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Volume 5 - 2000


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1-8

A novel expression vector induced by heat, γ- radiation and chemotherapy

Author(s): Dr. David Harris,

Abstract: In many gene therapy applications and molecular biology manipulations it is desirable to be able to control the expression of the therapeutic gene. In this study a new expression vector, pHOT-MCS, was constructed using a 451 bp fragment from the human heat shock protein 70B (HSP 70) promoter. The vector has a large multiple cloning site and the neomycin selectable marker, making it more user-friendly. Using the human Interleukin-2 (IL-2) gene as a marker, it was demonstrated that heat can induce the pHOT-IL-2 plasmid to express 2 fold more IL-2 than levels obtained with the human cytomegalovirus (CMV) promoter. Using the Enhanced Green Fluorescence Protein (EGFP) gene as a reporter gene, a human breast carcinoma cell line (MCF-7) was transfected with pHOT-EGFP and stably transfected cells selected with neomycin. The stable transfectants were subjected to three different experimental conditions; heat treatment at 42°C for one hour, treatment with geldanamycin at 1μg/ml (an anti- leukemic drug) or 3000 rads of γ-radiation. EGFP expression was measured for up to 72 hours by flow cytometry. The non-treated cells expressed a basal level of EGFP that increased 387% above background at 24 hours after heat-shock. Cells treated with Geldanamycin had a 208% increase in EGFP intensity at 24 hours which was maintained up to 72 hours as compared to the non-treated cells. Exposure of cells to 3000 rads of γ-radiation had a 150% increase in EGFP expression at 48 hours post-treatment as compared to the non-treated cells. Induction of heat shock proteins by heat, radiation and geldanamycin was confirmed by Western blot analysis. This inducible gene expression system may be applicable to clinical use in synergy with other types of standard therapy (e.g, hyperthermia, radiation and chemotherapy).

Keywords: beta-galactosidase, (β-gal); cytomegalovirus, (CMV); Enhanced Green Fluorescence Protein, (EGFP); heat shock protein 70B, (HSP 70); Interleukin-2, (IL-2)



9-30

Misregulation of pre-mRNA splicing that causes human diseases. Concepts and therapeutic strategies

Author(s): Dr. Stefan Stamm,

Abstract: About one third of all human genes are subject to alternative splicing. The molecular mechanisms that regulate alternative splice site usage are beginning to emerge and show that transcription and pre-mRNA processing are integrated processes that can be modified by cellular signals. Several diseases are caused by mutations in sequences that regulate pre-mRNA processing. Their molecular characterization indicates that contributions of pre-mRNA splicing defects to human diseases have been underestimated and could account for pleiotropic phenotypes. The understanding of the molecular mechanisms allows the development of therapeutic strategies.

Keywords: alternative splicing, kinases, tau, FTDP-17, signal transduction



31-37

Th2-type immune response induced by a phage clone displaying a CTLA4-binding domain mimic- motif

Author(s): Dr. ,

Abstract: We have recently isolated a phage clone, F2, which displays the CTLA4-binding domain mimic from a phage display library. To investigate the in vivo effects of an F2 motif on the regulation of immune responses, we immunized Balb/c mice intraperitoneally with varying doses of an F2 phage in a phosphate buffered saline and followed the resulting antibody and cytokine responses. It was shown that the F2 phage enhanced the IgG antibody response to phage particles in comparison to control phages that were randomly selected from the library. When the antigen specificity of the induced antibody response was examined, the production of an anti-g3p antibody was preferentially increased while an anti-g8p antibody was slightly down-regulated by the immunization of F2 in comparison to control phage clones. The increase of an anti-g3p antibody response was found in the isotype of IgG1 but not in the IgM or IgG2a. When the cytokine production was examined by culturing spleen cells from these mice under stimulation with anti-CD3 mAb, IL-4 production was approximately twice higher in the F2-primed cells than in the L4-primed cells while IFN-γ production was higher in L4-primed cells than in the F2-primed cells. Thus, these results suggested that the F2 phage clone bearing g3p with the CTLA4-binding domain mimic-motif induced the Th2-type response when compared to control phage clones.

Keywords: CTLA-4, Th2, immune deviation, peptide mimic, phage library, molecular design, vaccine



39-46

Segregation of partly melted molecules and its application to the isolation of methylated CpG islands in human cancer cells

Author(s): Dr. Masahiko Shiraishi,

Abstract: Segregation of partly melted molecules is a convenient and efficient method for isolation of DNA fragments associated with CpG islands. DNA fragments digested with restriction endonucleases are subjected to denaturing gradient gel electrophoresis. DNA fragments derived from CpG islands are preferentially retained in the gel after prolonged field exposure because of their lower rate of strand dissociation. An independent technique, methylated-DNA-binding column chromatography, permits separation of DNA fragments on the basis of the number of methyl-CpG sequences in the fragment, and it enables separation of methylated CpG islands from those that are not methylated. Segregation of partly melted molecules and methylated-DNA-binding column chromatography were successfully combined to isolate CpG islands methylated in human adenocarcinomas of the lung. The methylated CpG island library will be valuable in order to elucidate epigenetic process in carcinogenesis.

Keywords: denaturing gradient gel electrophoresis, DNA methylation, methylated DNA binding column, adenocarcinomas of the lung, epigenetics



47-53

PNA (peptide nucleic acid) anti-gene/antisense can access intact viable cells and downregulate target genes

Author(s): Dr. Lidia C Boffa,

Abstract: PNA (peptide nucleic acid) anti-gene/antisense can access intact viable cells and downregulate target genes

Keywords: Peptide Nucleic Acid (PNA), anti-gene, antisense, cell /nuclear localization vectors, gene downregulation



55-60

Delivery of plasmid DNA by in vivo electroporation

Author(s): Dr. Loree Heller,

Abstract: For gene therapy, in vivo delivery of plasmid DNA offers an alternative to viral delivery methods. Since the efficiency of plasmid delivery to tissues is generally lower than viral delivery, several methods have been introduced to augment in vivo plasmid delivery including liposome conjugation, particle mediated delivery, and electroporation. In vivo electroporation has reached clinical trials for the delivery of chemotherapeutic agents to cancers, and a number of preclinical studies have been performed demonstrating that this technique also enhances plasmid delivery and expression of both reporter and therapeutic genes or cDNAs. This delivery has been performed to a number of tissues including skin, muscle, liver, testes and tumors employing a wide range of electrical conditions and electrodes. While this preclinical research is promising, further optimization of electrical conditions and electrodes may be necessary for clinical use. With the availability of a variety of therapeutic gene delivery techniques, it will be possible to tailor gene therapies to individual diseases.

Keywords: gene therapy, electroporation



63-79

Potential roles of p53 in recombination

Author(s): Dr. Lisa Wiesmüller,

Abstract: Functional inactivation of p53 by mutation or by interactions with viral tumor antigens is associated with a deficit to maintain the genomic stability. Until recently, p53 was believed to exclusively avoid the manifestation of DNA damage by transcriptionally signaling transient cell cycle arrest or apoptotic cell death in response to cellular stress situations. New ideas on a direct involvement of p53 in DNA repair originated from the discoveries of interactions with replication and repair proteins and of repair-related enzymatic activities, such as the 3′-5′ exonuclease activity. Moreover, physical and genetic links were established between p53 and factors involved in homologous DNA recombination processes, namely the initial strand transferase RAD51 and the RAD51 complex partner BRCA1 and BRCA2. Importantly, several groups reported that p53 suppresses spontaneous as well as radiation-induced inter- and intrachromosomal homologous recombination events by at least one to two orders of magnitude. The identification of cancer-related separation of function mutations demonstrated that p53 regulates homologous recombination processes independently of its activities in transcription and growth control, and suggested that functions in recombinative repair contribute to tumor suppression. Mechanistic studies indicated that the p53- dependent regulation of DNA-exchange events is tied to the generation of DNA strand exchange intermediates by RAD51. The recognition of certain mismatched bases within these heteroduplex joints by p53 has lead to a model suggesting that p53 monitors the fidelity of homologous recombination events in a manner complementary to the mismatch repair factor MSH2. The possibility of an involvement of p53 in replication-associated recombination processes is discussed.

Keywords: double strand breaks, homologous recombination, nonhomologous end-joining, strand exchange, tumor suppressor



81-86

Characterisation of the p53 gene in the rat CC531 colon carcinoma

Author(s): Dr. Rob C Hoeben,

Abstract: The p53 gene was characterised in CC531 colon carcinoma of the Wag/Rij rat, a model frequently used for the evaluation of anti-cancer treatments. The gene incurred a large in-frame deletion, with junctions in exons 4 and 8, and encodes a protein of approximately 32 kDa, lacking the entire DNA-binding domain. No wild-type p53 allele is retained. Functional analysis shows that the mutated protein can repress the function of wtp53 protein.

Keywords: colorectal cancer, mutated p53



87-100

Recombinant adenoviruses as expression vectors and as probes for DNA repair in human cells

Author(s): Dr. Andrew J. Rainbow,

Abstract: There is widespread interest in the use of recombinant adenovirus (Ad) vectors for gene therapy of cancer and as tools in molecular biology research. There are also potential benefits to be gained by combining strategies for Ad- based gene therapy of cancer with radiotherapy and chemotherapy. However, there is limited information available on the effects of cytotoxic agents on transgene expression which would allow a rational approach to combining these modalities. We have used recombinant nonrepilcating Ad expressing the lacZ gene under the control of the cytomegalovirus (CMV) immediate early promoter to assess the effects of cytotoxic agents on the expression of a reporter gene in human cells. Using this approach we are able to examine both constitutive and inducible expression of the reporter gene. Pretreatment of normal human cells with low UV fluence results in an enhanced expression of the reporter gene. The enhanced expression occurs at lower doses of the DNA damaging agent in cells deficient in the transcription coupled repair (TCR) pathway of nucleotide excision repair (NER) suggesting that the enhancement in human cells is triggered by persistent damage in actively transcribed genes. The enhancement is also reduced or absent in SV4O-transformed cells and cells expressing the human papilloma virus ('WV) E7 gene, but not in Li-Fraumeni syndrome (LFS) cells or cells expressing the 'WV E6 gene. Since SV4O-transformation and HPV E7 expression both abrogate the retinoblastoma (pRb) family of proteins, whereas 'WV E6 abrogates p53 and LFS cells express mutant p53, these results indicate that the enhanced expression depends on one or more of the pRb family of proteins, but not on p53. We have also used recombinant Ad expressing a lacZ reporter gene as a probe for DNA repair in human cells. Using this approach we have examined constitutive as well as inducible DNA repair of a UV-damaged reporter gene in human cells. We detected enhanced host cell reactivation (HCR) of a UV- damaged reporter gene in pre-heat-shock treated or pre-UV treated TCR proficient but not in pretreated TCR deficient human fibroblasts or LFS cells. These results suggest the existence of an inducible repair response for UV- damaged DNA in human cells which is dependent on the TCR pathway of NER and the wild type p53 tumour suppressor. These results have important implications for the use of recombinant Ad-based expression vectors under the control of the CMV promoter in gene therapy for cancer when used in combination with DNA damaging agents.

Keywords: recombinant adenovirus, host cell reactivation, nucleotide excision repair, enhanced reporter gene expression, inducible DNA repair, p53 tumour suppressor, ultraviolet light



101-110

Chromatin remodeling and developmental gene regulation by thyroid hormone receptor

Author(s): Dr. Yun-Bo Shi,

Abstract: Thyroid horruone (TH) receptors (TRs) are dual function transcription factors. They activate or repress transcription in the presence or absence of TH, respectively. Using the Xenopus laevis oocyte as an in vivo system to assemble TH target promoters into chromatin under conditions mimicking somatic cells, we have shown that transcriptional repression by unilganded TR involves histone deacetylase while transcriptional activation by TH- bound TR leads to chromatin disruption. Using Xenopus laevis development as a developmental model, we have demonstrated that TR is constitutively bound to its target genes in chromatin. Transcriptional activation induced by TH is accompanied by the release of at least one histone deacetylase and increase in local histone acetylation. These studies together with the developmental expression profiles of TR genes suggest that TH-induced changes in chromatin remodeling play an important role in the dual functions of TR in frog development: gene repression in premetamorphic tadpoles when TH is absent and gene activadon during metamorphosis, a process induced by the endogenously synthesized TH.

Keywords: Xenopus laevis, Amphibian metamorphosis, histone acetylation, chromatin remodeling, thyroid hormone receptor



111-120

Signal transduction pathways in cancer cells; novel targets for therapeutic intervention

Author(s): Dr. Christos A. Tsatsanis,

Abstract: Oncogenic proteins participate in signal transduction cascades that induce cell transformation. Understanding the molecular events that take place during oncogenesis is necessary to find novel, more effective therapeutic interventions. Signal transduction in cancer cells involves signaling from the extracellular environment, through the membrane, into the cytoplasm and towards the nucleus where transcription is initiated to generate proteins that will eventually contribute to the oncogenic phenotype. Alterations in such signaling cascades via mutations, gene amplifications or deletions frequently occur in human neoplasms. The result of such alterations has an impact in the cell cycle control, the cell morphology and the regulation of apoptosis. An overview on the signaling molecules altered in human tumors and their potential role as therapeutic targets is presented.

Keywords: Human neoplasms, signal transduction, oncogenes, transcription factors, kinases



121-130

Control of pre-mRNA processing by extracellular signals: emerging molecular mechanisms

Author(s): Dr. Stefan Stamm,

Abstract: Alternative splicing is an important mechanism to regulate gene expression. At least 30% of all human genes are alternatively spliced. This process can be regulated by extracellular signals that include stress and cellular activity. Splice site selection is regulated by a multiprotein complex Its composition can be regulated by either releasing proteins from nuclear storage sites or by changing protein:protein, as well as protein:RNA Interactions by serine and tyrosine phosphorylation.

Keywords: acetylcholinesterase, tra-2: transformer-2, N-methyl-D-aspartart receptor, 1, serine-arginine-rich protein, heterogenous ribonuclear protein, kinase G-1



131-145

Herpes Simplex Virus vector-based gene therapy for malignant glioma

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

Abstract: Conventional therapies have made little impact on the poor prognosis associated with malignant glioma. Recent advances in the construction of replication-defective Herpes Simplex Virus (HPV)-based vectors have offered an opportunity to explore the therapeutic affects of simultaneous multiple transgene delivery to these tumours. Identification of co-operative molecular targets has enabled the rational selection of therapeutic transgene combinations. Exploiting the large capacity of HSV for the insertion of multiple transgenes, the high infectivity of HSV for many cell types and the ability to manufacture vectors of high tire and purity, a series of combination gene therapy vectors have been developed and tested in animal models of malignant glioma. Recent work has been established the principle that multi-modal therapies, including both radiosurgery and combination multi-gene therapy, are superior to single molecular interventions. Eradication of some experimental gliomas has been possible using a multi-modal approach, which provides optimism that further developments may yield reagents that prove therapeutically useful in the neuro-oncology clinic.

Keywords: Herpes Simplex Virus vector, gene therapy, malignant glioma, HSV-TK, bystander effect, TNFα



147-155

Viral vectors carrying a marker-suicide fusion gene (TK-GFP) as tools for TK/GCV –mediated cancer gene therapy

Author(s): Dr. Jarmo Wahlfors,

Abstract: A herpes simplex thymidine kinase – green fluorescent protein (TK-GFP) fusion gene was constructed to couple a marker gene to a therapeutic gene. For testing the utility of the fusion gene, it was cloned into four different viral vectors: Semliki forest virus (SFV), Sindbis virus, adenovirus and lentivirus. The produced viral TK-GFP vectors were then used to test their transduction efficiency on different tumor cells and especially the relationship between TK gene transfer and treatment result with prodrug ganciclovir (GCV). When the efficiency of the other three viral vectors were compared to adenoviral vector, a commonly used tool in cancer gene therapy, the alphaviral vectors SFV and Sindbis performed better on glioma cells, but were less efficient on renal carcinoma cells. On the other hand, an HIV-1 –based, VSV-G pseudotyped lentiviral vector was efficient on all cell lines tested, suggesting the potential of this vector type for cancer gene therapy. GCV-sensitivity studies revealed that regardless of the vector type used, the treatment result was directly proportional to the gene transfer efficiency (not to the multiplicity of infection). However, good gene transfer rate alone is not always sufficient: twenty- percent efficiency was enough to cause adequate cytotoxicity with 5 μg/ml GCV in both of the glioma cell lines, whereas even higher than 80% transduction was not enough to successfully treat Caki-2 cells under the same conditions. When the bystander effect was examined, Caki-2 cells were shown to display a much weaker effect than the glioma cells. Our results demonstrate the benefit of TK-GFP fusion gene in cancer gene therapy research and emphasize the importance of finding an appropriate vector type for each tumor, as well as testing the presence of bystander effect before continuing with TK/GCV approach.

Keywords: gene transfer, cancer, viral vectors, GFP, HSV-TK, fusion construct



157-161

Aberrant DNA methylation of p16 onco- suppressor gene in human cervical carcinoma

Author(s): Dr. Paola Caiafa,

Abstract: The aim of this paper is to investigate the role played by DNA methylation in uterine cervical carcinoma. Since in a subset of human cancers the onco-suppressor CDKN2/p16 gene is an essential target for malignant transformation, its first and second exon regions were investigated for their DNA methylation levels by PCR methylation-dependent analysis. Our results on purified DNAs from cervical tissues show that, in spite of the diffuse DNA hypomethylation which characterizes neoplastic cells, specific DNA methylation of HpaII and CfoI sequences of the first exon occurs and increases with the grade of neoplastic transformation. These data support the idea that the p16 onco- suppressor gene is directly involved in malignant transformation through the methylation process.

Keywords: DNA methylation, p16 gene, cervical carcinoma




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