Stephen Herbert Howell - US grants

The studies accomplished during the first three years of this grant have established the feasibility of combination cisplatin-based intraperitoneal therapy with thiosulfate neutralization. Current results suggest that this therapy can salvage patients with small volume drug-resistant disease. Our aim is to further improve the therapeutic index of ip chemotherapy by investigating: 1) the use of selective intraperitoneal biochemical modulation; and 2) the mechanism by which sodium thiosulfate alters the metabolism of cisplatin (DDP) so as to provide selective protection of the kidneys. The ability of dipyridamole (DP) to enhance the activity of methotrexate (MTX) has been selected as a mode concentration-dependent synergistic interaction with which to test the feasibility of producing selective biochemical modulation in the peritoneal cavity where high local concentrations of dipyridamole can be attained. The specific aims of this project are: a) to determine the basic parameters of the DP/MTX interaction in human ovarian carcinoma cell lines, and test the concept of selective ip biochemical modulation in an ascitic xenograft model of human ovarian carcinoma; b) to perform a pharmacokinetic study of DP administered ip to patients with cancer involving the peritoneal cavity; and c) to undertake a phase I study of the combination of DP and MTX administered together by the ip route. In the second project, we will further investigate the use of iv thiosulfate as a neutralizing agent in combination with ip administration of DDP. We will determine how thiosulfate influences the generation of nephrotoxic species from DDP using new techniques that permit resolution and quantitation of 8 metabolites. The specific aims of this project are: a) to determine how thiosulfate affects the generation of DDP metabolites in vitro, and how it influences the pharmacokinetics of DDP metabolites in the plasma of patients receiving DDP; b) to determine whether iv thiosulfate, at doses sufficient to protect the kidneys, interferes with the antitumor activity of ip administered DDP in the Ehrlich ascites tumor model; c) to identify the metabolites which are most important with respect to toxicity to the kidneys, marrow and nerves; d) to investigate the changes that DDP produces in plasma glutathione, cyst(e)ine, and methionine in association with the generation of DDP metabolites; and e) to study the influence of thiosulfate on the generation of DDP metabolites inside human ovarian carcinoma cells.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

neoplasm /cancer pharmacology; biomedical facility; method development; model design /development; pharmacokinetics; chemical registry /resource; antineoplastics; chemical information system; high performance liquid chromatography; clinical chemistry; atomic absorption spectrometry;

DESCRIPTION: The underlying goal of this research program to identify the molecular mechanisms underlying acquired resistance to the platinum drugs. The investigator has demonstrated that selection for the cisplatin-resistant phenotype results in cross resistance to a family of metalloids including arsenite, antimonite, selenite and tellurite (RASP phenotype). This pattern of cross resistance is mediated by an ATP dependent membrane export pump coded for by the arsA and arsB genes in bacteria. The hypothesis is that the human homolog of the arsA (harsA) plays a role in the resistance to DDP CBDCA in mammalian cells. Using degenerate primers a fragment of the human arsA gene was cloned and used to isolate both cDNA and genomic clones of this novel gene from lambda-based and P1-based libraries respectively. The specific aims are to compare the expression, sequence and structure of this gene in human ovarian carcinoma 2008 cells and sublines selected with DDP, arsenite and antimonite for expression of the RASP phenotype and their revertant., to determine whether expression of harsA regulates sensitivity to DDP, arsenite and antimonite, to identify the location of the gene in the human genome and characterize its genomic structure, to examine the differences in expression harsA in various human tissues and whether expression of i induced by cisplatin or metalloids themselves or by signal transduction pathways known to influence cisplatin sensitivity, and finally to begin characterization of the gene product by preparing pure protein, generating antibodies and measuring the level and subcellular location of the harsA gene product in drug sensitive and RASP phenotype cells.

DESCRIPTION: The overall goal of this project is to determine the role of loss of DNA mismatch repair (IVIMR) in the development of cellular drug resistance in vitro and in vivo. This project focuses on the new discovery that loss of IVIIVIR causes the cells to become resistant to cisplatin and carboplatin. The Specific Aims are directed at the following questions: 1) does treatment with cisplatin enrich for pre-existing MMR-deficient cells in a tumor population growing in vivo?; 2) does treatment with cisplatin enrich for MMR-deficient cells in patients?; 3) does loss of MMR increase the spontaneous rate of mutation to resistance to other clinically important chemotherapeutic agents?; and 4) does loss of MMR increase the ability of cisplatin to mutagenize tumor cells to other chemotherapeutic agents?

The Cancer Pharmacology Program consists of 7 Participating Members, representing total peer-reviewed funding of $2.4 Million in annual direct costs ($3.3. Million total costs). During the last year, its members were responsible for a total of 55 cancer-relevant, peer-reviewed publications, 42% of which were intra- and inter-programmatic collaborations. The overall goal of the Cancer Pharmacology Program is to develop new drugs, delivery systems, and diagnostics for the treatment of cancer. The interests of the Members of this Program include: identification and validation of new drug targets; synthesis and preclinical development of novel classes of anti-cancer agents; investigation of the mechanisms of drug-induced apoptosis and drug resistance at the biochemical, molecular and genetic levels; the development of stem cell culture systems; and, investigation of microcirculatory regulation of drug delivery. The Cancer Pharmacology Program is highly interdisciplinary; its members have specific expertise in physiology and bio-engineering, medical oncology, pathology, medicinal chemistry, experimental therapeutics, and molecular pharmacology. In particular, the Program members have an established record of achievements in drug testing, development, and delivery and they have been successful in bringing novel drugs and drug delivery systems into the clinic.

DESCRIPTION (provided by applicant): Cisplatin (DDP) is highly effective for the initial therapy of a variety of human cancers, but resistance emerges quickly during treatment. We have discovered that human ovarian carcinoma cells selected for resistance to DDP are cross resistant to copper (Cu) and that cells selected for resistance to Cu are cross-resistant to DDP. Our hypothesis is that DDP enters the cell, is distributed to various subcellular compartments, and is exported from the cell by transporters and chaperones that normally mediate Cu homeostasis. Such transporters and chaperones normally protect Cu from reacting with other molecules during its distribution. Our hypothesis is that these pteins also protect DDP from reacting with intracellular thiols while it travels between the cell surface and the nucleus, thus explaining how DDP can kill cells even when its concentration is 3 orders of magnitude lower than that of intracellular thiols. This project focuses specifically on ATP7A, a pump that is known to sequester Cu from the cytoplasm into the trans-Golgi network for subsequent export from the cell, It is the overall aim of this project to determine how ATP7A regulates sensitivity to DDP and what role ATP7A plays in acquired resistance to this drug. The specific aims are directed at the following questions: 1) Does ATP7.A alter sensitivity by modulating the cellular pharmacology of DDP? 2) Is DDP actually a substrate for the ATP7A transporter? 3) What structural components of ATP7A are essential to its ability to mediate DDP resistance? 4) For what classes of Pt-containing drugs is ATP7.A a major determinant of sensitivity 5) Does increased expression of ATP7.A account for acquired DDP resistance in human tumor cells? The hypothesis that DDP is a substrate for Cu-binding proteins is an entirely new concept in the field. Ifit is correct, then alterations in many of the other transporters and chaperones that normally mediate Cu homeostasis may also play a role in resistance to DDP and several other heavy metals. The results of these studies are expected to be of fundamental importance in identifying novel strategies for overcoming intrinsic resistance and reversing acquired resistance to this very important chemotherapeutic agent.

Organ regeneration in plants has intrigued scientists for years because the developmental fate of regenerating tissue in culture can be controlled by two simple plant hormones, cytokinin and auxin. At high levels of cytokinin relative to auxin, shoots are produced, while at high levels of auxin relative to cytokinin, roots are formed. Shoot regeneration is not only an interesting developmental phenomenon but also an important commercial process for the propagation and improvement of plants. Understanding its regulation may help to solve inefficiencies in shoot regeneration that often impede efforts to improve commercially significant plant varieties.

Recent studies were conducted in this laboratory using oligonucleotide array (DNA chip) analysis to describe the global pattern of gene expression that underlies the shoot development process. More than 8000 genes were analyzed in this study, and several hundred were up or downregulated among them were genes involved in hormone signaling and shoot meristem formation.

In Arabidopsis, some ecotypes (isolates from different geographical locations) differ in their ability to form shoots under comparable tissue culture conditions. In the effort to understand the genes that determine the efficiency of shoot development, a search was conducted for genes (loci) that control the difference in regeneration ability between one ecotype (Columbia) that regenerates well and another ecotype (Landsberg) that does not. One major locus on chromosome 5 was identified, in which the Columbia allele enhances shoot formation. Two other minor loci were identified on chromosomes 1 and 4. Landsberg alleles at these loci enhance shoot production. The proposed project will examine how these loci impact the program of gene expression during shoot development and whether the major locus on chromosome 5 controls the expression of a specific set of genes. Oligonucleotide arrays (DNA chip) with over 20,000 Arabidopsis genes will be employed to identify the genes that are differentially regulated during shoot development by the Columbia or the Landsberg alleles at the major locus on chromosome 5.

In addition, the expression level of every gene in the Arabidopsis genome will be assessed in the two ecotypes to see if any genes show heritable patterns of gene expression during shoot development. If so, searches will be undertaken for loci controlling individual genes that show heritable changes in gene expression. This approach, termed "genetical genomics," treats the expression level of any gene on an oligonucleotide array as a metric trait and permits one to seek out genes that control these traits. This represents the first step in constructing causal pathways of gene expression during shoot development in Arabidopsis.

Finally, it is proposed to identify the gene(s) at the major locus on chromosome 5 that enhances the efficiency of shoot development in the Columbia ecotype. This will be done by a process called "chromosome walking," in which fine-scale gene mapping techniques are used to pinpoint candidate genes.

Abstract

This research will determine the function of subtilase genes in Arabidopsis. Subtilase genes encode subtilisin-like serine proteases, proteins that play important roles in plant growth and development. In mammalian systems, some subtilases are called "hormone convertases" because they cut other proteins releasing bioactive peptides that serve as hormones and/or function in cell-to-cell signaling. The properties of some plant subtilases suggest they may play similar roles in plant development and physiology.

The function of nearly 60 subtilase genes in Arabidopsis will be investigated through collaboration with European partners in The Arabidopsis Subtilase Consortium (TASC). A listing of all the genes in the subtilase gene family (and their subfamily identification) is found at http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb/main/mgenes.html. This research will focus on the role of ten subtilase genes that are highly regulated during shoot regeneration in tissue culture. Three of these subtilase genes appear to influence shoot development in that the expression of these genes is controlled by a major quantitative trait locus (QTL) that determines shoot regeneration efficiency. Shoot regeneration efficiency varies in Arabidopsis and in some important economic crops where it can impede the successful application of genetic engineering technologies. Another seven subtilase genes are differentially regulated during shoot or root regeneration.

A proteomics approach (the study of a large number of proteins using protein expression profiling) will be used to understand the function of the subtilases. The function of these genes in vivo will be examined by quantitatively analyzing 2D difference gel electrophoresis (DIGE) profiles of T-DNA insertion mutants and transgenics with induced expression of subtilase genes. Subtilase target sites will be determined by analyzing the products from the limited digestion of common substrate proteins by the recombinant enzyme. Optimal subtilase target sites will be determined by designing and testing the hydrolysis of synthetic peptides related to the cleavage sites for common substrates.

Research results will shared with the public and other members of the scientific community through http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb.html. Information at this site is edited by the Altmann lab and will be updated on a monthly basis. This research is significant to the goals of the 2010 project in that it contributes to our understanding of a group of important genes that influence plant growth and development. This research makes a unique contribution in that it uses new tools in proteomics to address gene function.

Broader impact

This project has broader impact because it contributes knowledge to the role of proteolysis and peptide signaling in plant development. Little is known about bioactive plant peptides from genomics because peptides are often embedded in other proteins and are not released until cleaved by proteases, such as subtilases.

The project also has broader impact because it provides lab research training for Iowa State University and Des Moines Area Community College undergraduates. Some of the undergraduate trainees will also have the opportunity to conduct research during the summer in international labs of partners associated with TASC. To encourage ISU minority undergrads to participate in this project, we will host a meeting each fall in the Roy J. Carver Co-Laboratory for the local chapter of Minorities in Agriculture, Natural Resources and Related Sciences (MANRRS).

DESCRIPTION (provided by applicant): The goal of this application is to plan for the development of an AP4 Center at the University of California, San Diego. The Center for Cancer Drug Development (C2D2) will be established at the Rebecca and John Moores UCSD Cancer Center to exploit enormously powerful new genomic, genetic, molecular, and computational tools to accelerate the development of novel cancer therapeutics, diagnostics, and molecular imaging techniques. Investigators at UCSD have identified a myriad of potential drug targets, drugs, and novel computational imaging and diagnostic technologies that are of interest to numerous pharmaceutical companies. The research program of the C2D2 will be focused on the use of new biomedical tools and the information they generate to accelerate completion of the critical preclinical steps required to advance these potential therapeutics toward commercial development. The C2D2 expects to make available to partners not only fundamental genomic and genetic information that support the rationale for potential therapeutics, but also novel drugs, imaging technology and diagnostics for their consideration for further development. Major areas of research interest include: identification of multigenic complex genetic host determinants that predict the probability of response and outcome, and that influence the toxicity of cancer drugs; novel molecular imaging technology for functional assessment of tumors and drug effect; novel techniques for tumor-specific drug delivery; identification and exploitation of kinase pathways uniquely activated in chronic lymphocytic leukemia cells; the biology of tumor neovasculature and the development of novel anti-angiogenic agents; new computational and informatics tools to identify drug targets and assess drug effect; and, new strategies for antibody-based treatment of acute leukemia.

[unreadable] DESCRIPTION (provided by applicant): The ability of the copper (Cu) exporters ATP7A and ATP7B to regulate tumor cell sensitivity to the platinum-containing drugs by altering their intracellular sequestration and efflux has now been demonstrated in multiple experimental systems. The overall goal of this project is to determine the mechanism by which ATP7A and ATP7B mediate the efflux of cisplatin, carboplatin and oxaliplatin from ovarian carcinoma cells. Our hypothesis is that at clinically relevant concentrations, the platinum drugs enter the cell, are distributed to various subcellular compartments and are exported from the cell using transporters and chaperones that have evolved to control Cu homeostasis. A corollary to this hypothesis is that, as for Cu, these PI-type ATPases function to detoxify the Pt drugs by sequestering them into or onto vesicles of the secretory pathway that are eventually exported from the cell. The specific aims are to: 1) determine whether ATP7A and ATP7B bind and transport cisplatin, carboplatin and oxaliplatin into secretory vesicles; 2) investigate the ability of the Pt drugs to bind to the Cu-binding motif in the metal binding sequences of ATP7A, ATP7B and ATOX1 proteins; and, 3) determine whether the Cu chaperone ATOX1 is essential to the ability of ATP7A and ATP7B to mediate Pt drug resistance, sequestration, and export in living cells. Careful dissection of the mechanism by which the Cu efflux transporters modulate the export of the Pt drugs is expected to offer insight into why cisplatin, carboplatin and oxaliplatin differ in their efficacy and toxicity, and to identify strategies for improving the therapeutic index of these agents. These studies will also further elucidate the mechanisms that mediate resistance to this important class of chemotherapeutic agents and suggest clinically relevant strategies for reversing resistance. [unreadable] [unreadable] [unreadable]

PI: David Hannapel Proposal # 0518902
"The 7th Plant Science Symposium, Meristems 2005"
June 2-5, 2005, Iowa State University, Ames, IA

Abstract

The 7th Plant Science Symposium, Meristems 2005, will focus on meristem biology. Meristems are the growth centers of plants and embody many exciting concepts for plant developmental biologists. Meristems are small but dynamic structures, where processes of cell signaling and coordination in growth occur. As the centers of plant growth, meristems are also of great importance to agriculture and crop production. For example, most of our current knowledge about the mechanisms that control flowering in plants has been obtained from studies of meristems. During this Symposium, the world's leading experts in the biology of meristems will gather on the Iowa State University campus to discuss the latest discoveries in plant development. In a venue designed for discussion and interaction, the symposium will focus on the developmental and genetic processes that encompass growth activity throughout the plant. The symposium will include keynote presentations by internationally recognized speakers, poster sessions, and workshops. This meeting will facilitate opportunities for plant biologists to meet, discuss and interact in an informal and open setting. The program also addresses the current and potential applications of meristem biology for gene discovery and crop improvement. Significant participation and support from researchers in the biotechnology industry is expected. Because of the important role that meristems play in overall growth and crop production, it is expected that scientists from a wide array of disciplines will be attracted. By bringing together researchers interested in various aspects of plant development, this meeting will facilitate the exchange of information and ideas and will encourage participants to reach out in new directions in their future research.

This core brings together three established teams for mouse imaging and orthotopic and genetically induced tumor models. It will not only provide the animal models but also in-vivo non-destructive imaging to detect tumors or devices. Providing animal models is essential for this Center grant as it includes investigators from the physical sciences with limited animal experience.The Core will be able to perform entire experiments for investigators. The Genetically Induced Murine Tumor Service staffed with trained personnel that provide all the needed services to study and/or image native breast tumors and leukemia in genetically susceptible mice. The Dept of Radiology and the Cancer Center at UCSD have established a Small Animal Imaging Resource (SAIR) to support cancer research. The dedicated rodent imaging facility (-650 sq.ft.) located adjacent to the vivarium houses optical, CT, PET and ultrasound imaging, as well as a high-resolution digital autoradiography and fluorescent imaging system for post-mortem analysis. The SAIR also provides rodent MRI on a 7T system located about one mile from the Cancer Center adjacent to another vivarium. Support and expertise include: imaging, animal care, image computation, MR &optical hardware and software, diagnostic agent chemistry, radiochemistry including cyclotron, analytical chemistry, kinetic modeling and parametric imaging, anatomic and histological confirmation. The imaging team is developing molecular imaging approaches to provide non-invasive biomarker detection, characterization and monitoring of tumors. There are three high priority goals for the imaging team: 1) Optimize image acquisition and blood sampling to enable kinetic modeling to better evaluate agent distribution;2) Co-register imaging data to post-mortem slices to provide accurate anatomic confirmation and accurate image-guided tissue sampling of regions of interest for further analysis;and 3) Complete automation of imaging data reduction for kinetic modeling including co-registration of the multiple volume acquisitions to accurately define the time-intensity-curve on a volumetric basis and the generation of parametric images to improve throughput and eliminate operator bias.

DESCRIPTION (provided by applicant): This renewal application seeks funding to continue the highly successful Cancer Therapeutics Training (CT2) Program at the Moores Comprehensive Cancer Center at the University of California, San Diego. The mission of the CT2 program is to train the PhD and physician-scientists who will become the next generation of leaders in the field of cancer therapeutics and diagnostics in each of the major steps required for successful translation of laboratory-based discoveries into safe and effective therapeutic agents and diagnostic modalities. The CT2 training is designed to position trainees to play key leadership roles in the field of cancer developmental therapeutics and diagnostics. The 22 faculty of the CT2 Program are all Members of the Cancer Center and are based in 10 departments in the School of Medicine, the Scripps Oceanographic Institute or the general campus. Each faculty mentor is an accomplished investigator and educator with a history of training superb post-doctoral fellows. Each has substantial peer-reviewed cancer or cancer-related research funding. All of the participating faculty are conducting translational research and have been selected because of their interest in new cancer therapeutics. This program is extensively integrated into the other cancer related activities of the Cancer Center and of UCSD. The goal is to recruit and retain 8 MD and PhD scientists in this two-year program that will position them for careers in the development of new cancer drugs or the diagnostics needed to guide the use of these drugs. The training program has 3 components: 1) the completion of formal didactic teaching sessions that cover tools essential to the drug development process;2) the conduct of a drug or diagnostic development research project under the direction of a faculty mentor;and, 3) required participation in the annual meeting of the American Association for Cancer Research or an equivalent national drug development meeting. Trainees are also expected to participate in Cancer Center and Departmental seminars, research rounds and journal clubs to expand the breadth of their understanding of cancer research, and prepare formal project plans and practice or real grant applications for review by the Executive Committee. Methods are in place to ensure that all trainees are properly instructed in the principles of responsible conduct of research and scientific integrity. Trainees are recruited nationwide and special efforts are made recruit and retain exceptional minority, women and disadvantaged candidates. During its first 4 years of operation the CT2 Program has drawn trainees from major research universities across the country as well as from the Gradate Biomedical Sciences, Hematology/Oncology, Surgery and Radiology fellowship programs UCSD. This Program has been so successful that the Cancer Center has established a parallel program of additional fellowships in cancer therapeutics funded by donations. PUBLIC HEALTH RELEVANCE: This application seeks funding to continue a training program in developmental therapeutics focused on cancer drugs at the Moores Cancer Center at the University of California, San Diego. Our vision is that cancer can be controlled with novel therapeutics directed at molecular targets critical for tumor cell survival when combined with biomarkers that allow individualization of treatment. The mission of the program is to provide training to post-doctoral PhD and physician-scientists in each of the major steps in the development of a novel cancer therapeutics so as to position these individuals to play leading roles in translating laboratory-based discoveries into safe and effective therapeutic agents.

Stephen H. Howell
Proposal number IOS-0919707
ER stress transducing transcription factors in plants

Plant stress tolerance is a highly valued trait given the demands for producing crops for food and fuel under all kinds of environmental conditions, particularly those exacerbated by climate change. The goal of this project is to better understand the mechanism by which plants perceive adverse environmental conditions and how they protect themselves from the stress caused by these conditions. This project will focus on two factors called transcription factors that "turn on" the expression of stress response genes. These transcription factors are unusual in that they are "dormant" and attached to membranes in the cytoplasm of plant cells under normal conditions, but are activated and released from the membranes and enter the nucleus under stress conditions. The two transcription factors are activated by different stresses -- one by heat and the other by salt stress -- and each turns on a different set of genes. The investigators will determine how the factors are activated, how they distinguish different stresses and how they are relocated from one compartment of the cell to another. The factors turn on different sets of genes, and so the investigators will also examine how the factors selectively activate different target genes. This system represents a newly discovered stress response pathway in plants and has prospects for manipulating plants to achieve greater stress tolerance. The project will also provide the opportunity to train George Washington Carver interns in the area of plant stress research.

DESCRIPTION (provided by applicant): Multiple lines of evidence indicate that the platinum (Pt)-containing drugs can enter cells, be distributed to various subcellular compartments and exported from cells via transporters that evolved to manage copper (Cu) homeostasis. We and others have shown that the major Cu influx transporter CTR1 mediates the import of cisplatin (DDP), carboplatin and oxaliplatin into mammalian tumor cells. Knockout of both alleles of CTR1 impairs Pt drug accumulation and results in resistance when tested in vitro; it also renders tumors completely unresponsive to DDP treatment in vivo in a xenograft model. Thus, irrespective of what other Pt drug influx mechanisms might exist, CTR1 is a key determinant of the sensitivity of tumors to these drugs. Like Cu, the Pt drugs trigger the rapid endocytosis and degradation of CTR1 and thus these drugs limit their own uptake. It is the overall goal of this project to identify novel strategies for selectively enhancing Pt drug accumulation in tumors. It is our hypothesis that this can be achieved by determining the mechanism by which CTR1 transports the Pt-containing drugs and identifying the factors that modulate CTR1 trafficking, endocytosis and degradation in response to Pt drug exposure. The specific aims are to: 1) determine the mechanism by which CTR1 transports the Pt-containing drugs including whether the Pt drugs enter tumor cells by transiting the pore formed by CTR1 or by endocytosis after binding to the extracellular domain, and if they enter by both routes, whether they make different contributions to cytotoxicity; b) determine the mechanism that controls the trafficking of CTR1 within the cell and its endocytosis and subsequent degradation following exposure to the Pt drugs including the motifs in CTR1 that mediate its delivery to and recovery from the plasma membrane, its phosphorylation and ubiquitination and the mechanism by which the Cu chaperone ATOX1 controls Pt drug- induced degradation of CTR1; c) determine what is wrong with CTR1 function in cells with acquired Pt drug resistance including whether there is a defect in glycosylation that disables the transport function or defects in the pathways that control trafficking of CTR1 in resistant cells that result in inadequate delivery to the plasma membrane. The Pt-containing drugs remain one of the most important and widely used class of chemotherapeutic agents. We have already succeeded in using the results of detailed studies of the interaction of DDP with the Cu influx transporter CTR1 to identify a strategy for improving the therapeutic index of these agents that is currently entering a Phase I clinical trial. This proposal is innovative in that it challenges dogma in the field, introduces new concepts regarding how the Pt drugs enter cells, and brings state-of-the-art technology to a careful dissection of the mechanism by which CTR1 mediates the transport of DDP that can be expected to identify strategies for further increasing the efficacy and selectivity of the Pt containing drugs.

DESCRIPTION (provided by applicant): DNA adducts are the hallmark and most common form of DNA damage in the cell. They result from environmental carcinogen exposure (such as UV) or during chemotherapy using DNA modifying agents like cisplatin (cDDP) or alkylators such as chlorambucil (CLB). While mechanisms underlying sensitivity, agent homeostasis, detoxification, DNA repair and apoptosis, have been well investigated, the central molecular event, the formation of adducts, is not well understood in vivo. Evidence suggests that the epigenetic landscape and the structure of the chromatin influences the formation of adducts and mediates drug sensitivity. Therefore, there is a need to better identify DNA adducts and understand the association between the epigenetic marks in the cell. Currently there is no method to determine the exact location of DNA adducts in vivo nor at a high-resolution across the genome. In order to address this, we propose to develop a method, TdT-Seq, that will identify these adducts genome-wide at the single base pair resolution. The expertise of the investigators include knowledge in cancer biology and platinum drug pharmacology (Drs. Howell and Abada) as well as experience in high-throughput genomic assays and computational analysis (Dr. Harismendy); expertise that will be needed to successfully develop the assay. The TdT-Seq assay relies on adduct-mediated inhibition of the DNA polymerase in vitro. The resulting single strand DNA will be captured by a specific TdT mediated ligation, enriched, then sequenced in high throughput. We propose to establish the technical validity of the assay by determining 1) sensitivity at various cDDP concentrations and read depth, 2) specificity by the development of a locus specific method (Strand Specific Adduct Detection) and independent analysis of 50 adduct loci, and 3) quantativity using increasing cDDP concentrations and known spike-in controls. We will also perform specific experiments to establish TdT-Seq's use for clinical cancer research. In particular, we will optimize the protocol for the identification of UVor chlorambucil (CLB) induced adducts to broaden its applicability. We will also develop the protocol for low amounts of DNA originating from mouse tissues or heterogeneous tissue specimens. Finally, we will analyze the ability of TdT-Seq to measure the kinetics of DNA repair using genetically modified cell lines. TdT-Seq's development will therefore lead to a robust and innovative assay, with demonstrated performance and utility for cancer research. TdT-Seq will generate an entirely new type of data, which can be used in combination of other whole genome datasets from the ENCODE or TCGA consortium to provide a more precise and comprehensive description of the mechanism of DNA damage and repair in vivo in various cell types and cancers. The long-term benefits of such research include the prediction of drug sensitivity or the study of epigenetic modifying compounds to rationalize combinations for optimal drug efficacy.

An award is made to Iowa State University to develop and construct an Enviratron, a facility to test and evaluate the performance of plants under variable environmental conditions. To date most research on the performance of plants under different environmental conditions has been conducted with a limited number of differences, such as a single environmental stress versus control (unstressed) conditions. The Enviratron will permit researchers to incrementally alter multiple critical variables to better simulate changing conditions that will be faced in the future. The Enviratron will be an important research and training tool for students in the plant sciences, particularly for underrepresented minorities who will participate in the project through the George Washington Carver Summer Research Internship Program. It will provide them with the experience of simulating different environmental conditions or different climates of the world and the opportunity to study and improve the performance of plants under those conditions. It will also inspire engineering students to learn how to work hand in hand with plant scientists. The Enviratron will also be a demonstration centerpiece open to farmers and other visitors to promote appreciation and a better understanding of agricultural research.

Understanding how organisms in the biosphere can adapt to climate change is one of the grand scientific challenges of these times. This project creates a phenomics platform that will enable researchers to non-destructively monitor the performance of plants throughout their lifecycle under variable environmental conditions. The Enviratron represents a revolutionary new design in plant phenomics facilities. It consists of an array of plant growth chambers to create different environmental conditions. Unlike commercial plant phenomics systems, plants will not be conveyed out of the growth chambers to monitor their growth performance, rather a rover with a robotically controlled arm will periodically visit each chamber to image and analyze the plants. In addition to more standard visible light, fluorescence, near infrared and infrared imaging, sensors on the rover will be capable of imaging and conducting analyses not available on commercial systems such as hyperspectral and holographic imaging and Raman spectroscopy. The robot-assisted sensing approach will enable precise location-specific data acquisition, resulting in improved sampling strategies and data quality. The Enviratron will be used for research as well as graduate and undergraduate student training and will be located in a facility where it can be used to educate the public about climate change research.

Diane C Bassham

Proposal number IOS- 1353867, Autophagy and ER stress in plants.

Tolerance of changing environmental conditions is critical for plant growth and survival as they are unable to seek refuge from the environment. A key unresolved issue is how plants adapt their cellular functions to respond to stress conditions such as heat and drought. Two pathways required for stress tolerance are autophagy, in which damaged cell components are broken down and recycled, and the unfolded protein response, in which misfolded proteins activate the expression of genes that mitigate stress damage. In this project, the relationship between these two pathways will be examined. Additional proteins that are required for the regulation of both of these pathways will be identified and their functions analyzed by a combination of biochemical, cell biology and genetic approaches. The mechanism by which cells select substrates for recycling by autophagy will also be determined. This will provide insight into how plants sense adverse environmental conditions, how stress response pathways are then activated and how different responses work together to produce stress tolerance. The results of this project could provide information leading to the production of stress tolerant varieties of agriculturally important plants. The project will provide research training in plant genetics, cell and molecular biology for graduate and undergraduate students. In addition, the results of the research will be incorporated into a module of the Meta!Blast educational video game to help undergraduate and high school students learn cell biology. Seed stocks and plasmids generated in the course of the research will be donated to the Arabidopsis Biological Resource Center for distribution to the scientific community.

This research will investigate strategies for protecting maize, the major U.S. food, feed and fuel crop, from environmental stress. Maize can perceive and respond to adverse environmental conditions through a process called the unfolded protein response (UPR). Folding is a critical, but delicate step in the biosynthesis of proteins, and it can be easily upset by adverse conditions, such as high temperature. When this happens, misfolded proteins accumulate and activate a cascade of stress response genes. One of the objects of this project is to characterize in depth the cascade of genes to better understand how they protect maize from stress. Investigations in other plants has revealed that stress conditions eliciting the UPR also activate a process called autophagy in which plant cells repair stress damage. Therefore, another aim of this project is to discover how stress signals activate the autophagy machinery. In response to stress, other plants slow down protein synthesis to prevent overburdening the process of protein folding. In this project an investigation will be conducted to determine whether maize does this by degrading some of the messenger RNAs encoding proteins. Finally, attempts will be made to modify the UPR and probe more deeply into its operation by using new gene editing techniques. In parallel, there will be a focus on links between learning and research at the undergraduate level. This will be done through the development of easily accessible training on line and peer-support learning communities.

In this project, it is expected that new gene targets involved in ER stress responses in maize will be revealed through extensive transcriptomic analysis. It is anticipated that those targets may provide a clearer picture of both cell survival activities and cell death mechanisms in response to stress. In addition, these analyses will aid in uncovering the signaling pathways by which stress elicits the proliferation of the ER and activation of autophagy. While some UPR responses involve the upregulation of stress response genes, other responses result from the degradation of specific RNA transcripts brought about by Regulated IRE1-Dependent RNA Degradation (RIDD)and microRNA action. In this regard, the degradome as well as the transcriptome will be used to determine the role of selective RNA transcript degradation in maize stress responses. This research project is also expected to reveal through 'translateome' analysis whether ER stress is also mitigated in maize by selective and/or global regulation of the translation of RNA transcripts. A compelling reason for the selection of maize as a model for these studies is that the UPR has already been demonstrated in maize in the field. Therefore, variation in stress response in different lines of maize will be studied both in the laboratory and in the field with the goal of identifying genetic determinants that condition the UPR.