1985 — 2010 |
Hankinson, Oliver |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Carcinogen Activation and Screening in Variant Cells @ University of California Los Angeles
The aryl hydrocarbon receptor (AHR) binds a variety of pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a) pyrene, and mediates the carcinogenic and toxic effects of these compounds. After binding ligand, AHR dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer activates transcription of CYP1A1 and certain other genes. Induction of CYP1A1 is involved in carcinogenesis by benzo(a) pyrene. In contrast, the mechanism of TCDD carcinogenesis and toxicity is obscure. However, it probably depends (at least in part) upon transcriptional activation of certain other (unidentified) genes. In this application three specific aims are proposed. 1) Preliminary evidences indicates that the transcriptional co-activators steroid receptor co- activators 1,2 and 3 (SRC-1, -2 and -3) and thyroid receptor/retinoblastoma protein interacting protein (Trip230) may act as co-activators for the AHR/ARNT dimer. This possibility will be investigated further, for examined by ascertaining whether the endogenous co-activators bind endogenous ARNT and/or AHR in mammalian cells, and whether negation of function of each co-activator compromises transcriptional activation by AHR/ARNT. In addition, three novel ARNT-interacting proteins will be analyzed further, and novel co- activator for co-repressor proteins for AHR and ARNT will be sought. These studies should provide important insights into the mechanism of transcriptional activation by the AHR/ARNT dimer, may reveal cross-talk between AHR/ARNT and other transcription factors, and may explain some of TCDD's toxic actions. 2) We previously isolated mutants of the Hepa-1 mouse cell line. Class "B" mutants are probably defective in a transcription factor for AHR or a chromatin remodeling factor. The B gene will be cloned and characterized. These studies should provide important insight into the regulation of expression of the AHR gene. 3) Six novel TCDD-inducible genes have been isolated from Hepa-1 cells. Additional TCDD-regulatable genes will be isolated from mouse liver, mouse thymocytes, and human breast cancer cells. Using model cellular systems of TCDD toxicity, experiments will probe whether the products of these genes mediated TCDD toxicity.
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1 |
1995 — 1997 |
Hankinson, Oliver None |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Arnt Protein, Development and Carcinogenesis @ University of California Los Angeles
The Ah receptor mediates all, or nearly all, of the toxicological effects of halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). These compounds act as tumor promoters in rodents. The Ah receptor is also directly involved in carcinogenesis by many initiating agents. The receptor may also play a role in embryonic development. The DNA-binding, transcriptionally active form of the Ah receptor in cultured liver cells is a heterodimer of the Ah receptor nuclear translocator (Arnt) protein and the ligand-binding subunit of the Ah receptor. However, it is not clear whether some activities of the ligand-binding subunit and of Arnt can be manifested independently of the other protein. This project proposes to use transgenic mouse technology to address the potential role of the Arnt protein in carcinogenesis by non-genotoxic and genotoxic carcinogens, and in developmental processes. One Arnt allele in an embryonal stem (ES) cell line will be inactivated by homologous recombination. The targeted ES cells will be used to generate mice homozygous for the disrupted Arnt allele. If they are inviable, the developmental abnormalities and defects in the reproductive system of homozygous fetuses or newborns will be determined, in order to provide insight into the role of Arnt in development. If Arnt deficient mice develop to a late enough stage, we will determine whether they are resistant to the toxic effects of TCDD on development. If Arnt-knockout mice are fully viable we will investigate whether the adult mice are resistant to acute toxic responses to TCDD, thus testing the hypothesis, proposed by others, that some toxic effects of TCDD are produced via an Arnt-independent pathway. If the Arnt- knockout mice are fully viable we will also generate derivatives which are homozygous for a concatemer of the lacI/q gene located on mouse chromosome 4. The lacI/q gene can be used to quantitate mutations in all cells of the body. We will use the lacI/q derivatives to test the hypothesis that TCDD (which is non-genotoxic in bacteria but a complete carcinogen to mouse liver), enhances spontaneous mutagenesis or mutagenicity of initiators in mouse fee, and if so, whether enhancement is dependent on Arnt. We will also ascertain whether the liver and colon carcinogen, 2- amino-3-methylimidazo[4,5-f]quinoxaline (IQ) increases the frequency of liver and colon mutations in mice fed with the compound, whether cofeeding with TCDD (and therefore stimulation of Ah receptor activity) affects mutagenicity, and if any effects of TCDD are observed, whether these are dependent on Arnt.
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0.958 |
1997 — 2000 |
Hankinson, Oliver None |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Dep Induction of Allergic Inflammation and Identification of Genes Involved @ University of California Los Angeles
respiratory hypersensitivity; enzyme induction /repression; carbopolycyclic compound; engine exhaust; inflammation; mucosal immunity; T lymphocyte; cooperative study; antibody formation; immunoglobulin E; human subject; laboratory mouse;
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0.958 |
2002 — 2006 |
Hankinson, Oliver |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Arnt:Roles in Tumor Induction and Growth, and Toxicity. @ University of California Los Angeles
DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) binds a variety of pollutants, including benzo(a)pyrene (BP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and 2,3,7,8-tetrachlorodibenzofuran (TCDBF), and mediates the carcinogenic and toxic effects of these compounds. After binding ligand, AHR dimerizes with the Aryl Hydrocarbon Nuclear Receptor Translocator Protein (ARNT). The AHR/ARNT dimer then activates transcription of several genes involved in xenobiotic metabolism. However, there is evidence that liganded AHR can trigger biological responses via signal transduction pathways that do not involve ARNT. This proposal takes advantage of a recently derived Arnt conditional knockout mouse (homozygous for a foxed Arnt allele) in which Arnt can be knocked out (in specific tissues) in adulthood, to investigate whether ARNT is required for (1) the induction of thymic involution by dioxin (which is known to be dependent on AHR), (2) the (AHR-dependent) complete carcinogenic activity of BP, (3) the AHR-dependent tumor promoting activity of TCDBF and/or dioxin, and (4) the tumor initiating activity of BP. These investigations should shed light on the molecular mechanisms whereby AHR ligands induce toxicity and cancer. Hypoxia-Inducible Factor (HIF1) is the master regulator of the hypoxic response, triggering many adaptive responses to hypoxia, including angiogenesis. HIF-1 consists of a dimer of ARNT and HIF-la. Many cells in solid tumors exist in a hypoxic state. It is controversial as to whether the HIF-l mediated hypoxic response accelerates or retards tumor growth. Utilizing the Arnt conditional knockout mouse, Specific aim 5 will address this issue by comparing the growth rate and angiogenic response of endogenous tumors induced in ARNT-negative and ARNT-positive host cells. Specific aim 6 will investigate when during tumor development HIF- 1 activity affects (either positively or negatively) tumor growth. This will be addressed by comparing growth kinetics and angiogenesis of endogenous tumors before and after inducing disruption of the foxed Arnt gene, and by studying tumor xenografts generated from tumorogenic cells in which ARNT expression can be modulated by tetracycline.
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1 |
2006 — 2010 |
Hankinson, Oliver |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Function and Regulation of Human Cytochrome P4502s1 @ University of California Los Angeles
[unreadable] DESCRIPTION (provided by applicant): CYP2S1 is a recently identified human cytochrome P450, expressed extensively in epithelial tissues. We propose that CYP2S1 plays a significant role in the metabolic activation of environmental procarcinogens, and the metabolism of pharmaceuticals and endogenous compounds. The proposal will address this hypothesis and characterize regulation of the enzyme. There are three specific aims: (i) We have expressed human CYP2S1 in bacteria, and demonstrated that it metabolizes several compounds that are toxic and/or carcinogenic to epithelial tissues. We will also over-express the enzyme in mammalian cells. Using these expression systems, we will screen for additional substrates, and also for procarcinogens that are activated to mutagenic (and therefore probably carcinogenic) derivatives by CYP2S1. The Km and Vmax values will be determined for representative compounds, and the metabolites that are formed will be identified. The degree to which CYP2S1 contributes towards the total metabolism of particular substrates in human epithelial tissues will be determined using an inhibitory antibody to the enzyme, (ii) We have shown that CYP2S1 is inducible by dioxin, carcinogenic polycyclic aromatic hydrocarbons (PAHs), and hypoxia. We will investigate whether the potential Xenobiotic Responsive Elements (XREs), or the potential Antioxidant Response Element (ARE) in the 5' flanking region of the human CYP2S1 gene mediate induction by dioxin and/or PAHs, and address the hypothesis that due to the particular nucleotide sequences of the above XREs, the gene responds better to PAHs than to dioxin in certain cells. We will also analyze the mechanism of hypoxic induction of the gene, (iii) We will generate a knockout mouse for Cyp2s1, and then generate a derivative of this mouse containing the human CYP2S1 gene, including its flanking regulatory regions. This "CYP2S1-humanized" mouse will be used to study the metabolism of substrates of human CYP2S1, the biological consequences of this metabolism, and the regulation of the human CYP2S1 gene by xenobiotics and hypoxia, thus complementing and extending specific aims 1 and 2. Our studies may demonstrate important roles for CYP2S1 in the metabolism of carcinogens, Pharmaceuticals and endogenous compounds, and may ultimately provide opportunities for reducing the deleterious effects of environmental carcinogens and the adverse effects of certain Pharmaceuticals in the human population. [unreadable] [unreadable] [unreadable] [unreadable]
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1 |
2008 — 2021 |
Hankinson, Oliver Nmn |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training in Molecular Toxicology @ University of California Los Angeles
DESCRIPTION (provided by applicant) This training grant will support pre-doctoral and postdoctoral students in the Molecular Toxicology Interdepartmental Program (IDP) at the University of California, Los Angeles (UCLA). Prior to the establishment of the IDP in 2000, toxicological research and training was scattered through many departments at UCLA, and lacked cohesion and coordination. Establishment of the IDP focused training in one program and stimulated interactions and collaborations among the participating faculty and their students and postdoctoral fellows. The training grant will help further consolidate, improve and expand the Molecular Toxicology Program, and signal the arrival of toxicology as an important player in the biomedical sciences at UCLA. The nine faculty of the training grant have their primary appointments in seven different departments in three different schools. Nevertheless their laboratories/offices are in close proximity to one another. The faculty members have substantial NIEHS funding. Most importantly, they have a common interest in the mechanisms whereby toxicants induce disease. Several of the faculty members investigate the role of air pollution particulates in the exacerbation of asthma. Others investigate the carcinogenic/mutagenic effects of these and other environmental pollutants. A new area for the program is the role of pesticides in the etiology of Parkinson's disease. Capitalizing on their experience with ambient air particles, some of the faculty have begun research on the emerging field of the toxicity of manufactured nanoparticles (nanotoxicology). One pre- and one postdoctoral position is requested in the first year. These numbers will progressively increase to four and two, respectively, in the fifth year. Pre-doctoral students will be supported for up to three years after they have completed their first year of course work. Postdoctoral fellows will receive two years of support. Five of the nine mentoring faculty are physician-scientists. These faculty members will provide an avenue for the recruitment of physicians to postdoctoral positions in the training grant. The grant is highly relevant to public health. A better understanding of the processes whereby air pollution, pesticides and other environmental pollutants cause diseases, including asthma, cancer, and/or Parkinson's disease, will lead to improved risk assessment as well as methodologies for reducing or eliminating the deleterious effects of these environmental agents. BACKGROUND This is a resubmission of an application for a training grant in Molecular Toxicology at UCLA. This revised application has undergone substantial revision to address concerns of the previous review. Training grant preceptors without R01 type funding have been omitted and the number of preceptors has decreased from fifteen to nine. Course requirements have been changed to reflect the addition of more toxicology courses. Previous incomplete sections/tables are now generally complete. Dr. Hankinson, the Program Director, presented that among the 9 faculty members included in this current training grant, 7 now have substantial NIEHS funding. In response to the concern that none of the mentors is affiliated with environmental health sciences, Dr. Hankinson points out that one of the new members of the faculty (Dr. Froines) is a Professor in the Department of Environmental Health Sciences, while two other members of the faculty (Drs. Ritz and Schiestl) have secondary appointments in that Department. He also points out that it should be noted that the Molecular Toxicology Program is an interdepartmental program, and believes that the resulting broad perspective represents one of the strengths of the program.
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1 |
2015 — 2018 |
Hankinson, Oliver Nmn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tcdd Inductions of Omega-6 and Omega-3 Pufa Metabolism Act Inversely On Tumor Progression @ University of California Los Angeles
? DESCRIPTION (provided by applicant): The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the prototype of a large number of non-genotoxic carcinogens, dietary phytochemicals and endogenous metabolites that act via binding the aryl hydrocarbon receptor (AHR). The TCDD-liganded AHR massively upregulates CYP1A1, CYP1A2 and CYP1B1 in many mammalian organs. We previously demonstrated that TCDD treatment markedly increases the levels of epoxides of both w-6 and w-3 polyunsaturated fatty acids (PUFA) in the liver and lungs of mice fed laboratory chow, most likely via the activities of the CYP1 family members. w-6 epoxides are known to stimulate tumor growth, angiogenesis, and metastasis in mice, while w-3 epoxides have the opposite effect. In this application, we propose the overarching hypothesis that TCDD will impact angiogenesis, growth and metastasis of tumors in either a positive or negative way, depending on the relative levels of w-6 epoxides and w-3 epoxides generated in the host and/or tumor cells. To address this overarching hypothesis, four specific aims are proposed. Specific Aim 1 will address the hypothesis that TCDD stimulation of w-6 and w-3 PUFA metabolism both in tissues of the host and in the tumor cells will enhance and inhibit, respectively, the above parameters of tumor progression. Mice will be fed either a high w-6/low w-3 or a low w-6/high w-3 PUFA diet, will be treated with or without TCDD, and then injected subcutaneously with cancer cells that are inducible by TCDD for the CYP1 enzymes. The above parameters of tumor progression will then be quantified. Studies will also address whether the differential effects of w-6 and w-3 PUFA on the induction of metastases by TCDD are independent of any effects of TCDD on primary tumors. An inhibitor of epoxide hydrolase will be utilized to ascertain whether the effects of TCDD are mediated by w-6 and w-3 epoxides. Specific Aim 2 will address the hypothesis that TCDD stimulation of w-6 and w-3 PUFA metabolism in tissues of the host in the absence of TCDD stimulation of PUFA metabolism in the tumor cells will stimulate and inhibit, respectively, the parameters of tumor progression. To ascertain whether the effects of TCDD are mediated by CYP1 enzymes, a Cyp1a1-/- :Cyp1b1-/- double knockout mouse will be utilized. Specific Aim 3 will address the hypothesis that TCDD stimulation of w-6 and w-3 PUFA metabolism within the tumor cells, in the absence of stimulation in the host, will enhance, and inhibit, respectively, tumor growth, angiogenesis and metastasis. Specific Aim 4 will utilize a model in which TCDD promotes the development of primary tumors in mice treated with the tumor initiator, diethylnitrosamine, to investigate whether tumor promotion by TCDD is enhanced by including w-6 PUFA in the diet and inhibited by w-3 PUFA, and to investigate whether promotion is dependent upon PUFA epoxides and CYP1 enzymes. These studies may establish a novel mechanism whereby TCDD stimulates tumor progression; suggest a novel mechanism of cancer protection by w-3 PUFA, and suggest novel mechanisms of action of beneficial dietary AHR agonists and endogenous AHR ligands.
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0.958 |
2016 — 2017 |
Hankinson, Oliver Nmn |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Crispr-Cas9 Screen For Novel Proteins Required For Induction of Cyp1a1 by Ahr @ University of California Los Angeles
? DESCRIPTION (provided by applicant): The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a large number of toxic effects, including carcinogenicity. All the toxic effects of TCDD and of related polychlorinated compounds are mediated by the Aryl Hydrocarbon Receptor (AHR) and depend upon transcriptional activation by the AHR of not yet fully identified downstream genes. AHR also mediates carcinogenesis by polycyclic aromatic hydrocarbons, (e.g. benzo[a]pyrene) which are important carcinogens in tobacco smoke and smog. CYP1A1, CYP1A2 and CYP1B1 are massively induced in many tissues and are likely responsible for mediating the carcinogenic effects of PAHs, via metabolizing them to electrophilic derivatives. After binding agonist, the AHR translocates to the nucleus and forms a dimer with the aryl hydrocarbon nuclear translocator (ARNT) protein, which then binds to the enhancer regions of responsive genes, thereby leading to their transcriptional activation. In the current proposal we will seek to identify novel gene products that play roles in the induction of gene transcription by AHR, by screening a CRISPR-Cas9 library. The CRISPR-Cas9 system can induce DNA double strand breaks at specific genomic loci through synthetic 20 nucleotide sequences within guide RNAs (gRNAs), which when targeted to coding regions of genes can generation of deletions or insertions, resulting in frameshift mutations which lead to loss of function at both alleles in a diploid cell. There are two specific aims. In Specific Aim 1, genes required for CYP1A1 induction will be identified by using the GeCKOv2 library, which targets 20,611 mouse genes (and thus nearly all protein coding genes) and also 1,178 microRNAs. The library is contained in a single high titer lentiviral vector (lentiCRISPRv2), which will be transduced into the mouse hepatoma cell line, Hepa-1. The transduced cells will be selected for 10 days in benzo[a]pyrene. (Benzo[a]pyrene-resistant clones exhibit loss of AHR-dependent induction of CYP1A1). PCR amplification of the integrated gRNAs and their sequencing will then be performed. Those genes represented by more than one gRNA, and/or identified in more than one transduced culture, and/or represented at high frequency, are likely to represent true positives. Specific Aim 2 will validate hits from the screen. Two gRNAs for each gene of interest will be inserted into lentiCRISPRv2 to determine if they confer benzo[a]pyrene resistance, reduce TCDD induction of CYP1A1, and reduce AHR and ARNT expression upon transduction into Hepa-1 cells. The corresponding endogenous genes will be sequenced in certain transductants to see if they contain deletions or insertions. Characterization of confirmed hits will be pursued in a future R01 application. Such hits may include gene products that affect AHR or ARNT expression or function, and/or that are required for transcriptional activation of CYP1A1. The current proposal therefore lays the foundations for a comprehensive analysis of the mechanism of AHR-dependent induction of gene transcription, and may lead to strategies for reducing the harmful effects of ligands of the AHR.
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0.958 |