FAMRI 2001-2002 Funding Cycle Awardees

Clinical Innovators

• Scott J. Antonia, MD, PhD: Despite many years of research with conventional treatment modalities (surgery, radiation and chemotherapy), the majority of patients diagnosed with lung cancer eventually die from their disease. Therefore, alternative treatment modalities need to be developed. Over the past decade there has been increasing interest in developing immunotherapy as an anti-cancer treatment modality, since it has been shown that tumor associated antigens exist, T cells reactive to these antigens are present within cancer patients, and objective tumor responses have been observed in clinical trials involving immunotherapeutic strategies. We propose to conduct a clinical trial involving the treatment of patients with advanced non-small cell lung cancer (NSCLC) with a therapeutic tumor vaccine. The vaccine consists of two components. The first is a cell line that we have created (GMCD40L) by transfecting the K562 cell line with both the GM-CSF and CD40 ligand genes. The second component is irradiated autologous tumor cells. The rationale for this vaccine is the GM-CSF secreted by the GMCD40L cells will recruit and differentiate dendritic cells (DCÕs), which are the most potent antigen presenting cells, and the CD40 ligand will be a powerful activation stimulus for the DCÕs. The irradiated autologous tumor cells, which will be admixed with the GMCD40L cells, will undergo apoptosis, and the resultant apoptotic bodies will be taken up and processed by the DCÕs as the source of tumor associated antigens. These loaded and activated DCÕs will then migrate to regional lymph nodes where they will be able to present the tumor antigens to effector T cells and activate them. The T cells will then be able to migrate to metastatic tumors and kill them. We propose to conduct a phase I/II clinical trial involving patients with stage IIIB and IV NSCLC who have malignant pleural effusions. The effusions will be drained and used as the source of the autologous tumor cell component of the vaccines, and we have already produced a GMCD40L lot sufficient to complete this trial. The primary endpoint of the phase I component of the trial will be toxicity, and the primary endpoint of the phase II component of the trial will be clinical efficacy (objective tumor response rate, time to progression and overall survival). The secondary endpoint of the trial will be assessment of the development of anti-tumor immune responses, both in vivo with DTH skin testing with autologous tumor cells, and with in vitro assays of T cell function.
  
  
  
  
  
• Danny Bluestein, PhD: Cigarette smoking has been recognized as a primary risk factor for cardiovascular diseases. There is a clear need for a simple, sensitive technique, to detect environmental tobacco smoke (ETS) effects on cardiovascular risk under flow conditions existing in the circulation. Enhanced function of the hemostatic system, in which platelets play a major role, is a significant underlying mechanism in cardiovascular disease and its progression. It is hypothesized that ETS significantly increases cardiovascular risk by predisposing platelets to activation in areas of elevated arterial flow stresses. The hypothesis will be tested by comparing the activation response of ETS-exposed platelets to normal and pathological flow stresses with that of non-exposed platelets. We have developed an innovative assay for platelet activation state (PAS), which will enable real-time measurement of ETS-induced platelet activity. Its broad based nature addresses the need for a clinical screening assay, and is particularly relevant to ETS induced cardiovascular risk. It will be used to establish the thrombogenic potential of platelets subjected to ETS and fractionated smoke products in vitro under normal and pathological flow conditions, and of platelets from individuals subjected to ETS, according to the following specific aims:
  
  1. Measure with PAS platelet activity under flow conditions of platelets directly exposed to pure nicotine, and fractionated, sidestream, and mainstream cigarette smoke extracts in vitro.
  
  2. Measure the base activity of platelets of non-smoking volunteers exposed and not exposed to ETS and define its distribution within individuals (intra-person) and among the population (inter-person).
  
  3. Measure the sensitivity of platelets exposed to ETS and derived fractions of S.A.1 and 2 to pathological flow conditions under conditions characterizing arterial stenosis, and in blood recirculating devices.
  
  Based on a modification of the prothrombinase assay method, the innovative feature of the PAS is the elimination of the feedback activation of platelets by thrombin, which enables segregating contributions to the hemostatic response, such as ETS induced platelet activation. Comparative measurements will be performed with flow cytometry. Normal and pathological arterial shear stresses will be closely replicated using an innovative cone-plate, computer-controlled, Dynamic Cell Shearing Device. Interaction with intact and damaged blood vessel wall such as found in arterial stenosis, will be achieved by incorporating endothelium and collagen-coated surfaces in the device. Measurements in Left Ventricular Assist Device model will test whether patients with such devices who are exposed to ETS may be at increased risk because of the possible enhanced reactivity of their platelets.
  
  
  
  
  
• Joseph A Califano, MD: Over 40,000 new cases of head and neck squamous cell carcinoma (HNSC) are diagnosed in the United States yearly, most of these due to tobacco exposure. Despite high cure rates for premalignant lesions and early stage cancers, the majority of late stage malignancies are fatal. Molecular genetic progression models and data on premalignant lesions has demonstrated that chromosomal loss occurs early in HNSC progression, and a high incidence of structural and numerical chromosomal aberrations are noted in HNSC. Recent investigation of genetic alterations in other malignancies has implicated chromosomal instability (CIN) as a means for genetic progression of malignancy. Chromosomal integrity is maintained by double stranded DNA break repair mechanisms; appropriate segregation of chromosomal material during mitosis is maintained via an intact spindle assembly checkpoint mechanism. Preliminary studies are presented indicating that CIN is present HNSC, and that both double-stranded DNA repair and spindle assembly checkpoint mechanisms may be altered in HNSC. CIN will be tested and then correlated with the integrity of the spindle assembly checkpoint in HNSC cell lines as determined by ability to undergo nocodazole-induced arrest. Fluorescence-in-situ hybridization (FISH) using centromeric probes will be performed on expanded single colonies from HNSC and appropriate control cell lines during a finite number of cell divisions and over extended passage in culture. Nocodazole-induced arrest in HNSC and appropriate control cell lines will be assayed by standard determination of mitotic index, and by flow cytometry to determine cell cycle fraction. Complementation of gene products commonly lost in HNSC cell lines and primary will also be performed concurrently with the assay described above to determine the effect of complementation on chromosomal instability and spindle assembly checkpoint integrity. The integrity of double-strand DNA repair mechanisms in HNSC cell will be investigated. A recombination substrate construct will be stably transfected into HNSC cell lines and appropriate control cell lines. Transient transfection of a plasmid expressing a restriction enzyme will cleave the recombination substrate at this unique restriction site. Endogenous repair of the substrate via homologous recombination results in expression of a reporter, green fluorescent protein, while non-homologous end-joining products will not express reporter protein. Fluorescence activated cell sorting, real-time and conventional polymerase chain reaction, and southern blot analysis will be used to analyze the resulting cell population and determine the relative integrity of each repair pathway. Complementation of gene products commonly lost in HNSC cell lines and primary tumors (e.g. p16 and p53 expressing vectors) will also be performed concurrently with the assay described above to determine the effect of complementation on double stranded DNA repair pathways. High throughput assays for evaluation of double-stranded DNA repair will be developed in a real-time PCR model. Investigation of the basic, most early events in HNSC development will provide insight into strategies for screening, prevention, and treatment of this tobacco-related cancer.
  
  
  
  
  
• Xingming Deng, MD, PhD: Lung cancer is the leading cause of death in the United States, and it demonstrates a strong etiological association with smoking. Nicotine is a major component in tobacco, and it can be generally found in the blood of smokers. Chronic exposure to nicotine can lead to sustained activation of growth-promoting pathways and result in the development of lung cancer, as well as decrease the efficacy of anti-cancer agents. Although nicotine has been implicated as a potential factor in the pathogenesis in human lung cancer, its mechanism of action in the development of this cancer remains unknown. Interestingly, nicotine, as a potential factor of lung cancer development, can activate physiological Bcl2 kinases PKC and MAPK/ERK1/2 and inhibit chemotherapeutic drugs-induced apoptosis in lung cancer cells. Thus, we hypothesize that the mechanism involved nicotine-induced inhibition of apoptosis may occur through phosphorylation of Bcl2. To critically test this hypothesis we have identified two specific aims: (1) To determine whether phosphorylation of Bcl2 is required for nicotine-induced lung cancer survival and chemoresistance, and identification of site(s) of nicotine-induced Bcl2 phosphorylation in vivo; (2) To determine whether inhibition of nicotine-induced Bcl2 phosphorylation enhances chemosensitivity in human lung cancer cells. State of the art molecular and biochemical methodologies will be employed including mutational analysis with in vitro and in vivo expression studies. The results are expected to fill in fundamental gaps in our knowledge regarding the signaling mechanism by which nicotine regulates Bcl2 and apoptosis in lung cancer cells. From the results it is expected that efficient development of new and novel anti-lung cancer strategies that target these mechanisms will occur.
  
  
  
  
  
• Ronald B. Gartenhaus, MD: There are estimated to be over 50,000 new cases of lymphoma in the United States over the past year. The incidence of lymphoma has inexplicably dramatically increased over the past two decades even when excluding those lymphomas that are HIV - associated. While some of this increase has been associated with chemical carcinogens in herbicides and pesticides, there is scant data linking most lymphomas with etiologic factors. Recently, there have been several reports suggesting an increased incidence of follicular non-Hodgkin's lymphoma (NHL) with cigarette smoking. In approximately 40% of patients with follicular lymphoma the disease evolves to a histologic pattern consistent with a diffuse large cell lymphoma. While the vast majority of follicular lymphoma cases have the BCL-2 oncogene transposed adjacent to the site of the heavy immunoglobulin heavy chain gene t(14;18), the transformed follicular lymphoma is often associated with additional genetic alterations including mutations of the p53 tumor suppressor gene. Furthermore, in many cases of transformation a high level of microsatellite instability is observed with this large cell transformation.
  
  The Specific Aims of this proposal are as follows:
  1. Comparison of p53 Mutations in Transformed Follicular Lymphoma from Smoking and Nonsmoking Individuals.
  2. Comparison of the Presence of a Mutator Phenotype in Transformed Follicular Lymphoma from Smoking and Nonsmoking Individuals.
  
  DNA will be prepared from follicular lymphomas that transformed to a diffuse large cell phenotype and medical records will be reviewed to determine smoking histories of patients. In Specific Aim 1 examination of exons 4-8 of p53 in our tumor samples will be analyzed by direct sequencing thus providing valuable information relating to the frequency and spectra of mutations that are present in smoker and nonsmoker tumors. This information may ultimately prove to have prognostic value for example, if a specific genetic profile is associated with rapid clinical progression or poor response to therapy. In Specific Aim 2 we will perform experiments to determine the incidence of the mutator phenotype in smoking and nonsmoking patients with transformed follicular lymphoma. Specifically, we will characterize microsatellite instability using a validated panel of 5 microsatellite markers, which will be analyzed using a fluorescence - based DNA sequencer technique. The loss of DNA replication repair function may lead to increased mutations in genes critical to cell growth regulation and possibly drug resistance.
  
  
  
  
  
• Sergei A. Grando, MD, PhD, DSc: Purpose. This proposal will identify a role of keratinocyte cholinergic system in mediating deleterious effects of environmental tobacco smoke (ETS) on human skin. It will demonstrate how ETS-induced structural and functional changes of the nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChR) expressed by keratinocytes (KC) translate into alterations of cell proliferation, differentiation and apoptosis.
  
  Background. A continuous cycle of cell birth and death in the epidermis is a self-sustaining process controlled, in part, by locally produced ACh through the signaling pathways that couple each keratinocyte nAChR and mAChR to regulation of a particular cellular function. Nicotine (Nic), too, may use these signaling pathways to produce its pathobiological effect in the skin.
  
  Preliminary Observations. Signaling from keratinocyte nAChRs initiates complex changes in cell cycle regulation, including proliferation-inducing effects, DNA repair and replication anomalies, and pro-apoptotic gene activation. Normal functioning of keratinocyte ACh axis is altered in tobacco users. Overstimulation of KC with Nic provides for an unopposed activation of nAChRs, which both elicit Ca2+ influx and increase the concentration of intracellular Ca2+ leading to alterations of normal cell cycle.
  
  Hypotheses. 1) Chronic exposure to Nic disturbs dynamic equilibrium between nicotinic and muscarinic pathways of keratinocyte control by ACh, leading to an aberrant cell cycle with precocious squamatization. 2) Nic-induced alterations in the keratinocyte cell cycle progression and differentiation stem, at least in part, from reciprocal alterations in the expression of ACh receptors that control cell functions in KC.
  
  Specific aims. To identify long-term effects of ETS vs. pure Nic on: 1) the cell cycle progression and differentiation of normal human KC; and 2) expression/function of keratinocyte nAChRs, mAChRs, and the calcium signaling pathways subserving ACh signaling in KC.
  
  Methodology. A combination of molecular biological, immunochemical, pharmacological and biological assays will be employed to quantitate EST/Nic-induced changes in the cell cycle progression and differentiation regulators, and expression of ACh receptors due to chronic exposures of KC to tobacco products in vivo and in vitro. To conclude that an effect of ETS is mediated by nAChRs, it should be abolished in the presence of receptor antagonist, and, vise versa, reproduced by equivalent dose of nicotinic agonist.
  
  Significance. Obtained results will advance the knowledge about pathobiologic effects of Nic in the epithelium, lay a groundwork for future mechanistic studies of the receptor-mediated ETS toxicity, and provide a scientific basis for new approaches to ameliorate consequences of ETS exposure by interceding in disease pathways.
  
  
  
  
  
• Randolph H. Hastings, MD, PhD: The broad objectives of this project are to study fundamental mechanisms involved in progression of lung cancer and to provide the background for developing new therapies. Environmental tobacco smoke is a known cause of lung cancer according to the EPA. Our preliminary data suggest that parathyroid hormone-related protein (PTHrP), a growth factor made by many lung cancers, may predispose lung cancer cells to apoptosis if it is transported to the cell nucleolus. Furthermore, a specific isoform, PTHrP 1-173, is made by about 30% of lung cancers and is highly localized to the nucleolus. Thus, PTHrP 1-173 expression and nucleolar PTHrP may slow progression of lung cancer. The aims of this project are to evaluate PTHrP 1-173 expression and nucleolar PTHrP as favorable prognostic signs for non-small cell lung cancer (NSCLC) in patient and cell culture studies. To study prognostic significance, nucleolar PTHrP and PTHrP 1-173 mRNA will be evaluated in archival lung tumor specimens by immunocytochemistry and in situ hybridization, respectively. Clinical and demographic parameters will be collected from the VA tumor registry database. The effects of PTHrP expression, tumor histology, stage, type of therapy, age and gender will be analyzed with a Cox proportional hazards regression model. The mechanism by which nucleolar PTHrP may inhibit NSCLC progression will be investigated in cell culture. NSCLC cells will be stably transfected to express PTHrP 1-173, which localizes to the nucleolus, PTHrP 1-173 mutants and PTHrP 1-141, with nuclear but limited nucleolar distribution, and PTHrP 1-87, which is predominantly cytoplasmic. Cells will be treated with chemotherapeutic agents and apoptosis will be measured by evaluating nuclear morphology, terminal uridine nucleotidyl transferase end nick labeling (TUNEL) and flow cytometry for cell cycle distributions. Our research is innovative in investigating the importance of expression of a specific isoform of PTHrP and its distribution to the nucleolus on prognosis and response to therapy. These studies could lead to development of a serum PTHrP assay for diagnosis and prognosis in lung cancer. In addition, they could form the groundwork for developing therapeutic modalities for treating lung cancer with PTHrP protein or nucleic acid reagents.
  
  
  
  
  
• Jesse P. Joad, MD: Environmental tobacco smoke (ETS) exposure is associated with increased incidence and severity of lower airway infections in infants. Respiratory syncytial virus (RSV) is the most common virus causing severe bronchiolitis in infants and is associated with an increase in cysteinyl leukotrienes and interferon gamma. ETS exposure also appears to be associated with a T helper lymphocyte-2 phenotype which increases eosinophil recruitment and survival. Eosinophils release cysteinyl leukotrienes that produce airway narrowing, microvascular leak, and mucus production, the hallmarks of bronchiolitis. The hypothesis of the study is that the increased severity of RSV infections in infants exposed to ETS is due to the excess production of cysteinyl leukotrienes and thus a leukotriene receptor antagonist should be of therapeutic benefit. Infants and children who are hospitalized for RSV bronchiolitis (60 patients recruited over a 2 year period) will be randomly assigned to receive montelukast, a leukotriene receptor antagonist, 0.25 mg/kg daily for 7 days. A history of ETS exposure will be obtained and urine will be tested for cotinine, a metabolite of nicotine. An infant will be assumed to be exposed to ETS if the urinary cotinine level is more than 30 ng/ml. Nasal secretions will be analyzed for interleulkin-4 (IL-4), IL-5, leukotriene C4 (LTC4), and interferon gamma. The primary outcome variable will be duration of hospitalization. The secondary outcome variable will be a bronchiolitis scoring system including respiratory rate, oxygen requirement, auscultatory findings, retractions, and dyspnea. If the hypothesis is correct, the infants with RSV bronchiolitis who are exposed to ETS will have higher concentrations of IL-4, IL-5, and LTC4 and lower concentrations of interferon-gamma in their nasal secretions. Montelukast will decrease the hospital stay and the bronchiolitis score for all patients with RSV but more so for those who are exposed to ETS.
  
  
  
  
  
• Lester Kobzik, MD: The Problem: Exposure to cigarette smoke leads to an increased rate of respiratory tract infections. The molecular mechanism(s) for this important health effect of tobacco smoke are unknown, but they are likely to target the scavenger receptors on alveolar macrophages (AMs) ---receptors, which mediate the AMÕs primary function of defense against inhaled infectious agents and particulate matter.
  
  Hypotheses: 1) cigarette smoke down-regulates levels of class A scavenger receptors (i.e.,MARCO) on human AMs; 2) cigarette-smoke mediated decreased expression of MARCO results in diminished ability to bind and kill bacteria; 3) the effect of cigarette smoke particulates on MARCO levels is mediated by oxidant-dependent pathways and will be ameliorated by anti-oxidants, providing theoretical grounds for potential therapeutic interventions.
  
  Experimental plan: Normal human AMs (obtained by bronchoalveolar lavage of healthy, nonsmoking subjects) will be studied in vitro. The effects of side-stream and mainstream smoke components will be measured in 3 aims that test the hypotheses above. Aim 1 will use a novel monoclonal antibody specific for human AM MARCO to measure expression levels after exposure to smoke components by immunofluorescence and flow cytometry. Aim 2 will evaluate the effects of smoke on AM binding of fluorescent bacteria (flow cytometry) and subsequent bacterial killing. Aim 3 will measure intracellular oxidant production in AMs exposed to smoke and test the ability of antioxidants (e.g., nacetylcysteine) to block changes in MARCO expression and bacteria binding and killing functions.
  
  Significance: The proposed studies address specific molecular mechanisms of smoke-mediated AM dysfunction and will provide new insights about personal therapeutic targets to reduce smoke-related respiratory infections.
  
  
  
  
  
• David J. Lee, PhD: AIM: The proposed study is an expansion of the Florida Youth Cohort Study (FYCS), a study designed to monitor the impact of Florida's youth-focused anti-tobacco campaign in a representative sample drawn from 4th-7th grade students in 1998 (n=1219). This proposed study will continue to monitor these youth with an exclusive focus on the vast majority of children in the cohort who do not use tobacco (i.e. 92%), but who are exposed to environmental tobacco smoke (i.e., > 40%). representative sample drawn from 4th-7th grade students in 1998 (n=1219). This proposed study will continue to monitor these youth with an exclusive focus on the vast majority of children in the cohort who do not use tobacco (i.e. 92%), but who are exposed to environmental tobacco smoke (i.e., > 40%). The overall study aim is: determine the association between biologically verified exposure to environmental tobacco smoke (ETS) and health status, academic performance and quality of life in adolescents who do not use tobacco products.
  
  RATIONALE: Exposure to maternal prenatal ETS is associated with the development of respiratory illnesses, middle ear infections, and possibly developmental problems in infants and young children. Relatively few studies have addressed the impact of ETS on the health of older children. Furthermore, most of these studies failed to control for prenatal exposure to ETS. Many of these studies also relied on self-reported exposure to ETS, which is subject to reporting error and other limitations. The use of salivary cotinine, a metabolite of nicotine, is a valid and reliable marker of ETS exposure.
  
  METHODS: If funded, this study will measure cotinine levels in all FYCS participants during the fifth round of the survey. The interview will be expanded by asking a series of questions on school performance, ETS exposure, health status and psychosocial functioning. Both children and parents will participate in separate interviews; cotinine analyses will be limited to youth only.
  
  
  
  
  
• David T. Levy, PhD: Public policies may be directed at treating or preventing ETS exposure. Prevention policies, such as clean indoor air laws, limit smoking in the presence of nonsmokers. Other policies, such as taxes and media campaigns, indirectly reduce ETS by changing norms or by influencing smokers to quit or reduce the quantity of cigarettes smoked.
  
  This project proposes to incorporate the health effects of ETS into SimSmoke, a computer simulation model of tobacco control policies. SimSmoke simulates the effects of tax, clean indoor air laws, youth access, cessation, and mass media policies on smoking rates and smoking-attributable deaths by gender, age, and racial/ethnic groups. Specifically, we will:
  
  - Review existing literature on the effects of ETS exposure and examine factors affecting exposure. Specifically, we will examine how the different ETS health outcomes, including lung cancer, heart, stroke, emphysema, asthma, and pregnancy complications are related to ETS exposure. We also will empirically examine how exposure relates to smoking and demographic characteristics of the population.
  
  - Examine the effect of policies on ETS exposure. Specifically, we will consider the direct effect of clean indoor air policies and home smoking bans, and the indirect effects of other tobacco control policies, such as taxes and media policies taxes. We will also conduct an empirical analysis to examine the relationship between voluntary smoking bans and public tobacco policies.
  
  - Incorporate the effects of smoking on ETS-attributable health outcomes into SimSmoke. We will estimate their prevalence for the total population and different demographic groups.
  
  - Track the effects of public policies on ETS-attributable health outcomes using SimSmoke. We will report direct effects and demonstrate any indirect effects by encouraging voluntary home and work smoking bans.
  
  Modeling these phenomena as part of a dynamic social system helps researchers better understand the effects of policies and of limitations in our knowledge. In addition, it enables policy planners to better justify, plan, and predict the effects of tobacco control policies. The model and research will be used to disseminate information on effective policies to reduce ETS and its health consequences.
  
  
  
  
  
• Matthew L. Meyerson, MD, PhD: Carcinoma of the lung is the leading cause of cancer death in the United States, with most cases attributed to tobacco smoking and many to environmental tobacco smoke. We have recently generated a provisional classification scheme for lung adenocarcinoma, the most common sub-type of lung carcinoma, based on gene expression analysis. In parallel, we have pioneered the use of single nucleotide polymorphism (SNP) arrays for analyzing cancer loss-of-heterozygosity (LOH). We now propose to determine whether there are distinct patterns of LOH that correspond to the gene expression-derived lung adenocarcinoma classes. The discovery of such patterns would help to validate the adenocarcinoma classification and would be useful in the identification of lung cancer-associated tumor suppressor genes. Specifically, we propose the following aims: 1. To analyze patterns of chromosome loss of heterozygosity (LOH) in lung adenocarcinoma sub-classes defined by gene expression profiles, using single nucleotide polymorphism (SNP) array hybridization for over 1,400 markers. 2. To generate a genome-wide map of LOH in these samples. 3. To compare classification by LOH with classification by gene expression. Improvements in understanding the classification and pathogenesis of lung carcinoma are essential steps towards better diagnostic and treatment approaches. The combination of gene expression and allelotype classifications for lung adenocarcinoma should aid in fighting this deadly disease.
  
  
  
  
  
• John L. Pauly, PhD: The long-term objective of this investigation is to define bioassays for correlating environmental tobacco smoke (ETS) exposure with the early detection of lung pathology of never smokers, particularly members of a cohort of airline attendants. Lung macrophages (Mf) ingest numerous tobacco smoke microparticles ("tar"). The particles fluoresce; this is due to absorbed polycyclic aromatic hydrocarbons (PAH) that are generated during tobacco pyrolysis. Tar-pigmented Mf can be quantified by fluorescence activated cell sorting (FACS). Fluorescent Mf accumulate in large numbers in response to long-term smoking, and reside in the lung for many years (T1/2 = 5 years) after smoking cessation. It is theorized that the Mf are activated by the tobacco particles ("foreign bodies"), and that the activated Mf respond by secreting cytokines and other immunoregulatory molecules that mediate chronic pulmonary inflammation. Noteworthy is that inflammation is an early event in lung disease that precedes tobacco-associated tissue dysplasia and cytogenetic alterations. The hypothesis to be tested is that the smoke-activated Mf generate a unique array of pro-inflammatory cytokines. The specific aim, therefore, is to develop an assay for measuring an array of pro-inflammatory cytokines produced by Mf. The rationale is to use the test in future studies of Mf from induced sputum samples and/or bronchoalveolar lavage specimens of ETS-exposed personnel. In the experimental design, Mf will be isolated from fresh, surgically excised, human lung tissues of patients with lung cancer and sudden death cadavers with known smoking histories. Subjects will include current smokers, former smokers and never smokers Ð never smokers will be classified according to their lifetime exposure to ETS. Mf will be cultured in the absence (control; non-elicited) or presence (test; induced) of diverse stimulants (e.g., airborne pollutants: particles, fibers and tar). The array (i.e., cytokine type, dose and duration) produced will be assayed using a novel high throughput (N ³ 24 cytokines, 200 _L sample) technology that utilizes cytokine-capture fluorescent latex microspheres and high-speed FACS analysis. Biological activity of the human lung Mf-derived cytokines will be measured using human liver reporter cells that react to certain pro-inflammatory cytokines by producing acute phase proteins (APP; type I, a1-acid glycoproteins; type II, a2-macroglobulin). The APP will be assayed using rocket gel electrophoresis and Western blots. The significance of this investigation is that it will identify a unique array of pro-inflammatory cytokines that will serve as a biomarker for defining tobacco exposure and medical intervention required for ETS-related diseases.
  
  
  
  
  
• Roberto Pili, MD: Background: Vitamin A and is crucial for maintaining normal functions in epithelial cells and its analogs, retinoids, have been shown antitumor activity in preclinical tumor models. However, retinoids demonstrated limited clinical benefit in the prevention and treatment of solid tumors. A major hurdle of retinoid therapy is represented by the retinoid resistance. Loss of retinoic acid receptor b__RAR-b)_expression, due to either loss of RAR-b loci (genetic changes), or DNA methylation and repressive chromatin remodeling (epigenetic changes) at the RAR_ b _ has been associated with retinoid resistance. Epigenetic changes are reversible. Our preclinical data suggest that combining histone deacetylase inhibitors (HDACIs) and retinoids may revert epigenetic repression at RAR_b and consequently enhance/ restore retinoid sensitivity. Interestingly, selected loss of expression of RAR-b has been also detected in pre-cancerous lesions of the lung, oral cavity, cervix and skin.
  
  Hypothesis: In this proposal we hypothesize that: 1) retinoid resistance in tumors is associated primarily with epigenetic loss of expression of RAR-b 2) retinoid resistant tumors with epigenetic changes at RAR-b _may benefit from a combined therapy with RAR-b _agonists and chromatin-remodeling drugs (HDACIs); 3) loss of RAR_b expression due to epigenetic changes is a general phenomenon occurring in epithelial pre-cancerous lesions.
  
  Specific aims: Our goals are: A) to test a targeted transcriptional therapy to enhance/restore retinoid response in patients with metastatic solid tumors by combination of an HDAC inhibitor (SAHA) with 13-cis retinoic acid (CRA); B) in a parallel study, to assess the role of RAR-b loss during epithelial tumor cancer development by analyzing pre-cancerous lesions in breast and prostate samples obtained from our tissue bank.
  
  Study design: The specific aim 1) is directed to determine the maximum tolerated dose of SAHA in combination with CRA. The specific aims 2) and 3) are directed to analyze tissue expression and epigenetic changes at RAR-b _in tumors and pre-cancerous lesions by in situ hybridization and other specific molecular techniques.
  
  Relevance: This project is the first clinical attempt to evaluate this novel molecular targeted therapy in patients with epithelial tumors Thus, if combination of the HDAC inhibitor SAHA with the retinoid CRA is shown to modulate RAR-b expression in metastatic solid tumors, and progressive loss of RAR-b is documented in breast and prostate pre-cancerous lesions, this proposal will provide not only a novel therapeutic approach for the treatment of epithelial tumor patients, but also a chemoprevention strategy in smoke-related and high incidence tumors, such as breast and prostate cancer.
  
  
  
  
  
• Ronald Rodriguez, MD, PhD: Tumors derived from the urothelial lining of the urinary system, often referred to as transitional cell epithelium, account for almost 50,000 newly diagnosed cancers per year in the United States. Fortunately, the majority of these tumors are superficial bladder tumors, which can be managed endoscopically; however, bladder cancer is still the fourth most common cause of cancer death in males and the second most common malignancy associated with tobacco exposure. Bladder cancer treatment failures are both common and morbid. Superficial disease is known to have relapse rates as high as 50-70%, and metastatic bladder cancer is incurable, with only modest susceptibility to chemotherapy. Thus novel new treatment methods are necessary to approach these treatment failures. We propose that biologically based and optimized gene therapy utilizing an adenoviral vector may prove an efficacious method of treating this disease. However, initial analyses of bladder cancer tissue culture lines have shown an increased rate of down regulation of the receptor for adenovirus (CAR, for Coxsackie-Adenovirus Receptor). We propose that CAR is down regulated in clinical specimens of bladder cancer as well. Such a down regulation would normally render bladder cancer a poor target for adenoviral gene therapy. However, we additionally propose that by utilizing certain differentiating agents, such as trichostatin (TSA) and sodium phenylbutyrate (PB), the CAR expression can be re-established, thus sensitizing the bladder cancer cells to adenoviral mediated gene transfer. Although no normal biologic function has been identified for CAR, structural homology reveals that it is highly similar to cellular adhesion molecules. As such, loss of expression of such an adhesion molecule may allow an increased invasiveness and potentially an increased metastatic potential. We hypothesize that differentiating agent induction of the CAR expression may also affect the natural history and progression of bladder cancer in general. Thus in this proposal we intend to fully characterize the susceptibility of clinical specimens to adenoviral gene transfer by analyzing the receptors necessary for binding and internalization in both superficial and invasive disease. We will assess the ability to reconstitute CAR expression utilizing a panel of differentiating agents, including TSA and PB, as well as the persistence of CAR re-expression. We will then assess and the biologic effect of CAR re-expression on cellular invasiveness in vitro. These studies will provide the framework for potentially new and novel methods of bladder cancer treatment, both in adenoviral gene therapy and potentially in intra-vesical treatments as well.
  
  
  
  
  
• Lei Xiao, PhD: The overall objectives of the current proposal are to investigate the relationship between expression of the protein kinase C-epsilon (PKC-ε) isoform and the outcome of patients in response to chemotherapy. This relationship may have a potential use as a prognostic factor in treatment of patients with lung cancer. We have recently discovered a correlation of the expression of specific PKC isoforms with chemo-resistance of lung cancer cells. A striking differential expression profile of the PKC-ε isoform was found in human lung cancer cell lines. PKC-ε expression was detected in non-small cell lung cancer (NSCLC) cell lines, but not in small cell lung cancer (SCLC) cell lines. Importantly, our preliminary studies demonstrated that induction of PKC-ε expression in SCLC cells conferred resistance to clinically useful chemotherapeutic drugs, etoposide and doxorubicin. Down-regulation of PKC-ε expression in NSCLC cells increased the sensitivity to etoposide treatment. Based on our studies using lung cancer cell lines, we hypothesize that the difference in the regulation of PKC-ε expression is important with respect to cellular response to chemotherapy in lung cancer. The proposed studies are designed to extend our in vitro findings to pre-clinical studies in order to determine the clinical significance of PKC-ε expression on chemotherapy in patients. In addition, we wish to determine the genetic basis of the PKC-ε mediated chemo-resistance. Towards these ends we intend to:
  
  Specific Aim 1: Evaluation of PKC-ε expression in clinical samples obtained from patients with different stages of lung cancer, including SCLC and NSCLC; and investigation of the relationship between the status of PKC-ε expression and the outcome of chemotherapy in patients.
  
  Specific Aim 2: Investigation of the effect of PKC-ε expression on the overall patterns of gene expression, with an emphasis on the identification of differentially expressed genes and defining relationships among these genes in response to chemotherapeutic drugs.
  
  
  
  
  

Young Clinical Scientists

• Rhoda M. Alani, MD: The objectives of this proposal are to determine the role of histone acetyltransferases (HATs) in the development of human papillomavirus (HPV)-associated malignancies including cervical and head and neck cancers, and to develop HAT inhibitors as therapies for HPV-related tumors.
  
  Over the past several years the notion of chromatin remodeling as a means for coordinated regulation of gene expression has become widely accepted. HATs have been found to function as transcriptional coactivators through their ability to modify chromatin structure. DNA tumor viruses like the HPVs have evolved mechanisms to exploit cellular transcription programs in order to promote cellular proliferation and cell cycle progression and have been shown to specifically activate HATs as a means of promoting cell growth. The specific aims of this proposal are: (1) To develop a series of HAT inhibitors (HATIs) that will efficiently inhibit oncogenic HPV activation of these enzymes with minimal toxicity. The organic chemical synthesis of HATIs will take place in the laboratory of Dr. Philip Cole. In vitro analysis of compounds generated will be performed in HPV-positive and HPV-negative cervical cancer cell lines. (2) To determine the effect of HATIs on cell cycle progression and cell viability in HPV-positive and HPV-negative cell lines and identify HPV-specific inhibitors of cell growth. Various regulators of cell cycle progression will be examined in the presence or absence of the HATIs developed to identify compounds with the greatest potency and specificity for HPV-positive cancer cell lines. Cells will also be examined for apoptosis in the presence of HATIs to determine whether such a cell death program is initiated in the presence of the compounds tested. (3) To determine the precise mechanism of action of HPV-specific HATIs and their ability to affect cell cycle regulatory proteins. We will examine HAT-associated transcription factors involved in tumorigenesis to identify those most functionally affected by the HATIs tested to determine the most likely mechanistic targets of HATIs. Ultimately, HATIs that effectively inhibit the growth of HPV-positive tumor cell lines will be considered for further development as therapies for cervical and head and neck cancers.
  
  
  
  
  
• Wael K. Al-Delaimy, MD, PhD: Efforts to determine the associations between ETS and related illnesses have been undermined by the lack of accurate measures of ETS. The aim of this study is to assess the association between ETS and coronary heart disease (CHD) by using toenail biomarkers of nicotine in addition to questionnaire measures of exposure. We aim to determine the magnitude of effect of ETS exposure in relation to the risk of developing CHD among women participating in the Nurses Health Study (NHS). We also aim to explore the toenail biomarker as a measure of overall tobacco exposure in relation to lung cancer. A nested case-control study design will be carried out in the NHS cohort by selecting all CHD cases that were diagnosed between 1982 and 1992 and had available toenail samples collected in 1982. We will also assess long-term effect of tobacco smoke by selecting lung cancer cases diagnosed between 1986 and 1998 and who have available toenail samples from 1982. For each CHD case, two controls will be selected from a pool of controls for both CHD and lung cancer, out of those, three controls will be selected for each lung cancer case, matched by the time period of diagnosis of the cases and the date of return of toenail samples. Exposure will be assessed among cases and controls by analyzing toenail samples collected in 1982 from a subpopulation of 62,641 women participating in NHS. The toenail samples will be digested and analyzed for nicotine levels by using the High Performance Liquid Chromatography laboratory method to provide an objective measure of ETS exposure. In addition, questionnaire measures of ETS exposure were recorded in 1982. The dose-response relationship between toenail nicotine levels and incidence of CHD and lung cancer will be evaluated by logistic regression analysis adjusting for other CHD and lung cancer risk factors. Separate analyses by smoking history (non-smokers, active smokers), and by place of exposure to ETS among passive smokers (home, or work) will be carried out in secondary analyses.
  
  
  
  
  
• Ruth M. Belin, MD: Associations between active cigarette smoking and several forms of thyroid disease have been demonstrated. Smoking confers increased recurrence of Graves’ disease and substantially increased risk of associated Graves’ orbitopathy. This recognized environmental factor suggests the presence of an unrecognized, ubiquitous source of preventable thyroid morbidity—environmental tobacco smoke (ETS). Prior work shows that nearly nine of ten nonsmoking Americans have measurable biochemical markers of tobacco smoke. Ten percent of the population experiences some form of autoimmune, hyperplastic, or neoplastic thyroid disease. One in five adult women develop one or more of these thyroid conditions. Due to the high prevalence of both ETS and thyroid abnormalities, finding an association between ETS and thyroid disease could have tremendous implications for preventing thyroid disease and its complications, defining populations for thyroid screening, and decreasing costs associated with monitoring and treating thyroid abnormalities. The proposed study would evaluate the relationship between ETS and thyroid abnormalities, employing data from a large national cross-sectional investigation, the Third National Health and Nutrition Examination Survey (NHANES III). It uses multivariate analysis to examine the contribution of ETS on the risk of thyroid disease independent from potential influences of age, gender, diet, and race/ethnicity. This method would serve as a model for future work on ETS and other adverse endocrine effects, including changes in bone mineral density, fertility, or time of menopause.
  
  
  
  
  
• Shyam Biswal, Ph.D: Tobacco smoke (including second hand smoke) is generally accepted to be responsible for 85-90% of lung cancer cases which continues to be the leading cause of cancer death in most developed countries. Chemoprevention of tobacco smoke related cancer involves activation of antioxidant protective enzymes, which are involved in detoxification, and elimination of carcinogens. The goal of this proposal is to determine the functional characterization, distribution of known single nucleotide polymorphisms (SNPs) and identification of new SNPs for Nrf2. In recent years, Nrf2 has been established as the transcription factor which plays a central role in induction of a family of phase II antioxidant enzymes and proteins such as glutathione S- transferase, glutathione biosynthesis enzymes, UGT, NQO1, heme oxygenase and thioredoxin which provides chemoprotection against carcinogens by their detoxification and elimination from the cells. Studies using Nrf2 knockout mice have clearly demonstrated that a functional Nrf2 is indispensable for transcriptional activation of genes for almost all cancer protective enzymes. Lack of transcriptional activation of protective antioxidant enzymes in the Nrf2 knock out mouse has been shown to cause increased DNA damage in lungs after exposure to polyaromatic hydrocarbons. The knockout mice show increased sensitivity to benzo(a)pyrene, a potent carcinogen of tobacco smoke, and do not respond to chemopreventive agents, which act by induction of phase II proteins. It has been rationally predicted that single nucleotide polymorphisms that could decrease the transcriptional activator function of Nrf2 will be an important factor in (1) determining the cancer susceptibility after exposure to environmental carcinogens such as Tobacco smoke and (2) prediction of success of chemoprevention based on inducers of antioxidant enzymes. Functional assays using site directed mutagenesis and invitro transcription-translation of Nrf2 will be developed for functional characterization of the known Nrf2 SNPs. Exon sequencing of Nrf2 using genomic DNA from lymphoblastoid cells derived from 100 Caucasian and 100 African American will yield the preliminary information on the distribution of known SNPs and identification of new SNPs. This completion of the project would set the foundation for a broader study to understand the functional correlation of cSNPs of Nrf2 with cancer susceptibility and help in prediction of the target population where chemoprevention by phase II enzyme induction is likely to succeed.
  
  
  
  
  
• Paul Cairns, PhD: Because successful treatment of bladder and renal cancer depends upon early detection, there is a critical need for new and improved diagnostic tools. My long term goal is to identify genetic alterations and determine any translational utility. Genetic alterations are very promising markers for molecular diagnosis and prognosis because they can precede obvious cancer, are highly specific, can be detected by PCR-based techniques at extremely sensitive levels, can provide diagnostic and prognostic information simultaneously, and represent an absolute, rather than quantitative, change. My goal is to use these alterations as a target to detect neoplasia in bodily fluids that drain from the diseased organ using exciting new approaches such as Methylation Specific PCR (MSP). Our specific aims are 1) to validate the MSP molecular detection assay for the identification of cancer cell DNA in urine from 50 bladder and 50 renal cancers for simultaneous early diagnosis and molecular staging; 2) to construct progression models for bladder and renal cancer by determining the frequency and timing of hypermethylation of a panel of cancer genes in defined pathological stages from precursor lesions to tumors of all grades and stages from patients with and without a history of exposure to tobacco smoke; 3) to investigate the inclusion of new markers and application of chip technology to the panel to better address the heterogeneity of genetic alterations in, and behavior of, bladder and renal cancer. MSP-based approaches have been successful in the detection of several cancer types but have not been tested in bladder and renal cancer. Our preliminary results indicate that sensitive and specific molecular detection of bladder and renal cancer in urine is indeed feasible. Validation of a simple, non-invasive test for bladder and renal cancer would be of immense benefit towards the earlier diagnosis, and thereby cure, of these diseases. The progression models will identify the most useful markers for both early diagnosis and for molecular prognosis and may add to our understanding of the biologic basis of the disease. Molecular staging may improve the ability to assign pathological stage and to determine prognosis leading to more appropriate treatment and therefore better survival. Testing MSPs suitability for chip technology provides a basis for high-throughput analysis. Because genetic alterations can precede obvious cancer, eventually it may be possible to not only detect early stage cancer by MSP but to detect pre-cancer and respond with chemoprevention.
  
  
  
  
  
• Elizabeth J. Franzmann, MD: Head and neck squamous cell carcinoma (HNSCC) accounts for approximately 500,0000 incident cancers worldwide. The primary treatment modalities available today, surgery, radiation, and chemotherapy, have significant functional and aesthetic ramifications resulting in devastating patient morbidity, mortality, and substantial healthcare costs. Some of the most perplexing problems characteristic of HNSCC are its propensity to occur at multiple sites, to recur, and to metastasize distally. Establishing markers that identify HNSCC at an early stage and assess its potential to recur and metastasize would facilitate treatment delivery. Furthermore, development of more effective treatment strategies for patients with HNSCC will extend life and preserve quality of life. Studies in other tissues have shown that CD44, its ligand HA and the HA degrading enzyme hyaluronidase (HAase) are useful tumor markers. One main project goal is to determine if these molecules are useful markers for HNSCC. A second goal is to investigate the interactions between CD44 and its associated molecules in HNSCC progression in search of novel treatment targets. Our aims for achieving these goals are as follows: In Aim 1 we will use RT-PCR, western blot, ELISA-like assays, pH activity curves and tissue staining to examine the expression of CD44, HA, and HAase in tissues and saliva of HNSCC patients and cell lines. In Aim 2 we will study the CD44 gateway to oncogenic signaling using a stable CD44 transfectant, growth and migration assays, and CD44 blocking antibody. The HA barrier will be studied using HNSCC cell lines, ELISA-like assays, growth and migration assays, immunofluorescence staining, confocal microscopy, cocultures with fibroblasts and western blots. The results of these experiments will provide a better understanding of the role of HA and its associated molecules in HNSCC prognosis and disease progression. Accomplishing these goals will aid the principle investigator in further development as a physician scientist and preparation for a more comprehensive research plan in the future.
  
  
  
  
  
• Andrew J. Hyland, PhD: Smoke-free rules reduce people’s exposure to SS; therefore, they are an important component of reducing the disease caused by SS. A number of unresolved scientific questions remain concerning the impact of smoke-free rules on important measures such as behavior change and disease risk. The purpose of this project is to provide answers to the following broad study question:
  
  What economic, behavioral, and disease impact do smoke-free rules have?
  
  This question will be answered by examining several available data sources using statistical techniques to assess specific study questions from the following three research domains:
  Economic studies will include updating existing work on the economic impacts of smoke-free rules on hospitality business and examining the impact of smoke-free worksite rules on lost worker productivity using data from a 13-year follow-up survey of thousands of smokers.
  Attitude and behavioral studies will examine the impact of smoke-free work, home, automobile, and community rules on smoking behavior using data from the Community Intervention Trial for Smoking Cessation (COMMIT). Endpoints examined will include smoking cessation, quit attempts, amount smoked, level of dependence, attitudes and beliefs about secondhand smoke, and the type of nicotine-containing product consumed. Trends in people’s attitudes and beliefs about SS and smoke-free rules will be assessed using a variety of public opinion polls conducted since the 1950’s.
  Disease studies will use the COMMIT dataset to examine the association between smoke-free rules and self-reported tobacco-related morbidity such as emphysema, chronic obstructive pulmonary disease, and heart disease and death certificate confirmed mortality obtained through a National Death Index Mortality search.
  
  
  
  
  
• Francine Laden, ScD: There is a large body of epidemiologic evidence supporting an association of adult exposures to environmental tobacco smoke (ETS) with adverse effects on respiratory health. Studies have considered exposure in the home and the workplace, including office environments and service industry locations, but no study has focused on industrial environments. Although smoking rates have been declining among adults in the U.S., the smoking rates among blue-collar workers remain high. Thus, this population has a high potential for ETS exposure at work. We propose to study the association of ETS exposure with chronic respiratory tract symptoms in a cross-sectional study of the U.S. trucking industry. We will also measure air-borne nicotine as a marker of exposure to ETS in a large sample of workplaces throughout the industry, permitting us to validate the self report of exposure and to identify factors that determine exposure. To date, few studies have combined measurements of levels of ETS in the workplace with study of health effects.
  
  The study will be performed in conjunction with an ongoing National Cancer Institute funded national study of diesel exhaust exposure and lung cancer risk in four large unionized trucking companies. Detailed information on industrial exposures to diesel exhaust and other particulates will be available through the primary study. Therefore, we will be able to assess confounding or synergism of ETS with other industrial exposures. Access to this population provides a unique opportunity to combine research regarding ETS exposure and its association with respiratory symptoms with exposure assessment in a well-characterized blue-collar population.
  
  
  
  
  
• Daqing Li, MD: Chemotherapy plays an important role in the neoadjuvant or adjuvant setting and management of advanced and metastatic disease for the treatment of head and neck squamous cell carcinoma (HNSCC) (including oral cancer). The drug Cisplatin has played a major role in the chemotherapy and has been the most promising single chemotherapuetic agent used against HNSCC to date. The failure of Cisplatin monotherapy is apparently due to development of Cisplatin resistance or systemic toxicity, as high doses are required in order to obtain a significant antitumor effect. Enhanced DNA double-strand breaks (DSB) repair could be the primary cause for development of resistant in tumor cells to drugs, which induce DNA crosslinks or DSBs.
  
  Recently, a protein complex made up of hMre11, hRad50 and Nbs1 (MRN) has been shown to be a critical component of the cellular response to the repair of DNA DSBs. The proposed study will evaluate if the repair blockage hypothesis can interrupt the MRN complex in the tumor cells and therefore enhance the chemosensitization in Cisplatin-based chemotherapy for the treatment of HNSCC. Dominant negative recombinant adenovirus constructs that express domains of hMre11, hRad50 and Nbs1 will be developed and used to disrupt the normal function of the MRN complex. MRN-targeted chemosensitization to Cisplatin and Cisplatin cytotoxicity in human HNSCC cell lines will be examined. Fibroblast growth factor 2 (FGF2)-retargeted adenovirus delivery strategy will be used for enhancing outcome of dominant negative effects in human HNSCC cell lines.
  
  In order to achieve our goal, we proposed the following specific aims:
  
  Specific aim 1: To create recombinant adenoviruses that carry mutant hMre11, hRad50 and Nbs1 genes and express domains of hMre11, hRad50 and Nbs1.
  
  Specific aim 2: To determine the ability of expressing domains of hMre11, hRad50 and Nbs1 to sensitize human HNSCC cell lines following Cisplatin-based chemotherapy.
  
  Specific aim 3: To determine the efficacy of dominant negative construct alone and in combination with FGF2-retargeted strategy following Cisplatin-based chemotherapy against human HNSCC
  
  
  
  
  
• David M. Loeb, MD, PhD: WT1 is a transcription factor that is aberrantly expressed in breast cancer. Some genes that are regulated by WT1 that play a role in the disease have already been identified, but further work remains before the role of WT1 in the etiology and pathology of breast cancer is understood. The objectives of this research proposal are to identify new WT1 target genes that might be important for the development of breast cancer, and to investigate a correlation between WT1 expression and clinical outcomes in breast cancer patients. The specific aims are 1) to identify new WT1target genes important for proliferation and cell death, 2) to determine the effect of expressing WT1 in mammary epithelial cells, and 3) to utilize a breast tumor bank to correlate WT1 expression with both the expression of putative target genes and with clinical outcomes such as metastatic disease, response to chemotherapy, and survival. Some new target genes have been preliminarily identified, and initial work will focus on confirming these preliminary findings. A customized cDNA array generated based on data from an earlier gene expression profiling experiment will be used to identify other WT1 target genes important for the neoplastic transformation of mammary epithelial cells. Immortalized but not transformed mammary epithelial cells will be transfected with WT1 both to help confirm that these genes are bona fide targets in this system, and to determine the role of WT1 in neoplastic transformation of these cells. Finally, a breast tumor bank has been established, and this will provide the raw material for correlating WT1 expression and clinical outcomes, which will be necessary for translating these basic science results into a clinical intervention.
  
  
  
  
  
• David P. Miller, Sc.D., SM: The project, Molecular Epidemiology of Environmental Tobacco Smoke (ETS)-Related Lung Cancer aims to examine the role of genetic variants (known as polymorphisms) and environmental and occupational factors (such as asbestos) in the development of ETS-related lung cancer. The association between lung cancer and ETS is not as strong as lung cancer and mainstream smoking. Genetic polymorphisms may play an important role in determining how susceptible an ETS-exposed individual is to developing lung cancer. In a case-control study, genetic polymorphisms of the following classes of genes will be evaluated: carcinogen-metabolizing enzymes, such as the glutathione s-transferase family of enzymes (GST-mu, GST-theta, GST-pi), microsomal epoxide hydrolase, N-acetyltransferase 2, manganese superoxide dismutase; genetic polymorphisms of cell cycle genes (p53); and polymorphisms in certain DNA repair genes, XRCC1 and ERCC2. Each polymorphism will be evaluated using PCR (polymerase chain reaction) based genotyping techniques. The combined effect of asbestos and primary smoking on lung cancer risk is significantly greater than each risk factor alone. Using a semi-quantitative asbestos score, the combined effect of asbestos and ETS on lung cancer development will be evaluated. In this understudied area of research, we will also explore other techniques for estimating ETS exposure, gene-ETS relationships, and the role of ETS versus mainstream smoking. A better understanding of the role of genes and ETS in lung cancer may ultimately help in tailoring screening and prevention programs.
  
  
  
  
  
• Archana Mishra, MD, MS: Environmental Tobacco Smoke Exposure (ETS) is the third leading preventable cause of death. Among children it has been shown to cause lower respiratory infections, middle ear disease, sudden infant death syndrome, and asthma. Adults have the largest cumulative exposure to ETS and yet the effects of ETS exposure on adult respiratory health has not been investigated extensively and available data are fraught with methodological problems such as incomplete and /or inaccurate measurements of ETS exposure and inadequate ascertainment and control of potential confounders. In the proposed research project we will first investigate the relation between environmental tobacco smoke exposure and respiratory health in adult nonsmokers utilizing data of two cross-sectional studies. Our study is important because we propose to ascertain the impact of childhood and adulthood exposure, at work and home, looking at multiple outcomes to assess the overall impact on respiratory health. The proposed project offers two unique advantages. The first is, that the data have been collected already (in collaboration with the sponsors of this research proposal). The second is, that the collected information includes detailed ETS exposure and respiratory health outcome data not previously ascertained in other studies. At present, the first database includes 730 well-characterized nonsmokers who attended Roswell Park Memorial Cancer Institute for free cancer screening in 1986. The second database has 3693 subjects randomly selected from the adult population of Western New York. Data from both these study population will be used to calculate lifetime ETS exposure. Multiple linear regression models and other statistical techniques will be utilized to investigate the association of lifetime ETS exposure with respiratory health with adjustment for other covariates. The cross-sectional study will build the foundation for a longitudinal study to investigate impact of ETS exposure on lung function, respiratory symptoms, incidence of obstructive airways disease, quality of life, health care utilization and possibly mortality. This research could improve our understanding of the role of ETS exposure as modifiable factor in the etiology of impaired respiratory health. It will build the foundation for the development of future clinical research studies to better understand the etiologic mechanisms of ETS exposure and respiratory outcomes in the population that are found to be most susceptible to its effect.
  
  
  
  
  
• Neil Watkins, MD, PhD: Lung cancer caused by smoking results in more deaths than colon, breast and prostate cancer combined. Cytotoxic therapy of lung cancer is limited by side effects, and is rarely curative. Mechanism based therapies directed at tumor specific pathways offers hope for the development of novel treatments. Recapitulation of mammalian development pathways by human cancers may provide important insights into how such interventions can be achieved. The diffusible morphogen sonic hedgehog (Shh) is essential for lung development. I have shown that many human lung cancers recapitulate embryonic lung development by expressing components of the Shh pathway, and that inhibition of this pathway by the veratrum alkaloid cyclopamine inhibits the growth of lung cancer in vitro and in vivo. I hypothesize that Shh signaling is critical to the growth and malignant behavior of many human lung cancers, and that inhibition of this pathway may represent a novel therapy. To determine whether activation of Shh signalling is a common event in lung cancer, I first propose to look for evidence of Shh pathway activation in specimens of human lung cancer and pre-malignant bronchial airway epithelium using tissue array technology. Next, I will investigate the molecular and pharmacologic basis of cyclopamine in lung cancer cells. I then plan to examine the growth promoting effects of Shh signaling by overexpressing the transcriptional effector of Shh, Gli1, in immortalized human airway epithelial (BEAS2B) cells. Finally, I propose to use human lung cancer cell lines and Gli1 overexpressing airway epithelial cells to address the mechanisms of Shh signaling and cyclopamine mediated inhibition. This will involve the identification of novel downstream targets of the Shh pathway in lung cancer using microarray technologies. This research plan will firmly establish the importance of Shh signaling in lung cancer, will address the relationship between lung development and lung cancer, and provide a rational, mechanism based approach for the treatment of lung cancer with inhibitors of the Shh pathway.
  

Center of Excellence

• Neal Benowitz, MD: A FAMRI Center of Excellence on ETS will be established at the University of California, San Francisco in collaboration with UC Berkeley. The Center will be directed by Drs. Neal Benowitz and Rita Redberg. Scientific research is proposed in four areas - pulmonary disease, cardiovascular disease, exposure assessment and policy. The pulmonary disease research will examine the impact of ETS on the progression of chronic obstructive pulmonary disease and the progression of cardiopulmonary disease associated with aging. The cardiovascular research project will study abnormalities in pulmonary vascular resistance and gas exchange with exercise as an early manifestation of ETS-induced cardiovascular disease. The exposure assessment component will develop and validate improved or novel markers of ETS exposure and resultant injury, and will provide analytical chemistry support to the pulmonary and cardiovascular research components. The policy research group will study the development and implementation of smoking restriction policies, and will use a coronary heart disease population simulation model to estimate the cardiovascular health and economic consequences of ETS in the U.S. A new Clinical Practice devoted to the diagnosis and treatment of ETS-related diseases, as well as a site for ETS-related cardiovascular research, will be formed. Support for this center at a level of $1.5 million per year for five years is requested. This Center application brings together investigators from a broad range of disciplines (pharmacology, chemistry, epidemiology, policy analysis, clinical medicine) from two UC campuses with a long history of investigators involved in research into health effects and mechanisms related to exposure to ETS.
  
  
  
  
  Questions or problems regarding the use of this Web site should be submitted to famri@aibs.org.