"Anthracenyl isoxazole amides (AIMs) as novel G-quadruplex DNA-binding anticancer agents"
Dr. Howard Beall
Human telomeric DNA, located at the terminal ends of chromosomes, consists of guanine-rich single strands that can adopt folded conformations known as G-quartets. These G-quartets can stack in various arrangements to form G-quadruplexes (or G4 DNA). Recently, there has been considerable interest in telomeric G4 DNA as telomere maintenance is essential to drive the proliferation of cancer cells. The enzymatic activity of telomerase is responsible for telomere maintenance and adds the guanine-rich DNA sequence repeats to the 3' end of DNA strands in the telomere regions. High telomerase activity and expression have been correlated with a poor prognosis in many tumor types. Molecules that bind telomeric G4 DNA and inhibit telomerase have been found to possess anticancer activity, and there is a need to design and develop small molecules that selectivity target telomeric G4 DNA to circumvent the acute toxicity observed with duplex DNA interactions.
We have recently reported a novel series of anthracenyl isoxazole amides (AIMs) that demonstrate significant anticancer activity in the NCI-60 cell line panel, with some of the highest activity (nanomolar GI50 values) against brain cancer cells. We propose to further elucidate the mechanism of action of the AIMs and the structural requirements that confer anticancer activity. Using computational-based modeling we will examine the interactions of the AIMs with telomeric G4 DNA sequences. Data derived from the modeling studies will be used to direct the synthesis of further generations of AIMs that exhibit increased selectivity for telomeric G4 DNA and increased activity in brain cancer.
The hypothesis for this project is that novel AIMs that are selective for telomeric G4 DNA will exhibit significant activity against brain tumors. The objectives are (1) to utilize computational modeling and novel synthetic methods to design and produce bioavailable antitumor AIMs that bind G4 DNA with high affinity, (2) to establish the anticancer activity of the AIMs in a panel of commercial and primary brain tumor cell lines, and (3) to determine the ability of the AIMs to bind G4 DNA and inhibit telomerase activity.
For this project, the student will learn how to culture brain cancer cells using aseptic technique and how to carry out growth inhibition and cytotoxicity studies. In addition, the student will use imaging methods such as laser scanning cytometry and confocal microscopy to determine subcellular localization of the AIMs within cancer cells.
“Asthma, woodsmoke, and antioxidants”
Drs. Tony Ward and Megan Bergauff
Exposure to wood smoke particulate matter has been linked to increases in the number of asthma attacks and hospitalizations. It has been suggested that dietary factors such as antioxidants can have a positive effect on the incidence and severity of asthma. Supplementation with antioxidants has been suggested as a way to mediate asthma symptoms after exposure to airborne pollutants such as wood smoke, particularly in individuals with deficiencies in nutrients. The present study proposes to investigate the relationship between urinary vitamin E levels and markers of asthma severity. The results of this project will provide information about how dietary supplements could be used to help manage symptoms in asthmatic children, particularly those that are frequently exposed to high levels of particulate matter air pollution.
The summer intern will perform analysis for vitamin E and 8-isoprostane as a marker of asthma severity as well as assist in other aspects of the project. The student will gain experience in basic analytical chemistry lab techniques such as pipetting, organic solvent extraction, and calibration curve generation, techniques for colormetric and ELISA assays, biological sample handling, data analysis, and using GC-MS and UV plate reader instrumentation and software for sample analysis.
“Comparison of different types of ambient particulate matter”
Dr. Tony Ward and Matthew Ferguson
The U.S. Environmental Protection Agency (USEPA) currently regulates the levels of certain types of airborne particulate matter (PM), including PM2.5 and PM10. Recently, additional interest has been paid to the course fraction, PM10-2.5, due to a proposed National Ambient Air Quality Standard for this size of ambient PM. The aim of our current study is to determine the potential health effects of exposure to PM10-2.5 as compared to PM2.5 and PM10.
Using a particle concentrator (PC), the summer intern will assist with the collection of ambient particles at the University of Montana. Harvested particles will then be dosed on cell tissue cultures used to represent lung tissues. Levels of pro-inflammatory cytokines (e.g., IL-1β and TNFα) and cell viability are the tissue exposure endpoints to be investigated. Cell staining and microscopy will determine cell viability, and cytokine expression will determine inflammatory response. This study will aid in determining the future directions of this ongoing investigation.
“IL-10 family cytokines in the regulation of engineered nanomaterials (ENM)-induced intestinal inflammation”
Dr. Celine Beamer
Engineered nanomaterials (ENM) are anticipated for use in electronics, cosmetics, cleaning materials, coatings, food packaging, and medicines, with ever-increasing and inevitable human exposure. Hence, the potential health effects of ENM are a major concern that needs to be addressed. Although the likely route of ENM exposure is via inhalation or dermal contact, ingestion may also occur as a consequence of swallowing inhaled material following mucociliary clearance, as a result of hand-to-mouth contact in humans, and as a consequence of grooming during/after exposure in animal studies. Therefore, it is of great interest to examine the consequences of ENM exposure on extra-pulmonary tissues such as the gastrointestinal (GI) tract. Within the GI tract, intestinal epithelial cells (IECs) represent the initial point of contact with the external environment, including environmental contaminants such as ENM. In response to environmental contaminants, IECs initiate inflammatory responses resulting in the release of IL-1 family members (e.g. IL-1b/IL-18/IL-33), which have the potential to result in significant tissue damage. In contrast, IL-10 family cytokines (e.g. IL-10, IL-19, IL-22, IL-28) have critical functions in regulating inflammatory responses, as well as improving defense against environmental contaminants. Therefore, the two major goals of this project are to: 1) define the consequences of ENM exposure on IECs, and 2) to define the contributions of IL-10 family members to regulation of these responses.
This summer project will provide the student researcher with an opportunity to learn both the theory and techniques behind the immunotoxic effects of ENM. It will also give the student hands-on experience related to the design and execution of novel experiments intended to discover how ENM affect inflammatory responses of the GI tract. Technically, the student will learn: 1) how to grow and maintain human and mouse IECs using sterile tissue culture techniques, 2) how to expose IECs to ENM and assess immunotoxic responses, and 3) how to therapeutically manipulate these immunotoxic responses using IL-10 family members. In addition to expanding our knowledge of how ENM affect the GI tract using in vitro models, it is expected that the student will be exposed to ongoing animal experiments to evaluate the effects of ENM on inflammatory diseases in the GI tract. Collectively, these studies will generate novel data to improve our understanding of environmentally-induced diseases.
“Histological and molecular analysis of pulmonary macrophage subsets following particulate exposures”
Dr. Christopher Migliaccio
Macrophages are divided into subpopulations based on the pathway of activation and subsequent functions. Pathologies associated with environmental exposures have recently been linked with specific subsets. For example, in the silicosis model fibrosis has been linked with the M2 macrophage phenotype. To this end, the proposed project will attempt to define macrophage subsets and the population changes associated with different exposures. Using samples from silica, PM, and WS exposed mice, macrophages will be defined and compared. Subsets will be defined by DNA/RNA analyses of cells, as well as immunohistological assessment from different strains. Therefore, in this study we will analyze lung tissue from multiple strains of mice, including null and/or cellular depleted strains, in an effort to define changes in macrophage subpopulations following exposure to particulates.
In this project, the student will learn a variety of methods including sectioning and cell culture, DNA/RNA isolation and analysis, mounting of tissue, immunohistochemical staining procedures, and confocal microscopy.
“Investigation of NLRP-3 inflammasome inhibition by microbial isolates derived from the Berkeley Pit, Butte Montana”
Dr. Teri Girtsman
The Berkeley Pit Lake in Butte, Montana, is an acid mine waste reservoir rich in toxic metals and is part of the largest EPA Superfund site in North America. Drs. Don and Andrea Stierle have isolated over 70 unique microbes from this evolving extreme ecosystem and are in the process of studying the bioactive secondary metabolites as possible anticancer or antimicrobial agents. Several interesting compounds have been isolated from these microbes on the basis of their activities in different bioassay-guided fractionation schemes.
We have performed a battery of bioactivity and toxicity screenings of approximately 40 of the metabolites listed above using human monocyte cell line, THP-1. The THP-1 cells were differentiated to a macrophage like phenotype using a phorbol ester – PMA. The summer project would include a study comparing the bioactivity of the metabolite compounds using THP-1 cells differentiated with Vitamin D in comparison to the PMA induced cells. The endpoints evaluated will be activity of the NLRP3 inflammasome (IL-1beta and IL-18 cytokines) and measuring the toxicity of the metabolite compounds by two assays (MTS and LDH). Additionally, we would investigate the ability of these compounds to inhibit a third NLRP3 substrate, IL-33 in small airway epithelial cells (SAEC) in vitro.
In this project students would learn about a relatively newly discovered inflammatory pathway that may be the cause of many human diseases and how to do different cell cultures, ELISA assays and cytotoxicity assays.
“Determination of the non-volatile and re-entrained mass fractional composition of wood smoke as a function of dilution and wood type”
Dr. Maria Morandi
Controlled exposures of laboratory animals or humans to well characterized atmospheres of airborne contaminants is one of the main approaches for investigating potential health effects of air pollutants as well as for elucidating the underlying mechanisms leading to disease. This knowledge is critical for the protection of public health. Wood smoke is a significant indoor and outdoor airborne contaminant that has been linked to respiratory and other disease outcomes. We have available a system to perform controlled inhalation exposures to wood smoke that is undergoing characterization. This project is part of the characterization.
Similar to combustion of other materials and fuels, emissions from wood burning are dynamic and time-evolving so that the distribution between particle-bound and vapor phases and the chemical composition changes with distance from the combustion source. Close to the combustion zone, particles ≤ 0.01µm form rapidly as hot gases condense and mix with surrounding cooler air. Larger though still submicron-sized particles form further downstream as a result of particle coagulation (i.e., inelastic collision between two or more smaller particles) and the continued condensational growth resulting from further cooling and dilution. Consequently, the physical and chemical composition of the aerosol at the point of exposure, which is well downstream from the source, differs from that in the combustion zone. It is, therefore, critically important to determine the particle/gas phase mass partitioning of the aerosol present in the atmosphere at the point of exposure. This project will performed such characterization for two different types of wood combusted under similar conditions.
Pre-weighted amounts of two different types of woods will be burned in one of two wood stoves while monitoring combustion temperature. Emissions will be diluted with varying flows of pre-filtered ambient air. Wood smoke sampling will be performed immediately downstream from the combustion zone and further downstream in the exposure chamber using a combination filters (to collect the particle-bound phase of the aerosol) followed by impingers to collect the re-entrained gas phase (i.e., the combination of airborne chemicals originally present in the gas phase and those released from the particles collected by the filter). Mass determination of the particle phase will be done by gravimetric analysis; the gas phase mass will also be determined by gravimetric analysis of impinger solvent aliquots after evaporation at low flow. It is expected that the particle mass fraction will increase in a predictable manner as dilution flow increases, given the same type of wood and combustion temperature.
The student will learn basic principles of air pollution and aerosol dynamics, sampling of aerosols, determination of aerosol mass, and basic principles of dynamic chamber exposure systems.
"Oxidative Stress and Epigenetic Modulation"
Dr. Fernando Cardozo-Pelaez
Epigenetics is a major mechanism that accommodates gene-expression changes in response to gene-environment interactions. Epigenetics refers to modifications in gene expression that are influenced by DNA methylation and/or chromatin structure, RNA editing and RNA interference without any changes in DNA sequences. DNA methylation is one of the most studied aspects of epigenetic modifications. The addition or removal of methyl groups from cytosines can impact gene expression and alter cell and organism function. The methylation of cytosine can sometimes serve as a heritable code by the selective action of some methylases that act on cytosine nucleotides in a CG sequence. Methylation occurring predominantly at this symmetrical CG dinucleotide, due to the preferences of DNA methyltransferase for a hemi-methylated substrate, maintains specific heritable patterns of methylation. In CpG dinucleotides, the cytosine is the preferred base for DNA methylation, while the guanine is the site for oxidative damage. 8-oxo-dG is widely used as biomarker of oxidative DNA damage. In the absence of exogenous DNA-damaging reagents, endogenously formed metabolic reactive oxygen species (ROS) are able to create 105 8-oxo-dG daily in cells. Few studies have addressed DNA methylation and DNA oxidative damage simultaneously as an epigenetic phenomenon, and little is known on how DNA methylation and DNA oxidation interact with each other. The objective of the STEER project will be to test for variability in patterns of cytosine methylation in target promoters of gene highly regulated by methyl cytosine formation in DNA from wild-type mice and from mice lacking the DNA repair enzyme ogg1, responsible for removal of 8-oxo-dG. Results from these studies will help to advance a novel area of research that combines the relationship between oxidative stress and epigenetic programming. The STEER student will learn tissue preparation for DNA extraction, DNA extraction, HPLC analysis for methyl-cytosine and 8-oxo-dG. Also, the student will learn to design PCR primers to determine specific location of either methyl-cytosine and/or 8-oxo-dG in promoter regions of selected genes.
"The control of protein production related to lung fibrosis"
Dr. Elizabeth Putnam
Asbestos fibers are toxic substances that can cause serious health problems including cancer, asbestosis, and mesothelioma when they become airborne and are inhaled.
We have identified SPARC, a matricellular protein involved in tissue repair, extracellular matrix (ECM) regulation, cellular proliferation, and cellular adhesion, as a candidate for involvement in the fibrosis that occurs after asbestos exposure. Through our studies with wild type and SPARC knockout mice we found that expression of both SPARC and collagen (a major component of the ECM) is increased in asbestos treated mice compared to control and that SPARC knockout mice did not produce as much collagen in response to asbestos. We now want to see if we can control the expression of SPARC after asbestos exposure with RNA interference (RNAi) and thus decrease the amount of collagen produced. After establishing primary lung fibroblast cell strains, these cells will be exposed to previously prepared viral particles containing shRNA complementary to SPARC to inhibit collagen expression after asbestos exposure.
The student on this project will learn how to culture cells and titer virus. The student will also analyze the resulting changes in protein expression in test cell cultures treated with the inhibitory shRNA constructs.