DNA Repair In Cancer Biology And Therapy
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
Grant 5P01CA129186-02 from National Cancer Institute, IRG: ZCA1
Abstract: This is an application for a new Program Project grant entitled, "DNA Repair in Cancer Biology and Therapy." This is a laboratory-based, basic science program that features four inter-related projects that stress fundamental aspects of DNA repair, genome stability, cancer biology and tumor hypoxia, with a key long-term goal of developing novel anti-cancer therapies that target interconnected DNA repair pathways and/or exploit tumor hypoxia. Dr. Sartorelli will develop novel hypoxia-activated prodrugs that are designed to inhibit the repair factor, O6-alkylguanine-DNA alkyltransferase (AGT), as well as prodrugs that, upon activation in hypoxic cells, will damage and crosslink DNA. Dr. Glazer, the PI, will lead a project that focuses on the transcriptional regulation of the homology-dependent repair (HDR) genes, RAD51 and BRCA1, in hypoxic cancer cells. This project will probe how HDR is regulated in hypoxic cancer cells and will test the extent to which this regulation of HDR may render such cells vulnerable to agents that target interconnected repair pathways. Dr. Sweasy will study how repair factors in the base excision repair (BER) pathway vary in the normal population and in tumors. She will examine the phenotypes of BER variants in cells in culture and in mice using assays for mutagenesis, genetic instability, transformation, and tumor formation. She will examine how deficiencies in BER may play into the HDR pathway to guide the design of new cancer therapies. Dr. Sung will study how the HDR pathway is regulated at the level of protein-protein interactions, with a focus on the repair factors, BRCA2, FANCD2, and RAD51. One administrative and two scientific cores will provide essential services to the program. The Project leaders and Core directors are joined by common interests, a history of collaboration, and joint efforts in teaching and training that provide cohesiveness in support of this effort. The overall Program represents a significant commitment of the Yale University School of Medicine and the participating investigators to studies that have direct relevance to cancer biology and therapy
Project start date: 2007-08-06
Project end date: 2012-07-31
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Grants awarded to Peter M Glazer
RECOMBINATION INDUCED BY TRIPLE HELIX FORMATION
Peter M Glazer, Professor And Chair
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 5R01GM054731-08 from National Institute Of General Medical Sciences, IRG: MGN
Abstract: We propose experiments to test the hypothesis that oligonucleotide-mediated triple helix formation can stimulate homologous recombination in mammalian cells. The broad, long- term objective of these studies is to learn how triplex-forming oligonucleotides (TFOs) can be used as a tool for the genetic manipulation of mammalian cells for the ultimate purpose of gene therapy. Initial work has shown that TFOs can stimulate recombination between duplicated genes in an SV40 vector in mammalian cells, and preliminary studies with repair-deficient cell lines indicate that this induction may be dependent on nucleotide excision repair (NER). In recent studies using a mouse LTK- cell line carrying two mutant copies of the thymidine kinase (TK) gene as direct repeats in a single locus, we were able to demonstrate triplex-induced recombination at a chromosomal site. In the proposed work, factors that influence TFO-induced recombination will be studied using the established LTK- cell assay, with the focus on intracellular TFO delivery and TFO chemical modification. Novel recombination substrates will be developed to report recombination induced either by TFOs of by double-strand breaks generated by the I-Sce I nuclease. These novel vectors will be used in repair-deficient cell lines, with an emphasis on determining the influence of NER, transcription- coupled repair, and the Rad51-associated pathway of homologous recombination on triplex-induced recombination. Based on preliminary work demonstrating triplex-induced recombination in human cell-free extracts, we will further probe the roles of specific repair and recombination proteins, using a battery of antibodies and purified proteins to manipulate the in vitro reaction. These experiments will serve as a basis for designing additional cell lines and experimental protocols to test triplex-induced recombination between a chromosomal site and a separate homologous donor DNA fragment, in situations where the TFO is directed to bind either within or outside of the target gene. The possibility of a further enhancement in gene targeting will be tested using a novel strategy in which the TFO is covalently tethered to a short donor DNA fragment. With this hybrid molecule, target site recognition can be mediated by triplex formation, thereby positioning the donor fragment for efficient recombination and information transfer. We will build on positive initial results to pursue a systematic examination of this approach, using both episomal and chromosomal reporter gene constructs in mammalian cells. This work will provide the foundation for future efforts to correct mutations in disease- related human genes.
Keywords: DNA damage, DNA repair, genetic recombination, oligonucleotide, triple helix, chromosome, gene targeting, gene therapy, genetic manipulation, recombinant DNA, cell line, simian virus 40, tissue /cell culture, transfection /expression vector
Project start date: 1997-08-01
Project end date: 2006-07-31
5R01GM054731-08 (2004): $269173
5R01GM054731-07 (2003): $268938
2R01GM054731-05 (2001): $304623
Research Training: Radiation Therapy, Biology, Physics
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
Grant 5T32CA009259-24 from National Cancer Institute, IRG: NCI
Abstract: This program supports research training at the post-doctoral level in cancer biology, radiation biology, and radiological physics relevant to therapeutic radiology. The training program is of two years duration. The program is open to individuals who hold the Ph.D. degree in one of the basic biological sciences, biochemistry, molecular biology, cell biology, molecular genetics, radiation biology, or in physics or related sciences who wish to pursue cancer-related studies, particularly as applied to therapeutic radiology. The program is also open to individuals who hold the M.D. degree who wish to pursue careers in combining laboratory and clinical research in these same areas. The program takes advantage of the extensive laboratory and clinical research activity in the Department of Therapeutic Radiology, and in this revised application it has been expanded to include trainers in other departments who have interests relevant to radiobiology. Other key elements of this revised application are enhancement of the training environment by major renovations to the research space in the Department of Therapeutic Radiology (supported by both Yale University and the NIH) and expansion of the training opportunities by new faculty recruitments in radiobiology-related fields. The program includes required courses in radiobiology, radiological physics, and in biomedical ethics. Conferences, seminars and workshops cover a range of basic science and clinical topics designed to provide exposure not only to radiobiology, cancer biology, and radiation physics, but also to cancer as a disease. In this amended application, a new panel of faculty advisors has been set up to assist the program director and the training faculty in the selection of trainees and their assignment to research groups, as well as to provide trainee mentoring and supervision. Trainees devote more than 90% of their time to specific long-term research projects and interact with a wide variety of other biomedical science research programs throughout Yale University and the Yale Comprehensive Cancer Center
Project start date: 1978-09-30
Project end date: 2010-05-31
5T32CA009259-23 (2007): $232732
5T32CA009259-22 (2006): $229688
2T32CA009259-21A2 (2005): $186091
DNA Repair In Cancer Biology And Therapy
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
Grant 5P01CA129186-02 from National Cancer Institute, IRG: ZCA1
Abstract: This is an application for a new Program Project grant entitled, "DNA Repair in Cancer Biology and Therapy." This is a laboratory-based, basic science program that features four inter-related projects that stress fundamental aspects of DNA repair, genome stability, cancer biology and tumor hypoxia, with a key long-term goal of developing novel anti-cancer therapies that target interconnected DNA repair pathways and/or exploit tumor hypoxia. Dr. Sartorelli will develop novel hypoxia-activated prodrugs that are designed to inhibit the repair factor, O6-alkylguanine-DNA alkyltransferase (AGT), as well as prodrugs that, upon activation in hypoxic cells, will damage and crosslink DNA. Dr. Glazer, the PI, will lead a project that focuses on the transcriptional regulation of the homology-dependent repair (HDR) genes, RAD51 and BRCA1, in hypoxic cancer cells. This project will probe how HDR is regulated in hypoxic cancer cells and will test the extent to which this regulation of HDR may render such cells vulnerable to agents that target interconnected repair pathways. Dr. Sweasy will study how repair factors in the base excision repair (BER) pathway vary in the normal population and in tumors. She will examine the phenotypes of BER variants in cells in culture and in mice using assays for mutagenesis, genetic instability, transformation, and tumor formation. She will examine how deficiencies in BER may play into the HDR pathway to guide the design of new cancer therapies. Dr. Sung will study how the HDR pathway is regulated at the level of protein-protein interactions, with a focus on the repair factors, BRCA2, FANCD2, and RAD51. One administrative and two scientific cores will provide essential services to the program. The Project leaders and Core directors are joined by common interests, a history of collaboration, and joint efforts in teaching and training that provide cohesiveness in support of this effort. The overall Program represents a significant commitment of the Yale University School of Medicine and the participating investigators to studies that have direct relevance to cancer biology and therapy
Project start date: 2007-08-06
Project end date: 2012-07-31
1P01CA129186-01 (2007): $1573413
TARGETED MUTAGENESIS OF DNA VIA TRIPLE HELIX FORMATION
Peter M Glazer, Professor And Chair
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 1R01CA064186-01A1 from National Cancer Institute, IRG: CPA
Abstract: The overal goal of the work is to develop and test novel genetic methods to test the hypothesis that triple helix forming oligonucleotides can be used to deliver site-selective DNA damage and mutation in mammalian cells. The specific aims of the work are to develop new models to study triple helix- mediated mutagenesis (based upon shuttle vector technology), to investigate the physical parameters which affect targeted mutagenesis upon these shuttle vectors in vitro, to examine DNA damage and repair pathways which influence TFO mediated mutation, and finally to extend the work to study TFO mediated mutation in chromosomal genes of the mouse, rather than shuttle vectors.
Keywords: genetic manipulation, genetic technique, site directed mutagenesis, triple helix, DNA damage, chromosome, gene mutation, oligonucleotide, photochemistry, psoralen, ultraviolet radiation, laboratory mouse, simian virus 40, tissue /cell culture, transfection vector
Project start date: 1995-07-01
Project end date: 1999-04-30
1R01CA064186-01A1 (1995): $216032
5R01CA064186-04 (1998): $234109
5R01CA064186-03 (1997): $227423
5R01CA064186-09 (2003): $278184
5R01CA064186-08 (2002): $271853
5R01CA064186-07 (2001): $265705
5R01CA064186-06 (2000): $266288
2R01CA064186-05 (1999): $223064
Targeted Correction Of The Human Beta-globin Gene
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
Grant 5R01HL082655-02 from National Heart, Lung, And Blood Institute, IRG: ZRG1
Abstract: Targeted Correction of the Human p-globin Gene. The ability to genetically manipulate human stem cells has the potential to enhance the utility of stem cell therapy for human disease. One emerging approach to targeted genome modification is the use of triplex- forming oligonucleotides (TFOs). These molecules bind to duplex DNA in a sequence-specific manner, and this binding can be used to stimulate recombination and gene correction in mammalian cells. Initial work by our group and others has demonstrated that TFOs can stimulate recombination either by delivering a tethered psoralen adduct or by the ability of triple helices, themselves, to provoke DNA repair and thereby sensitize the target site to recombination. Using a series of chemical modifications, the intracellular effectiveness of TFOs has been progressively enhanced. In this revised A2 application, we propose to develop specific TFOs and TFO analogs such as peptide nucleic acids (PNAs) for intracellular binding to the human (3-globin gene with the intention of ultimately generating an optimized reagent set for molecular correction of mutations associated with thalassemia and sickle cell anemia. We will test promising TFOs or PNAs and donor DNA molecules for the ability to correct (3-globin mutations using specially designed reporter systems in cell lines in culture. New data now demonstrate our ability to target the (3-globin gene in human cells with modified TFOs. Gene correction in mouse hematopoietic stem cells (HSCs) will be studied in cells derived from transgenic mice engineered to incorporate human p-globin gene sequences in the context of a green fluorescent protein (GFP) reporter gene. We will also test targeting of the p-globin gene in primary human CD34+ cells enriched for HSCs in assays for both TFO-targeted psoralen crosslinks and for the generation of a targeted sequence change within the p-globin gene. In this revised application, continued optimization of transfection techniques in hematopoietic stem cells and new strategies to boost gene correction frequencies will enhance these efforts. The overall hypothesis driving this application is that high-affinity, site-specific DNA binding by TFOs can be used as a tool to stimulate gene targeting in stem cells. Our long-range goal is to develop such molecules as reagents for targeted genome modification of disease-related genes in human cells
Keywords: binding site, biomaterial development /preparation, chemical binding, chemical structure function, gene targeting, globin, hematopoietic stem cell, oligonucleotide DNA, analog, chemical substitution, gene therapy, hematopoietic tissue transplantation, intracellular, intron, peptide nucleic acid, reporter gene, stem cell transplantation cell line, genetically modified animal, laboratory mouse, tissue /cell culture
Project start date: 2007-09-01
Project end date: 2011-07-31
1R01HL082655-01A2 (2007): $412318
MUTAGENESIS IN DNA MISMATCH REPAIR DEFICIENT MICE
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 5R01ES005775-09 from National Institute Of Environmental Health Sciences, IRG: CPA
Abstract: Mutations in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer in humans. Transgenic mice in which the MMR genes have been inactivated are prone to cancer. However, the different knock-out mice develop different types of tumors, suggesting that the respective proteins may have non-overlapping roles in the preservation of genomic integrity and in tumor suppression. The principal investigator has developed a transgenic mouse assay system to study mutagenesis in vivo using a chromosomally-integrated, recoverable lambda phage shuttle vector carrying the bacterial supF gene as a mutation reporter. The broad, long-term objective of this proposal is to combine this in vivo mutation assay with the MMR-deficient animals to probe critical issues relating to genetic instability, carcinogenesis, and response to cancer therapy in the setting of MMR deficiency. The specific aims are 1) To generate second-generation transgenic mice with an improved supF reporter construct that is transcribed in mouse cells and that is modified to contain a signature sequence to identify sibling vs. independent mutations. 2) To examine spontaneous mutagenesis in hybrid mice, all carrying the XsupF shuttle vector, plus various combinations of alleles at the MMR loci, including PMS1, PMS2, MLH1, and MSH2. Each genotype will be compared for tissue-specific, age-related, and developmental differences. Mutation frequencies and spectra will be analyzed. 3) To examine induced mutagenesis in the mice by alkylating agents, UV light and base analogs in order to probe the role(s) of the MMR factors in cellular pathways other than mismatch correction, such as transcription-coupled repair. 4)To investigate the role of MMR in response to ionizing radiation, with emphasis on mutagenesis, cytotoxicity, and recognition of x-ray damage, based on the principal investigator
Keywords: s preliminary data showing that MMR-deficient cells are x-ray resistant. 5) To test the role of MMR in suppressing genetic instability secondary to errors made by polymerase during base excision repair. Use will be made of transgenic mice with a targeted disruption of the polymerase locus, as well as the availability of a mutator variant of the polymerase to test the influence of MMR on polymerase -mediated mutagenesis
Project start date: 1992-09-30
Project end date: 2003-01-31
5R01ES005775-09 (2001): $267796
5R01ES005775-08 (2000): $259992
2R01ES005775-06 (1998): $221791
Targeted Mutagenesis Of DNA Via Triple Helix Formation
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 5R01CA064186-14 from National Cancer Institute, IRG: CPA
Abstract: Molecules that bind DNA in a sequence-specific manner offer the potential to regulate gene structure and function. Triplex-forming oligonucleotides (TFOs) bind in the major groove of duplex DNA with high sequence specificity at polypurine sites. Initial work funded by this grant demonstrated that TFOs conjugated to psoralen could confer sequence specificity on the mutagenic effects of the psoralen in both episomal and chromosomal targets in mammalian cells. It was also found that triple helix formation, itself, constitutes a DNA lesion that can provoke DNA repair, leading to mutagenesis and stimulating recombination. The ability of TFOs to mediate targeted modification at chromosomal sites was demonstrated both in cultured mammalian cells and in mice following systemic administration of the TFOs. In this renewal application, we propose to further examine the repair of triplex structures, with an emphasis on aspects of the nucleotide excision repair (NER) pathway, including recognition by the transcription coupled repair (TCR) and global genome repair (GGR) sub-pathways and processing by repair endonuclease incisions. We will also test the roles of certain DNA mismatch repair (MMR) factors, the Werners´s and Bloom´s helicases, and selected polymerases in the metabolism of triplex DNA. In addition, we will continue to examine cellular processes that may affect gene targeting by TFOs, such as cell cycle phase, histone acetylation, and transcriptional activity. We will also examine selected TFO chemical modifications, including backbone, sugar, and base substitutions, for improved gene targeting in cells and for the ability of the associated triplexes to induce repair. Peptide nucleic acids (PNAs), a special class of DNA analogs with advantageous DNA binding properties, will also be investigated. These studies will lead to a greater understanding of the utility of TFOs and related molecules as gene targeting reagents for both research and possible therapeutic applications. In addition, an increased understanding of how triple helices and other unusual DNA structures disrupt genome integrity may provide clues to mechanisms of endogenous genomic instability associated with cancer and other diseases
Keywords: DNA damage, DNA repair, chemical structure function, gene targeting, oligonucleotide, site directed mutagenesis, triple helix DNA binding protein, acetylation, camptothecin, cell cycle, chromatin, enzyme activity, functional /structural genomics, gene mutation, genetic transcription, nucleotide analog, phosphoester ligase, reporter gene HeLa cell, autoradiography, biotechnology, gel electrophoresis, genetic mapping, polymerase chain reaction, southern blotting
Project start date: 1995-07-01
Project end date: 2009-02-28
5R01CA064186-13 (2007): $279050
3R01CA064186-12S1 (2006): $95929
5R01CA064186-12 (2006): $287384
5R01CA064186-11 (2005): $294300
2R01CA064186-10 (2004): $294300
Peter M Glazer, Professor And Chair
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 3R29ES005775-05S1 from National Institute Of Environmental Health Sciences, IRG: RAD
Abstract: In this proposal I describe an approach to study x-ray induced mutations in transgenic mice. The approach is based on an integrated lambda phage shuttle vector which carries target genes in which mutants can be scored and analyzed. This vector is amenable to study in vivo in transgenic mice as well as in cell culture systems. To produce transgenic mice, mouse oocytes are injected with lambda phage DNA containing the supF gene of E. coli and the c1857 gene of lambda as target genes for mutagenesis. Rescue of the lambda vector DNA for analysis is accomplished by addition of the transgenic mouse DNA (containing the integrated lambda DNA) to lambda in vitro packaging extracts which can identify, cut out, and package the lambda DNA, from within the mouse DNA, into viable phage particles. The availability of whole animals with recoverable shuttle vectors with target genes makes possible the study of physiologically relevant parameters in exposure to ionizing radiation. Some of these factors include dose-rate effects, tissue and organ specific differences, age-related effects, and tumor versus normal tissue differences. These factors have not yet been examined in vivo because no experimental system has been yet developed to evaluate them.
Keywords: gene frequency, gene mutation, ionizing radiation, laboratory mouse, nucleic acid sequence, transgenic animal, DNA, DNA repair, Escherichia coli, Escherichia coli k12 lambda, X ray, gene rearrangement, genetic mapping, molecular cloning, mutagen, transfection vector, viral rescue, microinjection
Project start date: 1992-09-30
Project end date: 1998-01-31
3R29ES005775-05S1 (1997): $1650
XRAY INDUCED MUTAGENESIS IN TRANSGENIC MICE
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 5R29ES005775-03 from National Institute Of Environmental Health Sciences, IRG: RAD
Abstract: In this proposal I describe an approach to study x-ray induced mutations in transgenic mice. The approach is based on an integrated lambda phage shuttle vector which carries target genes in which mutants can be scored and analyzed. This vector is amenable to study in vivo in transgenic mice as well as in cell culture systems. To produce transgenic mice, mouse oocytes are injected with lambda phage DNA containing the supF gene of E. coli and the c1857 gene of lambda as target genes for mutagenesis. Rescue of the lambda vector DNA for analysis is accomplished by addition of the transgenic mouse DNA (containing the integrated lambda DNA) to lambda in vitro packaging extracts which can identify, cut out, and package the lambda DNA, from within the mouse DNA, into viable phage particles. The availability of whole animals with recoverable shuttle vectors with target genes makes possible the study of physiologically relevant parameters in exposure to ionizing radiation. Some of these factors include dose-rate effects, tissue and organ specific differences, age-related effects, and tumor versus normal tissue differences. These factors have not yet been examined in vivo because no experimental system has been yet developed to evaluate them
Keywords: gene frequency, gene mutation, ionizing radiation, laboratory mouse, nucleic acid sequence, transgenic animal DNA, DNA repair, Escherichia coli, Escherichia coli k12 lambda, X ray, gene rearrangement, genetic mapping, molecular cloning, mutagen, transfection vector, viral rescue microinjection
Project start date: 1992-09-30
Project end date: 1997-09-29
5R29ES005775-03 (1994): $113278
Hypoxia, Genetic Instability And DNA Mismatch Repair
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 2R01ES005775-16 from National Institute Of Environmental Health Sciences, IRG: RTB
Abstract: Genetic instability is a hallmark of cancer. We have discovered that the hypoxic tumor microenvironment is an important cause of this instability. We have determined that hypoxic stress causes decreased expression at the transcriptional level of the DNA mismatch repair (MMR) factors, MLH1 and MSH2, and of the homology- dependent repair (HDR) factors, RAD51 and BRCA1. The MMR and HDR pathways are critical for maintaining genomic integrity. They play important roles in repairing environmental and endogenous DNA damage, and they influence the response of tumor cells to many cancer therapies, including ionizing radiation and alkylating and cross-linking agents. Mutations in MLH1 and MSH2 have been linked to hereditary colon cancer, while mutations in BRCA1 are associated with hereditary breast and ovarian cancers. In addition, MLH1 and BRCA1 levels are reduced in many sporadic cancers of these and other sites, a process associated with epigenetic silencing. Mechanistically, our initial studies have linked the Myc/Max network of transcription factors to the regulation of MLH1 and MSH2 expression and the E2F family of factors to the regulation of BRCA1 and RAD51. In this competing renewal application, we propose hypothesis-driven, mechanistic studies to elucidate the stress- response pathways that modulate the expression of these MMR and HDR repair genes. Using a battery of genetic and biochemical techniques, our work will focus on transcriptional regulation, epigenetic chromatin modification, and signal transduction events that occur in response to hypoxia and that may regulate DNA repair. We will also test specific hypotheses regarding the influence of hypoxia-induced changes in DNA repair on DNA damage signaling and on the efficacy of tailored therapeutic strategies that exploit these DNA repair changes. The altered regulation of DNA repair in response to cellular stresses such as hypoxia may be a key cause of genetic instability that promotes carcinogenesis and fosters tumor progression. At the same time, it may also offer the possibility of devising therapeutic strategies to which hypoxic cancer cells, but not normoxic, non- malignant cells, are especially susceptible. Hence, the proposed work will contribute to our understanding of basic cancer biology and may provide the basis for new approaches to cancer therapy
Project start date: 1992-09-30
Project end date: 2013-01-31
5R01ES005775-15 (2007): $310055
5R01ES005775-14 (2006): $319316
5R01ES005775-13 (2005): $327000
5R01ES005775-12 (2004): $327000
2R01ES005775-11 (2003): $321862
RECOMBINATION INDUCED BY TRIPLEX TARGETED DNA DAMAGE
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 5R01GM054731-04 from National Institute Of General Medical Sciences, IRG: MGN
Abstract: Experiments are proposed to test the hypothesis that the site-specific introduction of DNA damage via oligonucleotide-mediated triple helix formation can stimulate homologous recombination in mammalian cells. The broad, long-term objective of these studies is to learn how targeted DNA damage can be used as a tool for the genetic manipulation of mammalian cells for the ultimate purpose of gene therapy. Preliminary work has shown that by linking a triple helix-forming oligonucleotide to a mutagen, the sequence specificity of triplex formation can be imparted to the action of the mutagen, and so DNA damage can be directed to a specific site within mammalian cells. This laboratory has determined the conditions necessary for triple helix-mediated targeted mutagenesis of a selected gene in mammalian cells, and they have shown that a psoralen-linked TFO can stimulate recombination between duplicated genes in an SV40 vector in monkey cells. It is proposed to extend these results with a series of experiments to study the site-specific stimulation of recombination in mammalian cells. Novel SV40-based shuttle vectors will be constructed to investigate parameters that influence induced extrachromosomal recombination. The position of the triplex-targeted damage will be varied, and several types of DNA damage will be tested. Pathways of induced recombination will be examined by using these vectors in repair-deficient human cell lines. Cell lines will be established to contain duplicated thymidine kinase genes flanking a triple helix target site as a model substrate to examine induced recombination at a chromosomal locus. These experiments will serve as a basis for designing additional cell lines to test triplex-induced recombination between a chromosomal site and a homologous donor DNA fragment. This work will lead to experiments to provoke targeted recombination at the endogenous c-myc locus in rodent cells, as a model for gene therapy of a disease-related gene. The possibility of a further enhancement in gene targeting will be tested using a novel strategy in which the TFO is covalently tethered to the donor DNA fragment. With this hybrid molecule, target site recognition can be mediated by triplex formation, thereby positioning the donor fragment for efficient recombination and information transfer. The investigators will build on positive preliminary results to pursue a systematic examination of this approach, using a series of vectors and reporter gene constructs in mammalian cells. This work will provide the foundation for future efforts to correct mutations in disease-related human genes
Keywords: DNA damage, genetic recombination, triple helix chromosome, gene targeting, recombinant DNA cell line, simian virus 40, tissue /cell culture, transfection /expression vector
Project start date: 1997-08-01
Project end date: 2001-07-31
5R01GM054731-04 (2000): $226971
5R01GM054731-02 (1998): $216255
Cisplatin Damage Response And Cell-to-cell Communication
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 5R01CA113344-04 from National Cancer Institute, IRG: RTB
Abstract: Cisplatin is one of the most widely used chemotherapy agents, often in combination with radiation therapy. Although cells deficient in elements of the Ku/DNA-PK repair complex are extremely sensitive to ionizing radiation, we have found that such cells are markedly resistant to cisplatin. This resistance arises because these cells fail to activate a novel-signaling pathway that depends on the kinase activity of DNA-PK to generate a signal that is passed from cell to cell, inducing death in the recipient. Initial work has demonstrated that this signal is transmitted via gap junctions, since cells defective in gap junction communication are also resistant to cisplatin. These findings suggest that DNA-PK activity and gap junction communication may influence the clinical response to cisplatin in human cancers. We propose to test the hypothesis that the regulation of gap junction expression and function can influence the cellular response to cisplatin. We will investigate links between phosphorylation of connexins (the protein subunits of gap junctions) by selected kinases and cisplatin resistance. We will test the effect of factors that decrease connexin degradation (such as proteasome inhibitors) or enhance gap junction biogenesis (by altering connexin trafficking) on cell killing by cisplatin. To identify factors upstream or downstream of Ku/DNA-PK in response to cisplatin, we will probe candidate damage recognition and signaling factors, using co-immunoprecipitation techniques. We will also use mass spectrometry to identify proteins that show altered binding to Ku80 following cisplatin treatment. Using metabolic labeling and chromatography techniques, we will identify candidate molecules that may transduce the Ku/DNA-PK dependent cytotoxic signal through gap junctions. Finally, we will test the hypothesis that gap junction expression and DNA-PK function can influence cisplatin response in vivo using a series of genetically manipulated tumor models in mice. The long-term goal of this application is to elucidate the molecular mechanisms that mediate this novel cisplatin-response pathway. This work may lead to strategies to sensitize human cancers to cisplatin, providing the basis for new combined approaches to cancer therapy
Keywords: DNA repair, antineoplastic, biological signal transduction, cell cell interaction, cisplatin, drug resistance, enzyme complex, gap junction, membrane channel, neoplasm /cancer chemotherapy, protein kinase cytotoxicity, disease /disorder model, gene expression, growth factor receptor, membrane lipid, phosphorylation, platinum, protein protein interaction, protein transport athymic mouse, cell line, genetically modified animal, immunoprecipitation, mass spectrometry, proteomics
Project start date: 2005-04-15
Project end date: 2010-02-28
5R01CA113344-03 (2007): $270535
5R01CA113344-02 (2006): $283514
1R01CA113344-01 (2005): $290345
GENETIC INSTABILITY INDUCED BY VIRAL PROTEINS
Peter M Glazer, Professor And Chair
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 5P01CA016038-280026 from National Cancer Institute, IRG:
Abstract: Applicant s Description) Genetic instability is a critical factor in carcinogenesis and tumor progression. Recent work from the laboratory of Thomas Shenk has revealed that the human cytomegalovirus (CMV) proteins IE1 and IE2 can mediate cellular transformation in a manner associated with the induction of mutagenesis in the host cell genome. In this proposal, the nature and mechanism of the IE1- and IE2- induced genetic instability will be investigated. The induced mutation patterns will be characterized using a series of mutation reporter assays, with emphasis on the use of a chromosomally integrated lambda phage shuttle vector carrying the supF mutation reporter gene. The possibility that the proteins generated a hypermutable state will be tested by examining mutagen-induced as well as spontaneous. These studies will be facilitated by exploiting techniques for high efficiency gene transfer and for isolation of successfully transfected cells, so that the full effect of transient viral protein expression can be examined in bulk cell populations. Using reporter constructs developed by Dr. Glazer, the ability of IE1 and IE2 to promote recombination will also be determined as another measure of genetic instability. The influence of IE1 and IE2 expression on DNA repair will be investigated using assays for reactivation of damaged expression vector DNA, including lesions subject to nucleotide excision repair, base excision repair, and mismatch repair. Direct measurements of lesion removal from cellular DNA will also be performed, with analysis of both bulk DNA and actively transcribed regions. Possible protein-protein interactions between IE1 and IE2 and selected repair factors will be examined. A series of deletion mutants of IE1 and IE2 will be tested to identify protein domains associated with mutagenicity, for comparison with domains associated with the transcriptional regulatory and anti- apoptotic activities of the proteins. Pathways associated with IE1- and IE2-induced mutagenesis will be explored by testing mutagenesis in p53, c-fos, and c-jun knock out cells and by using genome array analyses to detect genes up- and down-regulated by the proteins. This work will build on Dr. Glazer s experience in the area of mutagenesis and DNA repair, and will benefit from a matrix of ongoing studies to examine other genetic and epigenetic causes of genetic instability in cancer. The studies will help to elucidate a novel pathway of viral protein- induced mutagenesis and transformation, and should help to identify fundamental cellular pathways contributing to genetic instability. As such, this project will serve as a direct bridge between the Dimaio and Sweasy projects.
Keywords: cytomegalovirus, genetic regulation, virus protein, DNA repair, gene expression, molecular oncology, mutation, neoplastic transformation, nucleic acid sequence, protein protein interaction, protein sequence, reporter gene, viral carcinogenesis, bacteriophage lambda, cell line, tissue /cell culture, transfection, transfection /expression vector
RESEARCH TRAINING: RADIATION THERAPY, BIOLOGY, PHYSICS
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 5T32CA009259-20 from National Cancer Institute, IRG: NCI
Abstract: Applicant´s Description) This program supports research training at the post-doctoral level primarily focused on cancer, biology, radiation biology, and radiological physics relevant to therapeutic radiology. The training program is of two years duration. The program in radiation biology is open to individuals who hold the Ph.D. degree in one of the basic biological sciences, biochemistry, molecular biology, cell biology, molecular genetics, classical radiation biology, etc. who wish to pursue cancer-related studies, particularly as applied to therapeutic radiology, and to individuals who hold the M.D. degree who wish to pursue careers in combining laboratory and clinical research in these same areas. The program in radiological physics is designed to train post- doctoral physicists in those aspects of dosimetry, radioactivity, diagnostic imaging, instrumentation and computers which are applicable to radiation therapy. The programs take advantage of the extensive laboratory and clinical research activity in the Department of Therapeutic Radiology. The program includes formal courses in therapeutic radiology, radiation biology, radiological physics, and in scientific ethics. Trainees are assigned to research groups and devote more than 90% of their time to specific long-term research projects. Trainees interact with a wide variety of other biomedical science research programs throughout Yale University and the Yale Comprehensive Cancer Center
Project start date: 1978-09-30
Project end date: 2003-07-31
5T32CA009259-20 (2001): $194968
5T32CA009259-19 (2000): $179713
5T32CA009259-17 (1998): $143407
RECOMBINATION INDUCED BY TRIPLEX TARGETED DNA DAMAGE
Peter M Glazer, Professor And Chair
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 1R01GM054731-01A1 from National Institute Of General Medical Sciences, IRG: MGN
Abstract: Adapted from the Investigator s ) Experiments are proposed to test the hypothesis that the site-specific introduction of DNA damage via oligonucleotide-mediated triple helix formation can stimulate homologous recombination in mammalian cells. The broad, long-term objective of these studies is to learn how targeted DNA damage can be used as a tool for the genetic manipulation of mammalian cells for the ultimate purpose of gene therapy. Preliminary work has shown that by linking a triple helix-forming oligonucleotide to a mutagen, the sequence specificity of triplex formation can be imparted to the action of the mutagen, and so DNA damage can be directed to a specific site within mammalian cells. This laboratory has determined the conditions necessary for triple helix-mediated targeted mutagenesis of a selected gene in mammalian cells, and they have shown that a psoralen-linked TFO can stimulate recombination between duplicated genes in an SV40 vector in monkey cells. It is proposed to extend these results with a series of experiments to study the site-specific stimulation of recombination in mammalian cells. Novel SV40-based shuttle vectors will be constructed to investigate parameters that influence induced extrachromosomal recombination. The position of the triplex-targeted damage will be varied, and several types of DNA damage will be tested. Pathways of induced recombination will be examined by using these vectors in repair-deficient human cell lines. Cell lines will be established to contain duplicated thymidine kinase genes flanking a triple helix target site as a model substrate to examine induced recombination at a chromosomal locus. These experiments will serve as a basis for designing additional cell lines to test triplex-induced recombination between a chromosomal site and a homologous donor DNA fragment. This work will lead to experiments to provoke targeted recombination at the endogenous c-myc locus in rodent cells, as a model for gene therapy of a disease-related gene. The possibility of a further enhancement in gene targeting will be tested using a novel strategy in which the TFO is covalently tethered to the donor DNA fragment. With this hybrid molecule, target site recognition can be mediated by triplex formation, thereby positioning the donor fragment for efficient recombination and information transfer. The investigators will build on positive preliminary results to pursue a systematic examination of this approach, using a series of vectors and reporter gene constructs in mammalian cells. This work will provide the foundation for future efforts to correct mutations in disease-related human genes.
Keywords: DNA damage, genetic recombination, triple helix, chromosome, gene targeting, recombinant DNA, cell line, simian virus 40, tissue /cell culture, transfection vector
Project start date: 1997-08-01
Project end date: 2001-07-31
1R01GM054731-01A1 (1997): $211129
Peter M Glazer, Professor And Chair
Yale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 1P01CA129186-016069 from National Cancer Institute, IRG: ZCA1
Project start date: 2007-07-01
Project end date: 2012-06-30
RESEARCH TRAINING: RADIATION THERAPY, BIOLOGY, PHYSICS
Peter M Glazer, Professor And Chair
Therapeutic Radiologyyale University
47 College Street, Ste 203
new Haven, Ct 065208047
Grant 2T32CA009259-16A1 from National Cancer Institute, IRG: NCI
Project start date: 1978-09-30
Project end date: 2002-07-31
2T32CA009259-16A1 (1997): $124965
Related Publications
Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids. Proc Natl Acad Sci U S A. 2008 Sep 9; 105( 36): 13514-9. Epub 2008 Aug 29. PMID: 18757759 Triplex-mediated gene modification. Methods Mol Biol. 2008; 435: 175-90. PMID: 18370076 MEN1 and FANCD2 mediate distinct mechanisms of DNA crosslink repair. DNA Repair (Amst). 2008 Mar 1; 7( 3): 476-86. Epub 2008 Feb 6. PMID: 18258493 Chronic hypoxia decreases synthesis of homologous recombination proteins to offset chemoresistance and radioresistance. Cancer Res. 2008 Jan 15; 68( 2): 605-14. PMID: 18199558 
Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids. Nucleic Acids Res. 2007; 35( 22): 7604-13. Epub 2007 Oct 30. PMID: 17977869 Repair and recombination induced by triple helix DNA. Front Biosci. 2007 May 1; 12: 4288-97. Review. PMID: 17485375 Regulation of DNA repair in hypoxic cancer cells. Cancer Metastasis Rev. 2007 Jun; 26( 2): 249-60. Review. PMID: 17415527 Co-repression of mismatch repair gene expression by hypoxia in cancer cells: role of the Myc/Max network. Cancer Lett. 2007 Jul 8; 252( 1): 93-103. Epub 2007 Feb 1. PMID: 17275176 Hypoxia-induced genetic instability--a calculated mechanism underlying tumor progression. J Mol Med. 2007 Feb; 85( 2): 139-48. Epub 2006 Dec 20. Review. PMID: 17180667 Basal repression of BRCA1 by multiple E2Fs and pocket proteins at adjacent E2F sites. Cancer Biol Ther. 2006 Oct; 5( 10): 1400-7. Epub 2006 Oct 30. PMID: 17106239 CHK2-dependent phosphorylation of BRCA1 in hypoxia. Radiat Res. 2006 Oct; 166( 4): 646-51. PMID: 17007555 Triplex-stimulated intermolecular recombination at a single-copy genomic target. Mol Ther. 2006 Sep; 14( 3): 392-400. Epub 2006 May 30. PMID: 16731047 
Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6. Carcinogenesis. 2006 Dec; 27( 12): 2402-8. Epub 2006 May 25. PMID: 16728433 Repair of DNA interstrand cross-links: interactions between homology-dependent and homology-independent pathways. DNA Repair (Amst). 2006 May 10; 5( 5): 566-74. Epub 2006 Mar 29. PMID: 16569514 Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide. Biochemistry. 2006 Feb 14; 45( 6): 1970-8. PMID: 16460044 Induction of aberrant crypt foci in DNA mismatch repair-deficient mice by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP). Cancer Lett. 2006 Nov 28; 244( 1): 79-85. Epub 2006 Jan 20. PMID: 16427736 Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance. Cancer Lett. 2006 Dec 8; 244( 2): 195-202. Epub 2006 Jan 19. PMID: 16426742 Targeted genome modification via triple helix formation. Ann N Y Acad Sci. 2005 Nov; 1058: 151-61. Review. PMID: 16394134 Site-specific gene modification by PNAs conjugated to psoralen. Biochemistry. 2006 Jan 10; 45( 1): 314-23. PMID: 16388608 Alterations in DNA repair gene expression under hypoxia: elucidating the mechanisms of hypoxia-induced genetic instability. Ann N Y Acad Sci. 2005 Nov; 1059: 184-95. PMID: 16382054