Progress 10/01/09 to 06/30/14
Outputs
Target Audience: Target Audience International Changes/Problems: Changes/Problems Not relevant for the final report. What opportunities for training and professional development has the project provided? Opportunities This project has provided continued training for doctoral student Trevor Fish, who has gained experience in new techniques, such as laser capture micro dissection, methyl-specific PCR, and bioinformatics. Additionally, undergraduates have gained experience in animal husbandry and general laboratory assays, such as PCR, RNA or DNA isolation, and cell culture. How have the results been disseminated to communities of interest? No publications or presentations related to Aim 2 were authored in 2013. In 2013, we published an article in Cancer Prevention Research related to Aim 2 (though not directly), in which we provided evidence that the estrogen receptor is critical for the anti-cancer effects of the bioactive food compound diindolylmethane in vivo. An additional publication of data obtained from Aim 1 is expected in 2015. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work During the last six months of this project, our key objective is to complete the analysis of the expansive datasets generated from Aim 1. Trevor Fish has received some training in R programming software, which should enable him to use the new tools available for analysis of methylation data. We expect to present this work at either the Society of Toxicology meeting in Spring 2014 or at the American Association of Cancer Research meeting in the Summer of 2014. We expect at least two additional publications directly from Aim 1 in the next 18 months. Regarding Aim 2, I think that our efforts are better focused on the interaction of basal diet and efficacy of bioactive foods and/or food chemicals. Thus, this will be the focus of a new project proposal to be submitted December 2013 for a new project starting July 2014. Not relevant for the final report.
Impacts
What was accomplished under these goals?
Aim 1. 1) Major activities completed: The in vivo study investigating polycyclic aromatic hydrocarbon(PAH) impact on DNA methylation in mouse lung was completed in 2012. Doctoral student Trevor Fish was responsible for execution of this project, under the direct oversight of laboratory technician Deanna Larson. Next, in 2013, we employed laser capture microdissection (LCM) to determine the timing of tumor suppressor gene silencing by assessing patterns of gene promoter methylation during the progression of preneoplastic lesions, atypical adenomatous hyperplasia (AAH), to lung adenocarcinomas in mice aged 15 to 45 weeks. Trevor completed LCM of lung samples to obtain tissues throughout the stages of lung cancer development (normal, pre-neoplastic, hyperplastic, cancerous). This work was performed at a core facility at the Univ. of Utah. Next, methylation of the promoter regions of Cdkn2a, Rarb, Dapk1, Mgmt and Cadh13 genes and expression of DNA methyltransferase enzymes 1, 3a and 3b in neonate lung were assessed by bisulfite sequencing and quantitative real time polymerase chain reaction, respectively. Genome wide methylation of lung tumors in the adult offspring mice was determined using the NimbleGen DNA Methylation array. Data obtained from sequenced LCM tissues aswell as the DNA Methylation array are currently being analyzed. 2) Objectives met: At this stage, all objectives regarding this aim have been met, save the final step of data analysis, which is ongoing. 3) Significant results achieved: We determined that lung tumor incidence varied depending on the potency of the chemical carcinogen used, with the extremely potent compound dibenzy[def,p]chrysene (DBC) causing 100% lung tumor incidence compared to only 30% incidence in mice initiated with benzo[a]pyrene (BAP). Interestingly, a gender difference was apparent in animals initiated with BAP, with females having a higher incidence. This finding warrants further exploration. 4) Key impacts or other accomplishments realized: While global methylation analysis was not part of the original experimental plan, we elected to include this element to the study design as a fallback option, should the targeted gene approach prove disappointing. We expect this additional data set will be useful for hypothesis generation and to correlate epigenetic changes in lung tissues of this animal model of non-small cell lung cancer to those profiles obtained from human patients with the disease. Aim 2 1) Major activities completed: To date, we have performed extensive cell culture assays to characterize the impact of a diverse set of bioactive compounds on the survival of human T-cell acute lymphoblastic lymphoma/leukemia (T-ALL) cells. Previously, my staff had completed a number of mixture experiments to determine whether combinations of these bioactive food chemicals (such as genistein and diindolylmethane) had greater efficacy than individual compounds). In 2013, we focused on establishing methods for analyzing activity of the enzyme DNA methyltransferase (DNMT), an assay that proved troublesome in our laboratory the prior year. 2) Objectives met: In 2013, the key objective was to get the DNMT assay working properly for our cell line. However, this objective was not achieved. 3) Significant results achieved: Results of the combination assays with bioactive food chemicals proved disappointing, as little evidence for additive or synergistic activities of combinations compared to individual compounds was obtained. The most promising pair of compounds was genistein (GEN) and diindolylmethane (DIM), which were tested in the DNMT enzyme assay. After repeated trials by two scientists (Deanna Larson and student Sean White), we were unable to obtain reproducible results using the only available kit for this assay. At this point, more effort on this aim was expected to lead to diminishing returns. Thus, overall, greater effort was given to completion of Aim 1 (and other pilot projects supported by UAES). 4) Key impacts or other accomplishments realized: In the 4th year of this project, I have become somewhat disenchanted with the notion of mixtures of specific bioactive food chemicals as an optimal strategy for cancer prevention, partly because of the disappointing results of the mixture studies we performed, but also because of a greater focus by federal funding agencies on the impacts of whole foods on health. Also, through pilot work (supported by the Utah Agricultural Experiment Station), my research team has obtained evidence that background nutrition plays an incredibly important role in assessing the efficacy of these bioactive food components. It is my expectation that our work in the future will shift toward understanding the interaction of these bioactive food chemicals (either alone, in extract or whole food form) and basal nutrition (typical consumption of macro- and micronutrients) for influencing cancer risk and/or prevention.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: International Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided continued training for doctoral student Trevor Fish, who has gained experience in new techniques, such as laser capture micro dissection, methyl-specific PCR, and bioinformatics. Additionally, undergraduates have gained experience in animal husbandry and general laboratory assays, such as PCR, RNA or DNA isolation, and cell culture. How have the results been disseminated to communities of interest? In 2013, we published an article in Cancer Prevention Research related to Aim 2 (though not directly), in which we provided evidence that the estrogen receptor is critical for the anti-cancer effects of the bioactive food compound diindolylmethane in vivo. No publications or presentations related to Aim 2 were authored in 2013. What do you plan to do during the next reporting period to accomplish the goals? During the last six months of this project, our key objective is to complete the analysis of the expansive datasets generated from Aim 1. Trevor Fish has received some training in R programming software, which should enable him to use the new tools available for analysis of methylation data. We expect to present this work at either the Society of Toxicology meeting in Spring 2014 or at the American Association of Cancer Research meeting in the Summer of 2014. We expect at least two additional publications directly from Aim 1 in the next 18 months. Regarding Aim 2, I think that our efforts are better focused on the interaction of basal diet and efficacy of bioactive foods and/or food chemicals. Thus, this will be the focus of a new project proposal to be submitted December 2013 for a new project starting July 2014.
Impacts
What was accomplished under these goals?
Aim 1. 1) Major activities completed: The in vivo study investigating polycyclic aromatic hydrocarbon (PAH) impact on DNA methylation in mouse lung was completed in 2012. Doctoral student Trevor Fish was responsible for execution of this project, under the direct oversight of laboratory technician Deanna Larson. Next, in 2013, we employed laser capture microdissection (LCM) to determine the timing of tumor suppressor gene silencing by assessing patterns of gene promoter methylation during the progression of preneoplastic lesions, atypical adenomatous hyperplasia (AAH), to lung adenocarcinomas in mice aged 15 to 45 weeks. Trevor completed LCM of lung samples to obtain tissues throughout the stages of lung cancer development (normal, pre-neoplastic, hyperplastic, cancerous). This work was performed at a core facility at the Univ. of Utah. Next, methylation of the promoter regions of Cdkn2a, Rarb, Dapk1, Mgmt and Cadh13 genes and expression of DNA methyltransferase enzymes 1, 3a and 3b in neonate lung were assessed by bisulfite sequencing and quantitative real time polymerase chain reaction, respectively. Genome wide methylation of lung tumors in the adult offspring mice was determined using the NimbleGen DNA Methylation array. Data obtained from sequenced LCM tissues aswell as the DNA Methylation array are currently being analyzed. 2) Objectives met: At this stage, all objectives regarding this aim have been met, save the final step of data analysis, which is ongoing. 3) Significant results achieved: We determined that lung tumor incidence varied depending on the potency of the chemical carcinogen used, with the extremely potent compound dibenzy[def,p]chrysene (DBC) causing 100% lung tumor incidence compared to only 30% incidence in mice initiated with benzo[a]pyrene (BAP). Interestingly, a gender difference was apparent in animals initiated with BAP, with females having a higher incidence. This finding warrants further exploration. 4) Key impacts or other accomplishments realized: While global methylation analysis was not part of the original experimental plan, we elected to include this element to the study design as a fallback option, should the targeted gene approach prove disappointing. We expect this additional data set will be useful for hypothesis generation and to correlate epigenetic changes in lung tissues of this animal model of non-small cell lung cancer to those profiles obtained from human patients with the disease. Aim 2 1) Major activities completed: To date, we have performed extensive cell culture assays to characterize the impact of a diverse set of bioactive compounds on the survival of human T-cell acute lymphoblastic lymphoma/leukemia (T-ALL) cells. Previously, my staff had completed a number of mixture experiments to determine whether combinations of these bioactive food chemicals (such as genistein and diindolylmethane) had greater efficacy than individual compounds). In 2013, we focused on establishing methods for analyzing activity of the enzyme DNA methyltransferase (DNMT), an assay that proved troublesome in our laboratory the prior year. 2) Objectives met: In 2013, the key objective was to get the DNMT assay working properly for our cell line. However, this objective was not achieved. 3) Significant results achieved: Results of the combination assays with bioactive food chemicals proved disappointing, as little evidence for additive or synergistic activities of combinations compared to individual compounds was obtained. The most promising pair of compounds was genistein (GEN) and diindolylmethane (DIM), which were tested in the DNMT enzyme assay. After repeated trials by two scientists (Deanna Larson and student Sean White), we were unable to obtain reproducible results using the only available kit for this assay. At this point, more effort on this aim was expected to lead to diminishing returns. Thus, overall, greater effort was given to completion of Aim 1 (and other pilot projects supported by UAES). 4) Key impacts or other accomplishments realized: In the 4th year of this project, I have become somewhat disenchanted with the notion of mixtures of specific bioactive food chemicals as an optimal strategy for cancer prevention, partly because of the disappointing results of the mixture studies we performed, but also because of a greater focus by federal funding agencies on the impacts of whole foods on health. Also, through pilot work (supported by the Utah Agricultural Experiment Station), my research team has obtained evidence that background nutrition plays an incredibly important role in assessing the efficacy of these bioactive food components. It is my expectation that our work in the future will shift toward understanding the interaction of these bioactive food chemicals (either alone, in extract or whole food form) and basal nutrition (typical consumption of macro- and micronutrients) for influencing cancer risk and/or prevention.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Benninghoff, A., Fish, T., Louderback, M., Williams, D. (2013). The role of estrogen receptor beta (ERb) in transplacental cancer prevention by indole-3-carbinol. Cancer Prevention Research. Cancer Prevention Research, 6(4), 339-348.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: In this study, a GWAS study was undertaken to identify genomic regions influencing several production traits in the Rambouillet breed. Genomic DNA was extracted from 259 animals in 35 flocks and genotyped with the Illumina Ovine SNP50 BeadChip. Phenotypes collected on the Rambouillet animals included both qualitative and quantitative measurements for wool, growth, reproduction, carcass, and conformation traits. PARTICIPANTS: Texas A&M AgriLife Research, University of Wyoming, CSIRO Livestock Industries TARGET AUDIENCES: Researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant associations for bent limb, a bone deformity manifested in yearling animals, were found on OAR18 (4 SNPs) and OAR10 (4 SNPs), as well as for scurs on OAR2 (2 SNPs)
Publications
- Shorey, L., Maughan, A., Wlliams, D., Dashwood, R., Ho, E., & Benninghoff, A.. (2012) 3,3-Diindolylmethane induces G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia cells: PLoS One, 7(4): e34975. (Published).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Aim 1. The in vivo study investigating PAH impact on DNA methylation in mouse lung is nearly complete. The study was begun at the end of 2010, and should be completed within the next month or so. Graduate student Trevor Fish is primarily responsible for execution of this project, under the direct oversight of laboratory technician Deanna Larson. They have developed methods for methyl-specific PCR for the candidate target genes. Since the last annual report, our department has acquired new instrumentation (Fluidigm AccessArray) that allows for more in-depth analysis of gene promoter methylation at a higher level of resolution. Thus, we have also devoted substantial effort this past year toward establishing methodologies using the AccessArray to perform ultra deep genome sequencing on the samples we are obtaining from the in vivo study. They are also working on methods for laser capture micro dissection to obtain abnormal lung cells in the very early stages of cancer development (i.e., pre-neoplastic). This work will be performed using the service center at the University of Utah. To date, I am satisfied with our progress on this aim of the UAES project. Aim 2 has progressed nicely as well. Amanda Maughn has been chiefly responsible for the large number of cell culture experiments to characterize the anticancer effects of a suite of nine bioactive food chemicals in multiple cell lines (normal an cancer lines for the lung and for T-cell lymphoma). We now have a very thorough dataset for these chemicals, with calculated IC50 values (that concentration that kills half the cells, a marker of the efficacy of the compound tested). From the initial survey of nine compounds, five were selected for the lung and lymphoma cancer lines, respectively, to be tested in binary combinations. Those experiments have also been completed, and we obtained evidence for some additive, and potentially synergistic, action of several combinations. Most notable is the combination of diindolylmethane (DIM) and genistein; the shape of the observed dose response curve suggested that lower concentrations of these compounds administered together were more effective than when administered separately. We are now moving on toward more specific biochemical assays to investigate the mechanism(s) by which these two particular bioactive food chemicals may be interacting. In addition, Ms. Maughn has also conducted a comparative study using multiple human T-cell lymphoma/leukemia (T-ALL) cell lines. The disease these cancer cells represent is particular heterogeneous, meaning that the cancer cells in patients develop from white blood cells (T-cells) at different stages of maturation. In order to demonstrate the broad applicability of cancer prevention by DIM, we evaluated its anticancer effects in four human T-ALL cell lines. DIM was a potent anticancer agent in all cell lines tested, causing reduced cell proliferation and increased apoptosis. These data will be presented at the international American Association of Cancer Research meeting next spring. PARTICIPANTS: Amanda Maughan, Laboratory Research Technician III, specializes in cell culture Deanna Larson, Laboratory Research Technician III, specializes in mouse models Brittany Packard, Undergraduate Researcher Trevor Fish, Graduate student Stephany Perez, Graduate student TARGET AUDIENCES: International PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Researchers with the Utah AES and in the USTAR Applied Nutrition Research Team have obtained data that suggesting that dietary intervention with the bioactive food component diindolylmethane (DIM) may be an effective strategy at preventing or suppressing T-cell lymphoblastic leukemia/lymphoma (T-ALL) in humans. Using a cell culture approach, this research team determined that low micromolar concentrations of DIM, reasonably achievable via a diet rich in cruciferous vegetables or through dietary supplementation, effectively blocked growth of four different human T-ALL cell lines by reducing cell proliferation and increasing apoptosis (programmed cell death). This finding is important because T-ALL is a heterogeneous disease resulting from the developmental arrest and abnormal proliferation of T-cells at different stages of maturation. The cell lines investigated in this study represent these different stages of T-cell development. The observation that DIM is effective in all lines tested broadens the applicability of dietary intervention with DIM to T-ALL in general, though it is interesting to note that cells derived from early developing T-cells were the most susceptible.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In my first year as a new faculty member of ADVS, I have established a functioning and productive research laboratory in the Center for Integrated BioSystems (CIB). By the Summer of 2010, my laboratory had been fully equipped with all of the necessary instrumentation, equipment and materials to facilitate my research program. Additionally, two laboratory research assistants have been hired this year and are now actively managing the laboratory space, equipment and research projects as well as the training of other personnel (chiefly, two undergraduate laboratory research assistants). We have also set up a BSL-2 research facility in the CIB, and we are in the final stages of obtaining University approval for its use. Aim 1. The in vivo study to investigate the impact of transplacental PAH exposure on the fetal epigenome are underway. During the in vivo exposure and grow out period (about 12 months), laboratory personnel are establishing protocols for genomic DNA isolation and methyl specific PCR (MS-PCR) for assessing methylation status of key tumor suppressor genes. Aim 2 - We have established culture systems for several cell lines in our laboratory, including lung cancer cells A549 and lymphoblastic leukemia cells CCRF-CEM. Preliminary experiments investigating the anticancer effects of nine different bioactive food components are underway using the A549 cells and a rapid cell viability assay (MTT assay). Additionally, we have established methods for assessing cell proliferation and viability by flow cytometry for combination experiments with two candidate bioactive food chemicals, epigallocatechin gallate and diindolylmethane. Finally, Mr. Fish is conducting in vitro experiments to determine the involvement of the estrogen receptor in diindolylmethane-induced inhibition of CCRF-CEM cell proliferation. While progress in year 1 may appear modest, we have accomplished many of the necessary preliminary steps to establish in vivo and in vitro models required for this project. PARTICIPANTS: Amanda Maughan, Laboratory Research Technician III, specializes in cell culture; Deanna Larson, Laboratory Research Technician III, specializes in mouse models; Brittany Packard, Undergraduate Researcher; Trevor Fish, Undergraduate Researcher TARGET AUDIENCES: International PROJECT MODIFICATIONS: We may select a different bioactive food component for testing in mixture studies in vitro than lycopene, which is reported to produce hydrogen peroxide in cell culture. This "side-effect" would likely confound any apparent anticancer effect of this chemical and complicate our data analysis. Otherwise, no significant modifications of the experiment plan are proposed at this time.
Impacts
None to date.
Publications
- No publications reported this period
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