required. These efforts will facilitate the development of gene therapy-based treatments, which will complement conventional approaches to manage and potentially cure blinding diseases of the eye.

Keywords: Eye diseases, gene therapy, retinitis pigmentosa, proliferative vitreoretinopathy, adenovirus vectors, retroviral vectors, lentivirus



209-219

The role of HSV amplicon vectors in cancer gene therapy

Author(s): Dr. Joseph D. Rosenblatt,

Abstract: Summary Recent progress in tumor biology, virology and immunology has led to new approaches to the gene therapy for cancer. Herpes Simplex Virus (HSV) based vectors are attractive vectors for gene therapy use due to a number of favorable biologic features. Several characteristics render HSV suitable for gene therapy, including high transduction efficiency, ability to transduce non-dividing cells, high packaging capacity, wide cellular tropism and the ability to package multiple copies of the same gene or several genes. Newer HSV vectors differ in replication potential, sensitivity to anti-viral agents, neurotoxicity, tumor-specific cytotoxicity and persistence in the host cell. So- called “oncolytic” HSV based vectors demonstrate selective replication in tumor cells relative to normal tissue. HSV amplicon based vectors allow genetic transfer of multiple transgene copies in the absence of viral genes. This degree of flexibility, relative to other viral vector systems, has allowed for the use of HSV vectors in a variety of antitumor strategies including oncolytic, as well as immune-based strategies. Successful immune based strategies in animal models have included transfer of cytokines, costimulatory molecules and/or chemokines. Phase I/II clinical trials using HSV based vectors have been initiated.

Keywords: Herpes Simplex Virus vectors, HSV amplicon, cancer gene therapy, cytokine, chemokine, immunotherapy



221-232

Quantitative detection of CFTR mRNA in gene transfer studies in human, murine and simian respiratory tissues in vitro and in vivo

Author(s): Dr. Giulio Cabrini,

Abstract: Pre-clinical and clinical studies aimed to correct the basic defect of cystic fibrosis (CF) by transferring the wild type gene into the airway cells have already shown promising results. One of the main unsolved questions of these studies is the quantitation of the amount of gene required to correct the defect. In this respect, suitable technologies able to measure how much gene is actually transferred into the airway cells are under development. In this article we present a series of application of a single-tube competitive RT-PCR assay to quantitate the vector-encoded CFTR mRNA after gene transfer in human, mouse or monkey respiratory cells. These assays are able to measure very low levels of mRNA, with advantages over traditional expression systems for vector-encoded transcript (Northern analysis) or protein (immunocytochemistry). In some instances they may indicate directly the ratio of vector-encoded versus endogenous transcript, being applicable with both viral-derived and synthetic vectors. The assays presented here are instrumental for future application in gene delivery pre-clinical and clinical trials designed to identify corrective doses of the vector, by providing the basic information on the amount of vector- encoded CF gene transcript expressed in the airway cells.

Keywords: cystic fibrosis, gene therapy, quantitative mRNA



233-248

Gene therapy for prostate cancer

Author(s): Dr. Mitchell S Steiner,

Abstract: The advent of recombinant DNA technology has sparked the age of molecular medicine. The ability to deliberately recombine pieces of DNA and then transfer these specific genes into diseased cells has revolutionized medical research. In fact, the ability to modify these genes in the living person is now possible. Several innovative approaches are being developed to circumvent the limitations of current vectors including more effective delivery routes for gene therapy, the incorporation of tissue specific promoters and other enhancers into vectors, and increasing cell death by a phenomenon known as the bystander effect. Gene therapy strategies are rapidly evolving as new gene targets, better vectors and improved gene expression systems become available. Innovative gene therapy strategies currently being employed for the treatment of prostate cancer include: immunotherapy, gene corrective therapy, exploitation of programmed cell death therapy, gene therapy to target critical biological functions of the cell, suicide gene therapy, oncolytic virus gene therapy, and finally combination gene therapy. At this time, 17 gene therapy trials have been approved by the NIH for the treatment of prostate cancer. Overall, current gene therapy to treat advanced localized prostate cancer has been shown to be safe and feasible. There are many challenges that lie ahead for gene therapy. Nonetheless, it is almost certain that gene therapy will be part of the armamentarium against prostate cancer and other human diseases in the next century.

Keywords: Prostate cancer, gene therapy, gene replacement, immunotherapy, oncolytic virus, suicide gene, prostatectomy, prodrug



249-260

The HSV-TK/GCV gene therapy for brain tumors

Author(s): Dr. Naoto Adachi,

Abstract: Herpes simplex virus-thymidine kinase (HSV-TK) gene transduction followed by ganciclovir (GCV) administration is widely used for cancer treatment as a "suicide gene" therapy. The present review describes the HSV-TK/GCV gene therapy for brain tumors (gliomas) through the eyes of a clinician, including a review of preclinical and clinical studies. In particular, the latest data on the first clinical trial are discussed and, moreover, the surgical procedures are depicted. The surgical technique is the most important for neurosurgeons; therefore, its improvement would be beneficial for further clinical developments as well as biological innovation of retroviral vectors or vector producer cells.

Keywords: HSV-TK/GCV, suicide gene therapy, brain tumor, kinase, retroviral vectors, vector producer cells, glioblastoma, bystander effect



261-274

Establishment of tumor cell lines by transient expression of immortalizing genes

Author(s): Dr. Liangping Li,

Abstract: Many basic and clinical studies on human cancers require establishing of tumor cell lines from fresh tumor tissues in a highly reproducible fashion. However, this goal has not been achieved since the first aneuploid epithelial cell line, HeLa, was established from cervix adenocarcinoma about 5 decades ago. A widely accepted concept is that malignant tumor cells are almost immortal so the difficulty in establishing new tumor cell lines is believed to arise from problems of cell culture techniques. Evidence from our experiments demonstrated that this difficulty has a genetic origin: many primary cancer cells have not completely lost their ‘tumor suppressor’ pathways. In this review the immortalizing genes, transient expression systems of foreign genes in mammalian cells, and the potential applications of transient expression of immortalizing genes are summarized.

Keywords: tumor cell line, immortalizing genes, cell culture, transient expression



275-284

Nuclear matrix and nucleotide excision repair: damage-recognition proteins are not constitutive components of the nuclear matrix

Author(s): Dr. Piotr Widlak,

Abstract: The nuclear matrix is a structure involved in organization of DNA structure and regulation of replication and transcription. Itisgenerallybelievedthatsomeenzymaticactivitiesofnucleotideexcisionrepairarelocalizedinthis nuclear subfraction and that the nuclear matrix anchorage affects the preferential repair of (potentially) active genes. Thus answering the question what is the role of nuclear matrix is very important to fully understand the DNA repair mechanisms. We have studied the in vitro interactions between nuclear matrices from rat liver cells and damaged DNA. A specific 36-bp DNA sequence was either UV-irradiated or damaged by benzo(a)pyrene diol epoxide and N- acetoxy-acetylaminofluorene. The data presented in this communication show that damaged DNA did not preferentially bind to nuclear matrices; damage-recognition proteins were loosely attached to the nucleoskeleton and were easily extracted from chromatin.

Keywords: DNA damage, damage recognition, damaged DNA-binding proteins, nuclear matrix



285-290

Functional improvement in ligament scar tissue following antisense gene therapy: A model system for in vivo engineering of connective tissues

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

Abstract: The present studies indicate that decorin antisense approaches and a HVJ-liposome delivery system can functionally impact on the mechanical properties of MCL scar tissue in a rabbit model. This is one of the first instances where such approaches have led to functional improvement in the tissue. While decorin antisense gene therapy has been shown to be effective at improving MCL scar tissue, the resulting tissue still remains deficient compared to normal tissue. Therefore, this study must be considered a first step towards tissue regeneration and additional experimentation is required to further move scar tissue down the path towards tissue regeneration.

Keywords: gene therapy of ligament healing, antisense therapy, in vivo tissue engineering, connective tissue healing



291-296

3-aminobenzamide: a novel drug to induce in vivo DNA hypermethylation

Author(s): Dr. Paola Caiafa,

Abstract: Both DNA methylation and core histone hypoacetylation are associated with gene silencing but only recent experiments allowed the interlocking of these two processes. Through such experiments it was shown that the two processes are united in inducing gene silencing through a “shuttle-system” involving the methyl CpG binding protein (MeCP2). In this scenario, it is not clear whether DNA methylation or histone deacetylation is the leader in inducing down regulation of gene expression. Trichostatin A (TSA), a potent inhibitor of histone deacetylase, is usually used to clarify this point. As far as DNA methylation is concerned, only the 5-azacytidine (5-AzaC), able to induce hypomethylation, has been described until now. The aim of this paper is to suggest the use of 3-aminobenzamide (3- ABA) as a method capable of inducing in vivo DNA hypermethylation, so that new experiments could be performed in both directions to clarify the chronology by which the influence on gene expression takes place and to pinpoint the structure of methylated condensed chromatin.

Keywords: histone hypoacetylation, gene silencing, histone deacetylation, 5-azacytidine, trichostatin A, chromatin



297-301

Mechanically stretching single chromatin fibers

Author(s): Dr. Jordanka Zlatanova,

Abstract: We have used the recently developed MAC Mode Atomic Force Microscope (AFM) that operates in aqueous solution to mechanically stretch single chicken erythrocyte chromatin fibers. The fibers contained the full complement of histones, or, alternatively, were depleted of linker histones. The AFM was used to produce the so- called force curves, by monitoring the cantilever deflection (proportional to force) as the distance between the AFM tip and the sample was experimentally manipulated. To that end, the AFM tip was pushed into the chromatin sample and then withdrawn, to mechanically stretch the fiber that was physically adsorbed to the tip. Pulling of the chromatin fiber produced complex sawtooth-like patterns of peaks that were characterized by unexpectedly large forces, in the range of several hundred picoNewtons. The distribution of forces in the linker histone-containing and linker histone-depleted fibers was remarkably different, possibly indicating that linker histone binding significantly changes the mechanical properties of the chromatin fiber.

Keywords: Atomic Force Microscopy, chromatin, histone



303-312

Gene potentiation: Forming long-range open chromatin structures

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

Abstract: Gene potentiation is the process of opening a chromatin domain, which in turn renders genes accessible to the various factors requisite for their expression. The formation of an open chromatin structure is central to the establishment of cell fate and tissue-specific gene expression. Both hematopoiesis and spermatogenesis serve as excellent models for examining gene potentiation. Each developmental pathway is governed by a unique differentiative program, which specifies a subset of potentiated genes enabling expression. A discussion of these contrasting potentiative cascades is presented illustrating that cell fate is ultimately determined by the selective opening and closing of gene containing domains. Elucidating the mechanism, which governs these perturbations in chromatin structure, will provide valuable insight into how differentiative decisions are made and whether commitment to a particular phenotype can be modified.

Keywords: chromatin domain, spermatogenesis, hematopoiesis, gene expression, chromatin structure



313-322

Glycine clock: Eubacteria first, Archaea next, Protoctista, Fungi, Planta and Animalia at last

Author(s): Dr. Edward N. Trifonov,

Abstract: Twenty-five different single-factor criteria and hypotheses about chronological order of appearance of amino acids in the early evolution are summarized in consensus ranking. All available knowledge and thoughts about origin and evolution of the genetic code are thus combined in a single list where the amino acids are ranked in descending order, starting with the earliest ones: G,A,D,V,P,S,E,L,T,I,N,F,H,K,R,Q,C,M,Y,W One may expect that in the composition of the ancient proteins the earliest amino acids would dominate. Indeed, when homologous prokaryotic and eukaryotic protein sequences are aligned, the most frequent residue amongst matching amino acids (presumably, what remains of the common ancestor sequence) is glycine that makes about 14% vs. glycine content of 6-7% in modern proteins. The glycine content of the matching residues may, then, serve as a measure of the time (glycine clock) since the separation of compared species. This approach is applied to 370 pairwise alignments of protein sequences from over 100 species of 6 major kingdoms. The evolutionary tree is derived, where the kingdoms separate consecutively from the central stem in the order: Eubacteria (13.5% G at the moment of separation), Archaea (11.5%), Protoctista (10.5%), Fungi (9%), Planta/Animalia (8%), largely consistent with common knowledge on the evolution of the kingdoms. The glycine content, thus, may serve as a time label that allows the tracing back of the separation of any two species with potential accuracy of the order of 50 to 100 million years, all the way to the very origin of species.

Keywords: evolutionary trees, triplet code, earliest proteins, amino-acid chronology, amino-acid composition, molecular evolution, codon chronology, primordial soup, multiple alignments,



323-338

Nuclear prostaglandin receptors

Author(s): Dr. Sylvain Chemtob,

Abstract: Prostaglandins and thromboxane are ubiquitous compounds and play important roles in cardiovascular homeostasis, inflammation, reproduction, respiration, mitogenesis and gene transcription and so on. These actions of prostanoids are presumed to be mediated by plasma membrane receptors belonging to the superfamily of G protein-coupled receptors. However, several lines of evidence suggest prostanoids may also act at the nuclear level. Nuclei contain cyclooxygenases and other intermediates required for prostanoid synthesis and receptor-mediated responses. This review focuses closely on various signal transduction cascades that exist in the nuclear membranes, including the presence of other nuclear G protein-coupled receptor, and discusses the discovery of functional nuclear prostaglandin E2 receptor. These data add new dimensions to the functions and signaling mediated by prostaglandin receptors.

Keywords: PGE2, EP receptors, G proteins, signal transduction, gene transcription, cyclooxygenase, prostanoid transporter



339-348

Target of rapamycin (TOR) signaling coordinates tRNA and 5S rRNA gene transcription with growth rate in yeast

Author(s): Dr. Michael C. Schultz,

Abstract: Transcriptional regulation of genes encoding ribosomal proteins, translation factors, ribosomal RNAs, and the tRNAs plays a critical role in cellular physiology by modulating the availability of key components of the protein synthetic machinery according to the need for cell growth. Recent work in yeast and mammalian systems has revealed that the target of rapamycin (TOR) signaling pathway functions in setting the translational output of the cell in response to nutrient/growth factor availability. The central components of the TOR pathway are the TOR kinases, which are inhibited by the macrolide antibiotic rapamycin. Using yeast as a model system we have tested if the control of translation by the TOR kinases includes an effect on transcription of two components of the translational apparatus, 5S rRNA and the tRNAs. Biochemical studies reveal that polymerase (pol) III transcription of the 5S rRNA and the tRNA genes is regulated by TOR signaling in yeast. Interference with TOR signaling inhibits the activity of RNA pol III and likely TFIIIB, core components of the pol III transcriptional machinery. The mechanism of inhibition involves an effect that is independent of the repression of translation that results when TOR signaling is impaired. We propose that the TOR kinases are components of a signaling network that ensures appropriate expression of the protein synthetic machinery under different growth conditions. Considering the conservation of the TOR pathway and the pol III transcription machinery between yeast and human, this regulatory mechanism is likely to be conserved in eukaryotes.

Keywords: TOR; target of rapamycin; transcriptional regulation by TORs; yeast; ribosomal RNA; tRNA; RNA polymerase I; RNA polymerase III; microarrays; transcription factors



349-362

DNA structural and sequence determinants for nucleosome positioning

Author(s): Dr. John N. Anderson,

Abstract: Positioned nucleosomes are thought to be regulators of genome function but the role of DNA sequence and structure in the control of positioning is poorly understood. We examined the intrinsic curvature of DNA sequences that are known to position histone octamers at single translational sites as a first step to investigate this problem and discovered a conserved pattern of intrinsic DNA curvature that was proposed to direct the formation of nucleosomes to unique positions. The pattern consists of two 50-60 base pair regions of curved DNA separated by preferred lengths of non-curved DNA. The conserved pattern was also seen in all 57 satellite sequences present in the GeneBank database and the distances between successive pairs of curved elements in repeated arrays of satellite monomers were similar to the average spacing of nucleosomes in chromatin. To test the significance of the pattern, ten synthetic DNAs were constructed which contain two regions of curved DNA that are separated by non-curved regions of variable length. Translational mapping of in vitro reconstituted nucleosomes demonstrated that two of the fragments positioned nucleosomes at a single site while most of the remaining fragments positioned octamers at multiple sites spaced at 10 base intervals. In support of the curvature- based model, the positioning sequences contained non-curved central regions of the same lengths that were seen in natural positioning sequences and displayed an affinity for histone octamers comparable to the strongest known natural positioning sequences. A detailed study was then carried out to identify the features that were responsible for high affinity and unique translational positioning activity. Nucleosomes assembled onto positioning fragments of different lengths shared a common upstream border suggesting that the positioning signals were located on the upstream half of these nucleosomal DNAs. In this region, the compressed minor grooves of the A-tracts did not assume the typical rotational orientation of facing the histone octamer. This unusual orientation was showed to be required for unique positioning since positioning activity was lost upon the insertion of 4 bp between the upstream tracts and the pseudo-dyad region. A permanganate hypersensitive site was also found in this region 1.5 turns from the pseudo-dyad at a site known to display DNA distortion in the nucleosome. The sequence of the hypersite contained a TA step flanked by an oligo-pyrimidine tract and the rotational orientation of the reactive TA step in the nucleosomal DNAs was such that the minor groove faces the histone octamer. Substitutions were made in the region of the hypersite and the resulting constructs tested for affinity for histone octamers and translational positioning. The results revealed that a single base change in the TA step and a few changes in the adjacent tract were sufficient to dramatically reduce affinity and positioning activity in a manner that appeared to be correlated with the presence of a permanganate hypersite. In addition, the rotational orientation of the sequence was shown to be important for function since altering the orientation of the site in a positioning fragment reduced positioning activity and octamer affinity while altering the orientation of the sequence in a nonpositioning fragment had the opposite effects. The 5S rDNA positioning sequence from L. variegatus also contained a permanganate hypersite at 1.5 turns from the pseudo-dyad and other natural positioning sequences were enriched in the sequence motifs that give rise to permanganate hypersensitivity in this location. These results suggest a model in which translational positioning is due to a concerted action between the stabilizing forces associated with the hypersite sequences occupying specific sites within the central three turns of nucleosomal DNA and destabilizing forces which appear when the upstream A- tracts with outward facing minor grooves occupy particular translational positions.

Keywords: DNA, nucleosome, histone octamers, GeneBank database, chromatin



363-368

Regulation of transcription by bent DNA through chromatin structure

Author(s): Dr. Ryoiti Kiyama,

Abstract: A class of bent DNA is present in the genomic DNA of higher eukaryotes as a repeating unit (Kiyama, R., Gene Ther. Mol. Biol. 1, 641-647, 1998). This bent DNA appears once every 680 bp on average, and often shows periodicity, suggesting biological significance. By having a higher affinity for histone core particles, it has a role in designating a key nucleosome, which initiates nucleosome alignment. This modulates the enhancer activity of β- LCR and the silencer activity of the human β-globin locus and other loci. This suggests that this type of bent DNA plays important roles in various biological functions by affecting chromatin structure.

Keywords: Transcription, chromatin, bent DNA, β-LCR, nucleosome, silencer, enhancer



369-385

Crosstalk between intra- and extracellular factors in the development of prolactinomas in the anterior pituitary

Author(s): Dr. Hélène Courvoisier,

Abstract: Prolactinomas are non invasive neoplasms resulting from an abnormal proliferation of the lactotroph cells in the anterior pituitary. Although prolactinomas have clearly been shown to be monoclonal tumors, the seek for the initial causal mutation has not been successful yet. In parallel to the recent advances made in the mutation analyses, this review aims at presenting the main extracellular factors, such as hypothalamic factors, estrogens, growth factors and cytokines, that could participate to the overproliferation of the lactotroph cells. In addition to their role in the development and maintenance of prolactinomas, it is proposed that the initial causal mutation could take place in the transduction pathways of such extracellular factors. Taking in account the complex regulation of the anterior pituitary could help in designing more specific and efficient treatments, especially for patients resistant to the most commonly used bromocriptine therapy.

Keywords: prolactinomas, anterior pituitary, prolactin, lactotroph, oncogenes, neuroendocrine tumor, signal transduction, G-protein coupled receptor, molecular pharmacology



387-395

Cks1 mediates the effects of mutant p53 proteins on the mitotic spindle cell cycle checkpoint

Author(s): Dr. Antonio Gualberto,

Abstract: The p53 tumor suppressor is the most frequently mutated gene in human cancer. Alteration of the p53 locus predisposes human cells to chromosomal instability. This is due in part to interference of mutant p53 proteins with the activity of the mitotic spindle and postmitotic cell cycle checkpoints. Recent data indicates that mutant p53 proteins affects the control of the metaphase-to-anaphase transition by up-regulating the expression of Cks1, a protein that mediates the activatory phosphorylation of the anaphase promoting complex (APC) by Cdc2. Cells carrying mutant p53 proteins overexpress Cks1 and are unable to sustain APC inactivation and mitotic arrest. These data implicate Cks1 in the onset of chromosomal instability in cells carrying mutant p53 proteins.

Keywords: p53, Cyclin B, Cks1, CksHs1, Cell Cycle Checkpoint



397-404

BRCA1 function in transcription

Author(s): Dr. Jeffrey D. Parvin,

Abstract: The breast and ovarian specific tumor suppressor protein, BRCA1, is associated with the transcriptional regulatory machine termed the RNA polymerase II (Pol II) holoenzyme. Experiments support a model in which molecules such as the BRCA1 protein and the CREB binding protein (CBP) function similarly as regulatory targets which link the enhancer-binding regulatory proteins to the basal transcriptional machinery. As an example, phosphorylated CREB protein interacts with the CBP molecule present in the holoenzyme complex and will activate transcription in vitro. We propose a similar model for the BRCA1 protein, but with a different subset of upstream activators functioning via BRCA1 to regulate the Pol II holoenzyme. We have established an in vitro transcriptional assay dependent upon the carboxy-terminal domain of BRCA1 fused to the DNA-binding domain of GAL4. We have also shown that both CBP and BRCA1 are linked to the Pol II holoenzyme complex via a common subunit -- the RNA Helicase A (RHA) protein. CBP binds to an amino terminal domain of RHA, and BRCA1 binds to a separate internal domain of RHA, and truncated RHA molecules have been shown to be dominant negative transcriptional repressors of either CBP or BRCA1. Since most tumor-associated BRCA1 mutations result in the truncation of the BRCA1 protein and loss of its carboxy-terminal holoenzyme interaction domain, it is suggested that an important component of the tumor suppressive function of BRCA1 occurs via the gene expression process.

Keywords:



405-410

Coding end resolution in scid recombination-inducible cell lines

Author(s): Dr. Yung Chang,

Abstract: VDJ recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by recombination activating gene (RAG1 and RAG2) proteins generates two types of broken DNA ends: blunt signal ends and hairpin coding ends. The standard joining of these ends to form signal joints and coding joints employs several proteins involved in double strand break (DSB) repair, including KU70/80, the catalytic subunit of DNA- dependent protein kinase (DNA-PKcs), XRCC4 and ligase IV. The cells from severe combined immunodeficient (scid) mice are defective in resolving recombination coding ends due to a point mutation in the DNA-PKcs gene. To study the effect of the scid defect on coding end resolution, we have established recombination-inducible cell lines from scid mice. These cells, at the nonpermissive temperature, actively initiate recombination at the endogenous light chain loci and produce large amounts of hairpin coding ends. After returning to the permissive temperature, scid cells are capable of resolving these coding ends. However, unlike the coding end resolution in normal cells, which is a rapid and regulated process, the resolution of hairpin coding ends in scid cells is slow and error prone. The resulting coding joints contain extensive nucleotide deletions. In addition, the interlocus recombination products are found at much higher frequency in scid cells than in their normal cell counterparts. Our results suggest that functional DNA-PKcs may play an important role in facilitating effective V(D)J recombination and minimizing chromosomal instability.

Keywords: RAG1, hairpin, scid, V(D)J recombination, chromosomal instability, Abelson murine leukemia virus, KU70/80, DNA- dependent protein kinase



411-416

Pak protein kinases as mediators of Ras signaling and cell transformation. A place for Pak on the Ras MAP: More than just another JNK bond

Author(s): Dr. Jeffrey Field,

Abstract: Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras>Raf>Mek>Erk (MAPK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase) which synthesizes several lipid second messengers that activate small G proteins such as Rac and Cdc42. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65Pak). Here we review studies documenting a novel Ras signal through PI 3-kinase and Rac/Cdc42 to Pak. The signal appears essential for maintaining cell transformation and Erk activation.

Keywords: Raf, signal transduction, PI3-kinase, cancer



417-424

Negative regulation of cytoplasmic protein tyrosine kinase activity by adaptor proteins

Author(s): Dr. Serge Roche,

Abstract: Adaptor proteins are cytoplasmic signaling molecules that lack intrinsic catalytic activity but they are nevertheless crucial for signal transduction. They bear homology domains (SH2, SH3, PH, PTB, ...) important for protein- protein interactions and for function. The first adaptors identified were positive regulators of cell responses, with some even having oncogenic activities. More recently, a new subfamily has emerged that negatively regulates signaling responses. This review will focus on adaptors of this genre and specifically, those that inhibit cytoplasmic tyrosine kinase function and which define a new mechanism for in vivo kinase regulation. They include the inhibitors of the Jak, Syk, Fak and Src kinase family and their mechanism for inhibition as well as their possible function in cellular regulation will be discussed.

Keywords: cytoplasmic protein, tyrosine, kinase, adaptor proteins, Jak, Syk, Fak, Src




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